Dual signal amplification for highly sensitive electrochemical detection of uropathogens via enzyme-based catalytic target recycling.

PubMed

Su, Jiao; Zhang, Haijie; Jiang, Bingying; Zheng, Huzhi; Chai, Yaqin; Yuan, Ruo; Xiang, Yun

2011-11-15

We report an ultrasensitive electrochemical approach for the detection of uropathogen sequence-specific DNA target. The sensing strategy involves a dual signal amplification process, which combines the signal enhancement by the enzymatic target recycling technique with the sensitivity improvement by the quantum dot (QD) layer-by-layer (LBL) assembled labels. The enzyme-based catalytic target DNA recycling process results in the use of each target DNA sequence for multiple times and leads to direct amplification of the analytical signal. Moreover, the LBL assembled QD labels can further enhance the sensitivity of the sensing system. The coupling of these two effective signal amplification strategies thus leads to low femtomolar (5fM) detection of the target DNA sequences. The proposed strategy also shows excellent discrimination between the target DNA and the single-base mismatch sequences. The advantageous intrinsic sequence-independent property of exonuclease III over other sequence-dependent enzymes makes our new dual signal amplification system a general sensing platform for monitoring ultralow level of various types of target DNA sequences. Copyright © 2011 Elsevier B.V. All rights reserved.

Detection of genetically modified organisms (GMOs) using isothermal amplification of target DNA sequences.

PubMed

Lee, David; La Mura, Maurizio; Allnutt, Theo R; Powell, Wayne

2009-02-02

The most common method of GMO detection is based upon the amplification of GMO-specific DNA amplicons using the polymerase chain reaction (PCR). Here we have applied the loop-mediated isothermal amplification (LAMP) method to amplify GMO-related DNA sequences, 'internal' commonly-used motifs for controlling transgene expression and event-specific (plant-transgene) junctions. We have tested the specificity and sensitivity of the technique for use in GMO studies. Results show that detection of 0.01% GMO in equivalent background DNA was possible and dilutions of template suggest that detection from single copies of the template may be possible using LAMP. This work shows that GMO detection can be carried out using LAMP for routine screening as well as for specific events detection. Moreover, the sensitivity and ability to amplify targets, even with a high background of DNA, here demonstrated, highlights the advantages of this isothermal amplification when applied for GMO detection.

Rapid detection of microbial DNA by a novel isothermal genome exponential amplification reaction (GEAR) assay.

PubMed

Prithiviraj, Jothikumar; Hill, Vincent; Jothikumar, Narayanan

2012-04-20

In this study we report the development of a simple target-specific isothermal nucleic acid amplification technique, termed genome exponential amplification reaction (GEAR). Escherichia coli was selected as the microbial target to demonstrate the GEAR technique as a proof of concept. The GEAR technique uses a set of four primers; in the present study these primers targeted 5 regions on the 16S rRNA gene of E. coli. The outer forward and reverse Tab primer sequences are complementary to each other at their 5' end, whereas their 3' end sequences are complementary to their respective target nucleic acid sequences. The GEAR assay was performed at a constant temperature 60 °C and monitored continuously in a real-time PCR instrument in the presence of an intercalating dye (SYTO 9). The GEAR assay enabled amplification of as few as one colony forming units of E. coli per reaction within 30 min. We also evaluated the GEAR assay for rapid identification of bacterial colonies cultured on agar media directly in the reaction without DNA extraction. Cells from E. coli colonies were picked and added directly to GEAR assay mastermix without prior DNA extraction. DNA in the cells could be amplified, yielding positive results within 15 min. Published by Elsevier Inc.

Microfluidic Devices for Forensic DNA Analysis: A Review

PubMed Central

Bruijns, Brigitte; van Asten, Arian; Tiggelaar, Roald; Gardeniers, Han

2016-01-01

Microfluidic devices may offer various advantages for forensic DNA analysis, such as reduced risk of contamination, shorter analysis time and direct application at the crime scene. Microfluidic chip technology has already proven to be functional and effective within medical applications, such as for point-of-care use. In the forensic field, one may expect microfluidic technology to become particularly relevant for the analysis of biological traces containing human DNA. This would require a number of consecutive steps, including sample work up, DNA amplification and detection, as well as secure storage of the sample. This article provides an extensive overview of microfluidic devices for cell lysis, DNA extraction and purification, DNA amplification and detection and analysis techniques for DNA. Topics to be discussed are polymerase chain reaction (PCR) on-chip, digital PCR (dPCR), isothermal amplification on-chip, chip materials, integrated devices and commercially available techniques. A critical overview of the opportunities and challenges of the use of chips is discussed, and developments made in forensic DNA analysis over the past 10–20 years with microfluidic systems are described. Areas in which further research is needed are indicated in a future outlook. PMID:27527231

A novel, sensitive and label-free loop-mediated isothermal amplification detection method for nucleic acids using luminophore dyes.

PubMed

Roy, Sharmili; Wei, Sim Xiao; Ying, Jean Liew Zhi; Safavieh, Mohammadali; Ahmed, Minhaz Uddin

2016-12-15

Electrochemiluminescence (ECL) has been widely rendered for nucleic acid testing. Here, we integrate loop-mediated isothermal amplification (LAMP) with ECL technique for DNA detection and quantification. The target LAMP DNA bound electrostatically with [Ru(bpy)3](+2) on the carbon electrode surface, and an ECL reaction was triggered by tripropylamine (TPrA) to yield luminescence. We illustrated this method as a new and highly sensitive strategy for the detection of sequence-specific DNA from different meat species at picogram levels. The proposed strategy renders the signal amplification capacities of TPrA and combines LAMP with inherently high sensitivity of the ECL technique, to facilitate the detection of low quantities of DNA. By leveraging this technique, target DNA of Sus scrofa (pork) meat was detected as low as 1pg/µL (3.43×10(-1)copies/µL). In addition, the proposed technique was applied for detection of Bacillus subtilis DNA samples and detection limit of 10pg/µL (2.2×10(3)copies/µL) was achieved. The advantages of being isothermal, sensitive and robust with ability for multiplex detection of bio-analytes makes this method a facile and appealing sensing modality in hand-held devices to be used at the point-of-care (POC). Copyright © 2016 Elsevier B.V. All rights reserved.

Global DNA methylation analysis using methyl-sensitive amplification polymorphism (MSAP).

PubMed

Yaish, Mahmoud W; Peng, Mingsheng; Rothstein, Steven J

2014-01-01

DNA methylation is a crucial epigenetic process which helps control gene transcription activity in eukaryotes. Information regarding the methylation status of a regulatory sequence of a particular gene provides important knowledge of this transcriptional control. DNA methylation can be detected using several methods, including sodium bisulfite sequencing and restriction digestion using methylation-sensitive endonucleases. Methyl-Sensitive Amplification Polymorphism (MSAP) is a technique used to study the global DNA methylation status of an organism and hence to distinguish between two individuals based on the DNA methylation status determined by the differential digestion pattern. Therefore, this technique is a useful method for DNA methylation mapping and positional cloning of differentially methylated genes. In this technique, genomic DNA is first digested with a methylation-sensitive restriction enzyme such as HpaII, and then the DNA fragments are ligated to adaptors in order to facilitate their amplification. Digestion using a methylation-insensitive isoschizomer of HpaII, MspI is used in a parallel digestion reaction as a loading control in the experiment. Subsequently, these fragments are selectively amplified by fluorescently labeled primers. PCR products from different individuals are compared, and once an interesting polymorphic locus is recognized, the desired DNA fragment can be isolated from a denaturing polyacrylamide gel, sequenced and identified based on DNA sequence similarity to other sequences available in the database. We will use analysis of met1, ddm1, and atmbd9 mutants and wild-type plants treated with a cytidine analogue, 5-azaC, or zebularine to demonstrate how to assess the genetic modulation of DNA methylation in Arabidopsis. It should be noted that despite the fact that MSAP is a reliable technique used to fish for polymorphic methylated loci, its power is limited to the restriction recognition sites of the enzymes used in the genomic DNA digestion.

Digital LAMP in a sample self-digitization (SD) chip

PubMed Central

Herrick, Alison M.; Dimov, Ivan K.; Lee, Luke P.; Chiu, Daniel T.

2012-01-01

This paper describes the realization of digital loop-mediated DNA amplification (dLAMP) in a sample self-digitization (SD) chip. Digital DNA amplification has become an attractive technique to quantify absolute concentrations of DNA in a sample. While digital polymerase chain reaction is still the most widespread implementation, its use in resource—limited settings is impeded by the need for thermal cycling and robust temperature control. In such situations, isothermal protocols that can amplify DNA or RNA without thermal cycling are of great interest. Here, we showed the successful amplification of single DNA molecules in a stationary droplet array using isothermal digital loop-mediated DNA amplification. Unlike most (if not all) existing methods for sample discretization, our design allows for automated, loss-less digitization of sample volumes on-chip. We demonstrated accurate quantification of relative and absolute DNA concentrations with sample volumes of less than 2 μl. We assessed the homogeneity of droplet size during sample self-digitization in our device, and verified that the size variation was small enough such that straightforward counting of LAMP-active droplets sufficed for data analysis. We anticipate that the simplicity and robustness of our SD chip make it attractive as an inexpensive and easy-to-operate device for DNA amplification, for example in point-of-care settings. PMID:22399016

On-chip isothermal, chemical cycling polymerase chain reaction (ccPCR)

NASA Astrophysics Data System (ADS)

Persat, Alexandre; Santiago, Juan

2008-11-01

We demonstrate a novel ccPCR technique for microfluidic DNA amplification where temperature is held constant in space and time. The polymerase chain reaction is a platform of choice for biological assays and typically based on a three-step thermal cycling: DNA denaturation, primers annealing and extension by an enzyme. We here demonstrate a novel technique where high concentration chemical denaturants (solvents) denature DNA. We leverage the high electrophoretic mobility of DNA and the electrical neutrality of denaturants to achieve chemical cycling. We focus DNA with isotachophoresis (ITP); a robust electrophoretic preconcentration technique which generates strong electric field gradients and protects the sample from dispersion. We apply a pressure-driven flow to balance electromigration velocity and keep the DNA sample stationary in a microchannel. We drive the DNA through a series of high denaturant concentration zones. DNA denatures at high denaturant concentration. At low denaturant concentration, the enzyme creates complementary strands. DNA reaction kinetics are slower than buffer reactions involved in ITP. We demonstrate successful ccPCR amplification for detection of E. Coli. The ccPCR has the potential for simpler chemistry than traditional PCR.

Protein Detection via Direct Enzymatic Amplification of Short DNA Aptamers

PubMed Central

Fischer, Nicholas O.; Tarasow, Theodore M.; Tok, Jeffrey B.-H.

2008-01-01

Aptamers are single-stranded nucleic acids that fold into defined tertiary structures to bind target molecules with high specificities and affinities. DNA aptamers have garnered much interest as recognition elements for biodetection and diagnostic applications due to their small size, ease of discovery and synthesis, and chemical and thermal stability. Herein, we describe the design and application of a short DNA molecule capable of both protein target binding and amplifiable bioreadout processes. As both recognition and readout capabilities are incorporated into a single DNA molecule, tedious conjugation procedures required for protein-DNA hybrids can be omitted. The DNA aptamer is designed to be amplified directly by either the polymerase chain reaction (PCR) or rolling circle amplification (RCA) processes, taking advantage of real-time amplification monitoring techniques for target detection. A combination of both RCA and PCR provides a wide protein target dynamic range (1 μM to 10 pM). PMID:17980857

Quantum dots for a high-throughput Pfu polymerase based multi-round polymerase chain reaction (PCR).

PubMed

Sang, Fuming; Zhang, Zhizhou; Yuan, Lin; Liu, Deli

2018-02-26

Multi-round PCR is an important technique for obtaining enough target DNA from rare DNA resources, and is commonly used in many fields including forensic science, ancient DNA analysis and cancer research. However, multi-round PCR is often aborted, largely due to the accumulation of non-specific amplification during repeated amplifications. Here, we developed a Pfu polymerase based multi-round PCR technique assisted by quantum dots (QDs). Different PCR assays, DNA polymerases (Pfu and Taq), DNA sizes and GC amounts were compared in this study. In the presence of QDs, PCR specificity could be retained even in the ninth-round amplification. Moreover, the longer and more complex the targets were, the earlier the abortion happened in multi-round PCR. However, no obvious enhancement of specificity was found in multi-round PCR using Taq DNA polymerase. Significantly, the fidelity of Pfu polymerase based multi-round PCR was not sacrificed in the presence of QDs. Besides, pre-incubation at 50 °C for an hour had no impact on multi-round PCR performance, which further authenticated the hot start effect of QDs modulated in multi-round PCR. The findings of this study demonstrated that a cost-effective and promising multi-round PCR technique for large-scale and high-throughput sample analysis could be established with high specificity, sensibility and accuracy.

Novel One-Tube-One-Step Real-Time Methodology for Rapid Transcriptomic Biomarker Detection: Signal Amplification by Ternary Initiation Complexes.

PubMed

Fujita, Hiroto; Kataoka, Yuka; Tobita, Seiji; Kuwahara, Masayasu; Sugimoto, Naoki

2016-07-19

We have developed a novel RNA detection method, termed signal amplification by ternary initiation complexes (SATIC), in which an analyte sample is simply mixed with the relevant reagents and allowed to stand for a short time under isothermal conditions (37 °C). The advantage of the technique is that there is no requirement for (i) heat annealing, (ii) thermal cycling during the reaction, (iii) a reverse transcription step, or (iv) enzymatic or mechanical fragmentation of the target RNA. SATIC involves the formation of a ternary initiation complex between the target RNA, a circular DNA template, and a DNA primer, followed by rolling circle amplification (RCA) to generate multiple copies of G-quadruplex (G4) on a long DNA strand like beads on a string. The G4s can be specifically fluorescence-stained with N(3)-hydroxyethyl thioflavin T (ThT-HE), which emits weakly with single- and double-stranded RNA/DNA but strongly with parallel G4s. An improved dual SATIC system, which involves the formation of two different ternary initiation complexes in the RCA process, exhibited a wide quantitative detection range of 1-5000 pM. Furthermore, this enabled visual observation-based RNA detection, which is more rapid and convenient than conventional isothermal methods, such as reverse transcription-loop-mediated isothermal amplification, signal mediated amplification of RNA technology, and RNA-primed rolling circle amplification. Thus, SATIC methodology may serve as an on-site and real-time measurement technique for transcriptomic biomarkers for various diseases.

Photocleavable DNA barcode-antibody conjugates allow sensitive and multiplexed protein analysis in single cells.

PubMed

Agasti, Sarit S; Liong, Monty; Peterson, Vanessa M; Lee, Hakho; Weissleder, Ralph

2012-11-14

DNA barcoding is an attractive technology, as it allows sensitive and multiplexed target analysis. However, DNA barcoding of cellular proteins remains challenging, primarily because barcode amplification and readout techniques are often incompatible with the cellular microenvironment. Here we describe the development and validation of a photocleavable DNA barcode-antibody conjugate method for rapid, quantitative, and multiplexed detection of proteins in single live cells. Following target binding, this method allows DNA barcodes to be photoreleased in solution, enabling easy isolation, amplification, and readout. As a proof of principle, we demonstrate sensitive and multiplexed detection of protein biomarkers in a variety of cancer cells.

Use of FTA elute card impregnated with cervicovaginal sample directly into the amplification reaction increases the detection of human papillomavirus DNA.

PubMed

Santos, Carla R; Franciscatto, Laura G; Barcellos, Regina B; Almeida, Sabrina E M; Rossetti, Maria Lucia R

2012-01-01

This study aimed to evaluate the use of the FTA elute card(TM) impregnated with cervicovaginal sample directly in the PCR amplification for detection of HPV-DNA. The results were compared to a reference technique. This method was more efficient than the protocol indicated by the manufacturer, identifying 91.7% against 54.2% of the positive samples.

Emergence of a replicating species from an in vitro RNA evolution reaction

NASA Technical Reports Server (NTRS)

Breaker, R. R.; Joyce, G. F.

1994-01-01

The technique of self-sustained sequence replication allows isothermal amplification of DNA and RNA molecules in vitro. This method relies on the activities of a reverse transcriptase and a DNA-dependent RNA polymerase to amplify specific nucleic acid sequences. We have modified this protocol to allow selective amplification of RNAs that catalyze a particular chemical reaction. During an in vitro RNA evolution experiment employing this modified system, a unique class of "selfish" RNAs emerged and replicated to the exclusion of the intended RNAs. Members of this class of selfish molecules, termed RNA Z, amplify efficiently despite their inability to catalyze the target chemical reaction. Their amplification requires the action of both reverse transcriptase and RNA polymerase and involves the synthesis of both DNA and RNA replication intermediates. The proposed amplification mechanism for RNA Z involves the formation of a DNA hairpin that functions as a template for transcription by RNA polymerase. This arrangement links the two strands of the DNA, resulting in the production of RNA transcripts that contain an embedded RNA polymerase promoter sequence.

Something from (almost) nothing: the impact of multiple displacement amplification on microbial ecology.

PubMed

Binga, Erik K; Lasken, Roger S; Neufeld, Josh D

2008-03-01

Microbial ecology is a field that applies molecular techniques to analyze genes and communities associated with a plethora of unique environments on this planet. In the past, low biomass and the predominance of a few abundant community members have impeded the application of techniques such as PCR, microarray analysis and metagenomics to complex microbial populations. In the absence of suitable cultivation methods, it was not possible to obtain DNA samples from individual microorganisms. Recently, a method called multiple displacement amplification (MDA) has been used to circumvent these limitations by amplifying DNA from microbial communities in low-biomass environments, individual cells from uncultivated microbial species and active organisms obtained through stable isotope probing incubations. This review describes the development and applications of MDA, discusses its strengths and limitations and highlights the impact of MDA on the field of microbial ecology. Whole genome amplification via MDA has increased access to the genomic DNA of uncultivated microorganisms and low-biomass environments and represents a 'power tool' in the molecular toolbox of microbial ecologists.

Simple System for Isothermal DNA Amplification Coupled to Lateral Flow Detection

PubMed Central

Roskos, Kristina; Hickerson, Anna I.; Lu, Hsiang-Wei; Ferguson, Tanya M.; Shinde, Deepali N.; Klaue, Yvonne; Niemz, Angelika

2013-01-01

Infectious disease diagnosis in point-of-care settings can be greatly improved through integrated, automated nucleic acid testing devices. We have developed an early prototype for a low-cost system which executes isothermal DNA amplification coupled to nucleic acid lateral flow (NALF) detection in a mesofluidic cartridge attached to a portable instrument. Fluid handling inside the cartridge is facilitated through one-way passive valves, flexible pouches, and electrolysis-driven pumps, which promotes a compact and inexpensive instrument design. The closed-system disposable prevents workspace amplicon contamination. The cartridge design is based on standard scalable manufacturing techniques such as injection molding. Nucleic acid amplification occurs in a two-layer pouch that enables efficient heat transfer. We have demonstrated as proof of principle the amplification and detection of Mycobacterium tuberculosis (M.tb) genomic DNA in the cartridge, using either Loop Mediated Amplification (LAMP) or the Exponential Amplification Reaction (EXPAR), both coupled to NALF detection. We envision that a refined version of this cartridge, including upstream sample preparation coupled to amplification and detection, will enable fully-automated sample-in to answer-out infectious disease diagnosis in primary care settings of low-resource countries with high disease burden. PMID:23922706

[Research status and prospects of DNA test on difficult specimens].

PubMed

Dang, Hua-Wei; Mao, Jiong; Wang, Hui; Huang, Jiang-Ping; Bai, Xiao-Gang

2012-02-01

This paper reviews the advances of DNA detection on three types of difficult biological specimens including degraded samples, trace evidences and mixed samples. The source of different samples, processing methods and announcements were analyzed. New methods such as mitochondrial test system, changing the original experimental conditions, low-volume PCR amplification and new technologies such as whole genome amplification techniques, laser capture micro-dissection, and mini-STR technology in recent years are introduced.

Use of FTA elute card impregnated with cervicovaginal sample directly into the amplification reaction increases the detection of human papillomavirus DNA

PubMed Central

Santos, Carla R.; Franciscatto, Laura G.; Barcellos, Regina B.; Almeida, Sabrina E. M.; Rossetti, Maria Lucia R.

2012-01-01

This study aimed to evaluate the use of the FTA elute cardTM impregnated with cervicovaginal sample directly in the PCR amplification for detection of HPV-DNA. The results were compared to a reference technique. This method was more efficient than the protocol indicated by the manufacturer, identifying 91.7% against 54.2% of the positive samples. PMID:24031844

Hybridization chain reaction: a versatile molecular tool for biosensing, bioimaging, and biomedicine.

PubMed

Bi, Sai; Yue, Shuzhen; Zhang, Shusheng

2017-07-17

Developing powerful, simple and low-cost DNA amplification techniques is of great significance to bioanalysis and biomedical research. Thus far, many signal amplification strategies have been developed, such as polymerase chain reaction (PCR), rolling circle amplification (RCA), and DNA strand displacement amplification (SDA). In particular, hybridization chain reaction (HCR), a type of toehold-mediated strand displacement (TMSD) reaction, has attracted great interest because of its enzyme-free nature, isothermal conditions, simple protocols, and excellent amplification efficiency. In a typical HCR, an analyte initiates the cross-opening of two DNA hairpins, yielding nicked double helices that are analogous to alternating copolymers. As an efficient amplification platform, HCR has been utilized for the sensitive detection of a wide variety of analytes, including nucleic acids, proteins, small molecules, and cells. In recent years, more complicated sets of monomers have been designed to develop nonlinear HCR, such as branched HCR and even dendritic systems, achieving quadratic and exponential growth mechanisms. In addition, HCR has attracted enormous attention in the fields of bioimaging and biomedicine, including applications in fluorescence in situ hybridization (FISH) imaging, live cell imaging, and targeted drug delivery. In this review, we introduce the fundamentals of HCR and examine the visualization and analysis techniques for HCR products in detail. The most recent HCR developments in biosensing, bioimaging, and biomedicine are subsequently discussed with selected examples. Finally, the review provides insight into the challenges and future perspectives of HCR.

In-gel multiple displacement amplification of long DNA fragments diluted to the single molecule level.

PubMed

Michikawa, Yuichi; Sugahara, Keisuke; Suga, Tomo; Ohtsuka, Yoshimi; Ishikawa, Kenichi; Ishikawa, Atsuko; Shiomi, Naoko; Shiomi, Tadahiro; Iwakawa, Mayumi; Imai, Takashi

2008-12-15

The isolation and multiple genotyping of long individual DNA fragments are needed to obtain haplotype information for diploid organisms. Limiting dilution of sample DNA followed by multiple displacement amplification is a useful technique but is restricted to short (<5 kb) DNA fragments. In the current study, a novel modification was applied to overcome these problems. A limited amount of cellular DNA was carefully released from intact cells into a mildly heated alkaline agarose solution and mixed thoroughly. The solution was then gently aliquoted and allowed to solidify while maintaining the integrity of the diluted DNA. Exogenously provided Phi29 DNA polymerase was used to perform consistent genomic amplification with random hexameric oligonucleotides within the agarose gels. Simple heat melting of the gel allowed recovery of the amplified materials in a solution of the polymerase chain reaction (PCR)-ready form. The haplotypes of seven SNPs spanning 240 kb of the DNA surrounding the human ATM gene region on chromosome 11 were determined for 10 individuals, demonstrating the feasibility of this new method.

Improved technique that allows the performance of large-scale SNP genotyping on DNA immobilized by FTA technology.

PubMed

He, Hongbin; Argiro, Laurent; Dessein, Helia; Chevillard, Christophe

2007-01-01

FTA technology is a novel method designed to simplify the collection, shipment, archiving and purification of nucleic acids from a wide variety of biological sources. The number of punches that can normally be obtained from a single specimen card are often however, insufficient for the testing of the large numbers of loci required to identify genetic factors that control human susceptibility or resistance to multifactorial diseases. In this study, we propose an improved technique to perform large-scale SNP genotyping. We applied a whole genome amplification method to amplify DNA from buccal cell samples stabilized using FTA technology. The results show that using the improved technique it is possible to perform up to 15,000 genotypes from one buccal cell sample. Furthermore, the procedure is simple. We consider this improved technique to be a promising methods for performing large-scale SNP genotyping because the FTA technology simplifies the collection, shipment, archiving and purification of DNA, while whole genome amplification of FTA card bound DNA produces sufficient material for the determination of thousands of SNP genotypes.

Loop-mediated isothermal amplification (LAMP): a versatile technique for detection of micro-organisms.

PubMed

Wong, Y-P; Othman, S; Lau, Y-L; Radu, S; Chee, H-Y

2018-03-01

Loop-mediated isothermal amplification (LAMP) amplifies DNA with high specificity, efficiency and rapidity under isothermal conditions by using a DNA polymerase with high displacement strand activity and a set of specifically designed primers to amplify targeted DNA strands. Following its first discovery by Notomi et al. ( Nucleic Acids Res 28: E63), LAMP was further developed over the years which involved the combination of this technique with other molecular approaches, such as reverse transcription and multiplex amplification for the detection of infectious diseases caused by micro-organisms in humans, livestock and plants. In this review, available types of LAMP techniques will be discussed together with their applications in detection of various micro-organisms. Up to date, there are varieties of LAMP detection methods available including colorimetric and fluorescent detection, real-time monitoring using turbidity metre and detection using lateral flow device which will also be highlighted in this review. Apart from that, commercialization of LAMP technique had also been reported such as lyophilized form of LAMP reagents kit and LAMP primer sets for detection of pathogenic micro-organisms. On top of that, advantages and limitations of this molecular detection method are also described together with its future potential as a diagnostic method for infectious disease. © 2017 The Society for Applied Microbiology.

One-step random mutagenesis by error-prone rolling circle amplification

PubMed Central

Fujii, Ryota; Kitaoka, Motomitsu; Hayashi, Kiyoshi

2004-01-01

In vitro random mutagenesis is a powerful tool for altering properties of enzymes. We describe here a novel random mutagenesis method using rolling circle amplification, named error-prone RCA. This method consists of only one DNA amplification step followed by transformation of the host strain, without treatment with any restriction enzymes or DNA ligases, and results in a randomly mutated plasmid library with 3–4 mutations per kilobase. Specific primers or special equipment, such as a thermal-cycler, are not required. This method permits rapid preparation of randomly mutated plasmid libraries, enabling random mutagenesis to become a more commonly used technique. PMID:15507684

Comparison of DNA Microarray, Loop-Mediated Isothermal Amplification (LAMP) and Real-Time PCR with DNA Sequencing for Identification of Fusarium spp. Obtained from Patients with Hematologic Malignancies.

PubMed

de Souza, Marcela; Matsuzawa, Tetsuhiro; Sakai, Kanae; Muraosa, Yasunori; Lyra, Luzia; Busso-Lopes, Ariane Fidelis; Levin, Anna Sara Shafferman; Schreiber, Angélica Zaninelli; Mikami, Yuzuru; Gonoi, Tohoru; Kamei, Katsuhiko; Moretti, Maria Luiza; Trabasso, Plínio

2017-08-01

The performance of three molecular biology techniques, i.e., DNA microarray, loop-mediated isothermal amplification (LAMP), and real-time PCR were compared with DNA sequencing for properly identification of 20 isolates of Fusarium spp. obtained from blood stream as etiologic agent of invasive infections in patients with hematologic malignancies. DNA microarray, LAMP and real-time PCR identified 16 (80%) out of 20 samples as Fusarium solani species complex (FSSC) and four (20%) as Fusarium spp. The agreement among the techniques was 100%. LAMP exhibited 100% specificity, while DNA microarray, LAMP and real-time PCR showed 100% sensitivity. The three techniques had 100% agreement with DNA sequencing. Sixteen isolates were identified as FSSC by sequencing, being five Fusarium keratoplasticum, nine Fusarium petroliphilum and two Fusarium solani. On the other hand, sequencing identified four isolates as Fusarium non-solani species complex (FNSSC), being three isolates as Fusarium napiforme and one isolate as Fusarium oxysporum. Finally, LAMP proved to be faster and more accessible than DNA microarray and real-time PCR, since it does not require a thermocycler. Therefore, LAMP signalizes as emerging and promising methodology to be used in routine identification of Fusarium spp. among cases of invasive fungal infections.

Development of Rapid Isothermal Amplification Assays for Detection of Phytophthora spp. in Plant Tissue.

PubMed

Miles, Timothy D; Martin, Frank N; Coffey, Michael D

2015-02-01

Several isothermal amplification techniques recently have been developed that are tolerant of inhibitors present in many plant extracts, which can reduce the need for obtaining purified DNA for running diagnostic assays. One such commercially available technique that has similarities with real-time polymerase chain reaction (PCR) for designing primers and a labeled probe is recombinase polymerase amplification (RPA). This technology was used to develop two simple and rapid approaches for detection of Phytophthora spp.: one genus-specific assay multiplexed with a plant internal control and the other species-specific assays for Phytophthora ramorum and P. kernoviae. All assays were tested for sensitivity (ranging from 3 ng to 1 fg of DNA) and specificity using DNA extracted from more than 136 Phytophthora taxa, 21 Pythium spp., 1 Phytopythium sp., and a wide range of plant species. The lower limit of linear detection using purified DNA was 200 to 300 fg of DNA in all pathogen RPA assays. Six different extraction buffers were tested for use during plant tissue maceration and the assays were validated in the field by collecting 222 symptomatic plant samples from over 50 different hosts. Only 56 samples were culture positive for Phytophthora spp. whereas 91 were positive using the Phytophthora genus-specific RPA test and a TaqMan real-time PCR assay. A technique for the generation of sequencing templates from positive RPA amplifications to confirm species identification was also developed. These RPA assays have added benefits over traditional technologies because they are rapid (results can be obtained in as little as 15 min), do not require DNA extraction or extensive training to complete, use less expensive portable equipment than PCR-based assays, and are significantly more specific than current immunologically based methods. This should provide a rapid, field-deployable capability for pathogen detection that will facilitate point-of-sample collection processing, thereby reducing the time necessary for accurate diagnostics and making management decisions.

[Assessment of two DNA extraction methods to amplify the pneumolysin gene (PLY) from blood culture samples of Streptococcus pneumoniae].

PubMed

Hernández, Carolina; Durán, Claudia; Ulloa, María Teresa; Prado, Valeria

2004-05-01

Streptococcus pneumoniae is a common etiologic agent of invasive respiratory infections among children under 5 years of age and older adults. Isolation rates of S. pneumoniae by traditional culture techniques are low. To study the sensitivity and specificity of two different DNA extraction methods to amplify the ply gene, applied to three different types of blood culture broths, experimentally inoculated with S. pneumoniae. DNA was extracted from the cultures using an organic method or a technique that consists in dilution, washing with NaOH and concentration of the sample. This was followed by PCR amplification of a 355 pb fragment of the pneumolysin gene (ply). The organic DNA extraction method inhibited the PCR reaction at all concentrations studied (0.6 to 10(6) colony forming units/mL). Using the NaOH extraction, ply gene amplification was positive in all three blood culture broths, but only at concentrations of 10(3) colony forming units/mL, or higher. Using the same DNA extraction method, PCR was negative when the broths were inoculated with seven other related bacterial species, which results in a 100% specificity. Detection of S. pneumoniae by amplification of ply gene from blood cultures using the protocol of NaOH for DNA extraction is specific and provides results in a short lapse. However, the diagnostic sensitivity is not optimal, which limits its clinical use.

Genome-wide single-nucleotide polymorphism arrays demonstrate high fidelity of multiple displacement-based whole-genome amplification.

PubMed

Tzvetkov, Mladen V; Becker, Christian; Kulle, Bettina; Nürnberg, Peter; Brockmöller, Jürgen; Wojnowski, Leszek

2005-02-01

Whole-genome DNA amplification by multiple displacement (MD-WGA) is a promising tool to obtain sufficient DNA amounts from samples of limited quantity. Using Affymetrix' GeneChip Human Mapping 10K Arrays, we investigated the accuracy and allele amplification bias in DNA samples subjected to MD-WGA. We observed an excellent concordance (99.95%) between single-nucleotide polymorphisms (SNPs) called both in the nonamplified and the corresponding amplified DNA. This concordance was only 0.01% lower than the intra-assay reproducibility of the genotyping technique used. However, MD-WGA failed to amplify an estimated 7% of polymorphic loci. Due to the algorithm used to call genotypes, this was detected only for heterozygous loci. We achieved a 4.3-fold reduction of noncalled SNPs by combining the results from two independent MD-WGA reactions. This indicated that inter-reaction variations rather than specific chromosomal loci reduced the efficiency of MD-WGA. Consistently, we detected no regions of reduced amplification, with the exception of several SNPs located near chromosomal ends. Altogether, despite a substantial loss of polymorphic sites, MD-WGA appears to be the current method of choice to amplify genomic DNA for array-based SNP analyses. The number of nonamplified loci can be substantially reduced by amplifying each DNA sample in duplicate.

Molecular Technique to Reduce PCR Bias for Deeper Understanding of Microbial Diversity

NASA Technical Reports Server (NTRS)

Vaishampayan, Parag A.; Venkateswaran, Kasthuri J.

2012-01-01

Current planetary protection policies require that spacecraft targeted to sensitive solar system bodies be assembled and readied for launch in controlled cleanroom environments. A better understanding of the distribution and frequency at which high-risk contaminant microbes are encountered on spacecraft surfaces would significantly aid in assessing the threat of forward contamination. However, despite a growing understanding of the diverse microbial populations present in cleanrooms, less abundant microbial populations are probably not adequately taken into account due to technological limitations. This novel approach encompasses a wide spectrum of microbial species and will represent the true picture of spacecraft cleanroom-associated microbial diversity. All of the current microbial diversity assessment techniques are based on an initial PCR amplification step. However, a number of factors are known to bias PCR amplification and jeopardize the true representation of bacterial diversity. PCR amplification of a minor template appears to be suppressed by the amplification of a more abundant template. It is widely acknowledged among environmental molecular microbiologists that genetic biosignatures identified from an environment only represent the most dominant populations. The technological bottleneck overlooks the presence of the less abundant minority population and may underestimate their role in the ecosystem maintenance. DNA intercalating agents such as propidium monoazide (PMA) covalently bind with DNA molecules upon photolysis using visible light, and make it unavailable for DNA polymerase enzyme during polymerase chain reaction (PCR). Environmental DNA samples will be treated with suboptimum PMA concentration, enough to intercalate with 90 99% of the total DNA. The probability of PMA binding with DNA from abundant bacterial species will be much higher than binding with DNA from less abundant species. This will increase the relative DNA concentration of previously "shadowed" less abundant species available for PCR amplification. These PCR products obtained with and without PMA treatment will then be subjected to downstream diversity analyses such as sequencing and DNA microarray. It is expected that PMA-coupled PCR will amplify the "minority population" and help in understanding microbial diversity spectrum of an environmental sample at a much deeper level. This new protocol aims to overcome the major potential biases faced when analyzing microbial 16S rRNA gene diversity. This study will lead to a technological advancement and a commercial product that will aid microbial ecologists in understanding microbial diversity from various environmental niches. Implementation of this technique may lead to discoveries of novel microbes and their functions in sustenance of the ecosystem.

Amplification and overexpression of cyclin D1 in breast cancer detected by immunohistochemical staining.

PubMed

Gillett, C; Fantl, V; Smith, R; Fisher, C; Bartek, J; Dickson, C; Barnes, D; Peters, G

1994-04-01

Immunohistochemical staining with a monoclonal antibody against human cyclin D1 can be used to identify breast cancers that have an amplification of the q13 region of chromosome 11. In general, the intensity of staining is directly proportional to the degree of DNA amplification. In two unusual tumors, in which the CCND1 locus is highly amplified but staining is relatively weak, it appears that the DNA has undergone rearrangement and that the amplified/rearranged CCND1 allele may have reduced transcriptional activity. More significantly, the immunohistochemical technique identifies additional tumors in which the cyclin D1 gene is overexpressed with only marginal or undetectable increases in copy number, implying that other mechanisms can lead to deregulated expression. These results suggest that the frequency of overexpression is much higher than previously concluded from DNA-based analyses and that more than one-third of human breast cancers may contain excessive levels of cyclin D1. The technique we describe should facilitate the detection of this abnormality in a clinical setting and clarify its prognostic significance.

Challenging loop-mediated isothermal amplification (LAMP) technique for molecular detection of Toxoplasma gondii.

PubMed

Fallahi, Shirzad; Mazar, Zahra Arab; Ghasemian, Mehrdad; Haghighi, Ali

2015-05-01

To compare analytical sensitivity and specificity of a newly described DNA amplification technique, LAMP and nested PCR assay targeting the RE and B1 genes for the detection of Toxoplasma gondii (T. gondii) DNA. The analytical sensitivity of LAMP and nested-PCR was obtained against10-fold serial dilutions of T. gondii DNA ranging from 1 ng to 0.01 fg. DNA samples of other parasites and human chromosomal DNA were used to determine the specificity of molecular assays. After testing LAMP and nested-PCR in duplicate, the detection limit of RE-LAMP, B1-LAMP, RE-nested PCR and B1-nested PCR assays was one fg, 100 fg, 1 pg and 10 pg of T. gondii DNA respectively. All the LAMP assays and nested PCRs were 100% specific. The RE-LAMP assay revealed the most sensitivity for the detection of T. gondii DNA. The obtained results demonstrate that the LAMP technique has a greater sensitivity for detection of T. gondii. Furthermore, these findings indicate that primers based on the RE are more suitable than those based on the B1 gene. However, the B1-LAMP assay has potential as a diagnostic tool for detection of T. gondii. Copyright © 2015 Hainan Medical College. Production and hosting by Elsevier B.V. All rights reserved.

[Quality of DNA from archival pathological samples of gallbladder cancer].

PubMed

Roa, Iván; de Toro, Gonzalo; Sánchez, Tamara; Slater, Jeannie; Ziegler, Anne Marie; Game, Anakaren; Arellano, Leonardo; Schalper, Kurt; de Aretxabala, Xabier

2013-12-01

The quality of the archival samples stored at pathology services could be a limiting factor for molecular biology studies. To determine the quality of DNA extracted from gallbladder cancer samples at different institutions. One hundred ninety four samples coming from five medical centers in Chile, were analyzed. DNA extraction was quantified determining genomic DNA concentration. The integrity of DNA was determined by polymerase chain reaction amplification of different length fragments of a constitutive gene (β-globin products of 110, 268 and 501 base pairs). The mean DNA concentration obtained in 194 gallbladder cancer samples was 48 ± 43.1 ng/µl. In 22% of samples, no amplification was achieved despite obtaining a mean DNA concentration of 58.3 ng/ul. In 81, 67 and 22% of samples, a DNA amplification of at least 110, 268 or 501 base pairs was obtained, respectively. No differences in DNA concentration according to the source of the samples were demonstrated. However, there were marked differences in DNA integrity among participating centers. Samples from public hospitals were of lower quality than those from private clinics. Despite some limitations, in 80% of cases, the integrity of DNA in archival samples from pathology services in our country would allow the use of molecular biology techniques.

Application of Locked Nucleic Acid (LNA) Primer and PCR Clamping by LNA Oligonucleotide to Enhance the Amplification of Internal Transcribed Spacer (ITS) Regions in Investigating the Community Structures of Plant-Associated Fungi.

PubMed

Ikenaga, Makoto; Tabuchi, Masakazu; Kawauchi, Tomohiro; Sakai, Masao

2016-09-29

The simultaneous extraction of host plant DNA severely limits investigations of the community structures of plant-associated fungi due to the similar homologies of sequences in primer-annealing positions between fungi and host plants. Although fungal-specific primers have been designed, plant DNA continues to be excessively amplified by PCR, resulting in the underestimation of community structures. In order to overcome this limitation, locked nucleic acid (LNA) primers and PCR clamping by LNA oligonucleotides have been applied to enhance the amplification of fungal internal transcribed spacer (ITS) regions. LNA primers were designed by converting DNA into LNA, which is specific to fungi, at the forward primer side. LNA oligonucleotides, the sequences of which are complementary to the host plants, were designed by overlapping a few bases with the annealing position of the reverse primer. Plant-specific DNA was then converted into LNA at the shifted position from the 3' end of the primer-binding position. PCR using the LNA technique enhanced the amplification of fungal ITS regions, whereas those of the host plants were more likely to be amplified without the LNA technique. A denaturing gradient gel electrophoresis (DGGE) analysis displayed patterns that reached an acceptable level for investigating the community structures of plant-associated fungi using the LNA technique. The sequences of the bands detected using the LNA technique were mostly affiliated with known isolates. However, some sequences showed low similarities, indicating the potential to identify novel fungi. Thus, the application of the LNA technique is considered effective for widening the scope of community analyses of plant-associated fungi.

Application of Locked Nucleic Acid (LNA) Primer and PCR Clamping by LNA Oligonucleotide to Enhance the Amplification of Internal Transcribed Spacer (ITS) Regions in Investigating the Community Structures of Plant–Associated Fungi

PubMed Central

Ikenaga, Makoto; Tabuchi, Masakazu; Kawauchi, Tomohiro; Sakai, Masao

2016-01-01

The simultaneous extraction of host plant DNA severely limits investigations of the community structures of plant–associated fungi due to the similar homologies of sequences in primer–annealing positions between fungi and host plants. Although fungal-specific primers have been designed, plant DNA continues to be excessively amplified by PCR, resulting in the underestimation of community structures. In order to overcome this limitation, locked nucleic acid (LNA) primers and PCR clamping by LNA oligonucleotides have been applied to enhance the amplification of fungal internal transcribed spacer (ITS) regions. LNA primers were designed by converting DNA into LNA, which is specific to fungi, at the forward primer side. LNA oligonucleotides, the sequences of which are complementary to the host plants, were designed by overlapping a few bases with the annealing position of the reverse primer. Plant-specific DNA was then converted into LNA at the shifted position from the 3′ end of the primer–binding position. PCR using the LNA technique enhanced the amplification of fungal ITS regions, whereas those of the host plants were more likely to be amplified without the LNA technique. A denaturing gradient gel electrophoresis (DGGE) analysis displayed patterns that reached an acceptable level for investigating the community structures of plant–associated fungi using the LNA technique. The sequences of the bands detected using the LNA technique were mostly affiliated with known isolates. However, some sequences showed low similarities, indicating the potential to identify novel fungi. Thus, the application of the LNA technique is considered effective for widening the scope of community analyses of plant–associated fungi. PMID:27600711

Whole genome amplification of Chelex-extracted DNA from a single mite: a method for studying genetics of the predatory mite Phytoseiulus persimilis.

PubMed

Konakandla, Bhanu; Park, Yoonseong; Margolies, David

2006-01-01

We developed and optimized a method using Chelex DNA extraction followed by whole genome amplification (WGA) to overcome problems conducting molecular genetic studies due to the limited amount of DNA obtainable from individual small organisms such as predatory mites. The DNA from a single mite, Phytoseiulus persimilis Athias-Henrot (Acari: Phytoseiidae), isolated in Chelex suspension was subjected to WGA. More than 1000-fold amplification of the DNA was achieved using as little as 0.03 ng genomic DNA template. The DNA obtained by the WGA was used for polymerase chain reaction followed by direct sequencing. From WGA DNA, nuclear DNA intergenic spacers ITS1 and ITS2 and a mitochondrial DNA 12S marker were tested in three different geographical populations of the predatory mite: California, the Netherlands, and Sicily. We found a total of four different alleles of the 12S in the Sicilian population, but no polymorphism was identified in the ITS marker. The combination of Chelex DNA extraction and WGA is thus shown to be a simple and robust technique for examining molecular markers for multiple loci by using individual mites. We conclude that the methods, Chelex extraction of DNA followed by WGA, provide a large quantity of DNA template that can be used for multiple PCR reactions useful for genetic studies requiring the genotypes of individual mites.

Ultrasensitive Visual Detection of HIV DNA Biomarkers via a Multi-amplification Nanoplatform.

PubMed

Long, Yuyin; Zhou, Cuisong; Wang, Congmin; Cai, Honglian; Yin, Cuiyun; Yang, Qiufang; Xiao, Dan

2016-04-01

Methodologies to detect disease biomarkers at ultralow concentrations can potentially improve the standard of living. A facile and label-free multi-amplification strategy is proposed for the ultrasensitive visual detection of HIV DNA biomarkers in real physiological media. This multi-amplification strategy not only exhibits a signficantly low detection limit down to 4.8 pM but also provides a label-free, cost-effective and facile technique for visualizing a few molecules of nucleic acid analyte with the naked eye. Importantly, the biosensor is capable of discriminating single-based mismatch lower than 5.0 nM in human serum samples. Moreover, the visual sensing platform exhibits excellent specificity, acceptable reusability and a long-term stability. All these advantages could be attributed to the nanofibrous sensing platform that 1) has a high surface-area-to-volume provided by electrospun nanofibrous membrane, and 2) combines glucose oxidase (GOx) biocatalysis, DNAzyme-catalyzed colorimetric reaction and catalytic hairpin assembly (CHA) recycling amplification together. This multi-amplification nanoplatform promises label-free and visual single-based mismatch DNA monitoring with high sensitivity and specificity, suggesting wide applications that range from virus detection to genetic disease diagnosis.

Selective whole genome amplification for resequencing target microbial species from complex natural samples.

PubMed

Leichty, Aaron R; Brisson, Dustin

2014-10-01

Population genomic analyses have demonstrated power to address major questions in evolutionary and molecular microbiology. Collecting populations of genomes is hindered in many microbial species by the absence of a cost effective and practical method to collect ample quantities of sufficiently pure genomic DNA for next-generation sequencing. Here we present a simple method to amplify genomes of a target microbial species present in a complex, natural sample. The selective whole genome amplification (SWGA) technique amplifies target genomes using nucleotide sequence motifs that are common in the target microbe genome, but rare in the background genomes, to prime the highly processive phi29 polymerase. SWGA thus selectively amplifies the target genome from samples in which it originally represented a minor fraction of the total DNA. The post-SWGA samples are enriched in target genomic DNA, which are ideal for population resequencing. We demonstrate the efficacy of SWGA using both laboratory-prepared mixtures of cultured microbes as well as a natural host-microbe association. Targeted amplification of Borrelia burgdorferi mixed with Escherichia coli at genome ratios of 1:2000 resulted in >10(5)-fold amplification of the target genomes with <6.7-fold amplification of the background. SWGA-treated genomic extracts from Wolbachia pipientis-infected Drosophila melanogaster resulted in up to 70% of high-throughput resequencing reads mapping to the W. pipientis genome. By contrast, 2-9% of sequencing reads were derived from W. pipientis without prior amplification. The SWGA technique results in high sequencing coverage at a fraction of the sequencing effort, thus allowing population genomic studies at affordable costs. Copyright © 2014 by the Genetics Society of America.

Embryo Sexing and Sex Chromosomal Chimerism Analysis by Loop-Mediated Isothermal Amplification in Cattle and Water Buffaloes

PubMed Central

HIRAYAMA, Hiroki; KAGEYAMA, Soichi; MORIYASU, Satoru; SAWAI, Ken; MINAMIHASHI, Akira

2013-01-01

Abstract In domestic animals of the family Bovidae, sex preselection of offspring has been demanded for convenience of milk/beef production and animal breeding. Development of the nonsurgical embryo transfer technique and sexing methods of preimplantation embryos made it possible. Sexing based on detection of Y chromosome-specific DNA sequences is considered the most reliable method to date. PCR enables amplification of a target sequence from a small number of blastomeres. However, it requires technical skill and is time consuming. Furthermore, PCR has the risk of false positives because of DNA contamination during handling of the PCR products in duplicate PCR procedures and/or electrophoresis. Therefore, for embryo sexing to become widely used in the cattle embryo transfer industry, a simple, rapid and precise sexing method needs to be developed. Loop-mediated isothermal amplification (LAMP) is a novel DNA amplification method, and the reaction is carried out under isothermal conditions (range, 60 to 65 C) using DNA polymerase with strand displacement activity. When the target DNA is amplified by LAMP, a white precipitate derived from magnesium pyrophosphate (a by-product of the LAMP reaction) is observed. It is noteworthy that LAMP does not need special reagents or electrophoresis to detect the amplified DNA. This review describes the development and application of an embryo sexing method using LAMP in cattle and water buffaloes. PMID:23965599

Dynamics and control of DNA sequence amplification

DOE Office of Scientific and Technical Information (OSTI.GOV)

Marimuthu, Karthikeyan; Chakrabarti, Raj, E-mail: raj@pmc-group.com, E-mail: rajc@andrew.cmu.edu; Division of Fundamental Research, PMC Advanced Technology, Mount Laurel, New Jersey 08054

2014-10-28

DNA amplification is the process of replication of a specified DNA sequence in vitro through time-dependent manipulation of its external environment. A theoretical framework for determination of the optimal dynamic operating conditions of DNA amplification reactions, for any specified amplification objective, is presented based on first-principles biophysical modeling and control theory. Amplification of DNA is formulated as a problem in control theory with optimal solutions that can differ considerably from strategies typically used in practice. Using the Polymerase Chain Reaction as an example, sequence-dependent biophysical models for DNA amplification are cast as control systems, wherein the dynamics of the reactionmore » are controlled by a manipulated input variable. Using these control systems, we demonstrate that there exists an optimal temperature cycling strategy for geometric amplification of any DNA sequence and formulate optimal control problems that can be used to derive the optimal temperature profile. Strategies for the optimal synthesis of the DNA amplification control trajectory are proposed. Analogous methods can be used to formulate control problems for more advanced amplification objectives corresponding to the design of new types of DNA amplification reactions.« less

Real-time DNA Amplification and Detection System Based on a CMOS Image Sensor.

PubMed

Wang, Tiantian; Devadhasan, Jasmine Pramila; Lee, Do Young; Kim, Sanghyo

2016-01-01

In the present study, we developed a polypropylene well-integrated complementary metal oxide semiconductor (CMOS) platform to perform the loop mediated isothermal amplification (LAMP) technique for real-time DNA amplification and detection simultaneously. An amplification-coupled detection system directly measures the photon number changes based on the generation of magnesium pyrophosphate and color changes. The photon number decreases during the amplification process. The CMOS image sensor observes the photons and converts into digital units with the aid of an analog-to-digital converter (ADC). In addition, UV-spectral studies, optical color intensity detection, pH analysis, and electrophoresis detection were carried out to prove the efficiency of the CMOS sensor based the LAMP system. Moreover, Clostridium perfringens was utilized as proof-of-concept detection for the new system. We anticipate that this CMOS image sensor-based LAMP method will enable the creation of cost-effective, label-free, optical, real-time and portable molecular diagnostic devices.

Initiator-catalyzed self-assembly of duplex-looped DNA hairpin motif based on strand displacement reaction for logic operations and amplified biosensing.

PubMed

Bi, Sai; Yue, Shuzhen; Wu, Qiang; Ye, Jiayan

2016-09-15

Here we program an initiator-catalyzed self-assembly of duplex-looped DNA hairpin motif based on strand displacement reaction. Due to the recycling of initiator and performance in a cascade manner, this system is versatilely extended to logic operations, including the construction of concatenated logic circuits with a feedback function and a biocomputing keypad-lock security system. Compared with previously reported molecular security systems, the prominent feature of our keypad lock is that it can be spontaneously reset and recycled with no need of any external stimulus and human intervention. Moreover, through integrating with an isothermal amplification technique of rolling circle amplification (RCA), this programming catalytic DNA self-assembly strategy readily achieves sensitive and selective biosensing of initiator. Importantly, a magnetic graphene oxide (MGO) is introduced to remarkably reduced background, which plays an important role in enhancing the signal-to-noise ratio and improving the detection sensitivity. Therefore, the proposed sophisticated DNA strand displacement-based methodology with engineering dynamic functions may find broad applications in the construction of programming DNA nanostructures, amplification biosensing platform, and large-scale DNA circuits. Copyright © 2016 Elsevier B.V. All rights reserved.

The problems and promise of DNA barcodes for species diagnosis of primate biomaterials

PubMed Central

Lorenz, Joseph G; Jackson, Whitney E; Beck, Jeanne C; Hanner, Robert

2005-01-01

The Integrated Primate Biomaterials and Information Resource (www.IPBIR.org) provides essential research reagents to the scientific community by establishing, verifying, maintaining, and distributing DNA and RNA derived from primate cell cultures. The IPBIR uses mitochondrial cytochrome c oxidase subunit I sequences to verify the identity of samples for quality control purposes in the accession, cell culture, DNA extraction processes and prior to shipping to end users. As a result, IPBIR is accumulating a database of ‘DNA barcodes’ for many species of primates. However, this quality control process is complicated by taxon specific patterns of ‘universal primer’ failure, as well as the amplification or co-amplification of nuclear pseudogenes of mitochondrial origins. To overcome these difficulties, taxon specific primers have been developed, and reverse transcriptase PCR is utilized to exclude these extraneous sequences from amplification. DNA barcoding of primates has applications to conservation and law enforcement. Depositing barcode sequences in a public database, along with primer sequences, trace files and associated quality scores, makes this species identification technique widely accessible. Reference DNA barcode sequences should be derived from, and linked to, specimens of known provenance in web-accessible collections in order to validate this system of molecular diagnostics. PMID:16214744

Effect of food processing on plant DNA degradation and PCR-based GMO analysis: a review.

PubMed

Gryson, Nicolas

2010-03-01

The applicability of a DNA-based method for GMO detection and quantification depends on the quality and quantity of the DNA. Important food-processing conditions, for example temperature and pH, may lead to degradation of the DNA, rendering PCR analysis impossible or GMO quantification unreliable. This review discusses the effect of several food processes on DNA degradation and subsequent GMO detection and quantification. The data show that, although many of these processes do indeed lead to the fragmentation of DNA, amplification of the DNA may still be possible. Length and composition of the amplicon may, however, affect the result, as also may the method of extraction used. Also, many techniques are used to describe the behaviour of DNA in food processing, which occasionally makes it difficult to compare research results. Further research should be aimed at defining ingredients in terms of their DNA quality and PCR amplification ability, and elaboration of matrix-specific certified reference materials.

Detection of DNA methylation changes in micropropagated banana plants using methylation-sensitive amplification polymorphism (MSAP).

PubMed

Peraza-Echeverria, S; Herrera-Valencia, V A.; Kay, A -J.

2001-07-01

The extent of DNA methylation polymorphisms was evaluated in micropropagated banana (Musa AAA cv. 'Grand Naine') derived from either the vegetative apex of the sucker or the floral apex of the male inflorescence using the methylation-sensitive amplification polymorphism (MSAP) technique. In all, 465 fragments, each representing a recognition site cleaved by either or both of the isoschizomers were amplified using eight combinations of primers. A total of 107 sites (23%) were found to be methylated at cytosine in the genome of micropropagated banana plants. In plants micropropagated from the male inflorescence explant 14 (3%) DNA methylation events were polymorphic, while plants micropropagated from the sucker explant produced 8 (1.7%) polymorphisms. No DNA methylation polymorphisms were detected in conventionally propagated banana plants. These results demonstrated the usefulness of MSAP to detect DNA methylation events in micropropagated banana plants and indicate that DNA methylation polymorphisms are associated with micropropagation.

Theory and applications of the polymerase chain reaction.

PubMed

Remick, D G; Kunkel, S L; Holbrook, E A; Hanson, C A

1990-04-01

The polymerase chain reaction (PCR) is a newly developed molecular biology technique that can significantly amplify DNA or RNA. The process consists of repetitive cycles of specific DNA synthesis, defined by short stretches of preselected DNA. With each cycle, there is a doubling of the final, desired DNA product such that a million-fold amplification is possible. This powerful method has numerous applications in diagnostic pathology, especially in the fields of microbiology, forensic science, and hematology. The PCR may be used to directly detect viral DNA, which may facilitate the diagnosis of acquired immune deficiency syndrome (AIDS) or other viral diseases. PCR amplification of DNA allows detection of specific sequences from extremely small samples, such as with forensic material. In hematology, PCR may help in the diagnosis of hemoglobinopathies or of neoplastic disorders by documenting chromosomal translocations. The new PCR opens exciting new avenues for diagnostic pathology using this new technology.

A Sequence-Independent Strategy for Detection and Cloning of Circular DNA Virus Genomes by Using Multiply Primed Rolling-Circle Amplification

PubMed Central

Rector, Annabel; Tachezy, Ruth; Van Ranst, Marc

2004-01-01

The discovery of novel viruses has often been accomplished by using hybridization-based methods that necessitate the availability of a previously characterized virus genome probe or knowledge of the viral nucleotide sequence to construct consensus or degenerate PCR primers. In their natural replication cycle, certain viruses employ a rolling-circle mechanism to propagate their circular genomes, and multiply primed rolling-circle amplification (RCA) with φ29 DNA polymerase has recently been applied in the amplification of circular plasmid vectors used in cloning. We employed an isothermal RCA protocol that uses random hexamer primers to amplify the complete genomes of papillomaviruses without the need for prior knowledge of their DNA sequences. We optimized this RCA technique with extracted human papillomavirus type 16 (HPV-16) DNA from W12 cells, using a real-time quantitative PCR assay to determine amplification efficiency, and obtained a 2.4 × 104-fold increase in HPV-16 DNA concentration. We were able to clone the complete HPV-16 genome from this multiply primed RCA product. The optimized protocol was subsequently applied to a bovine fibropapillomatous wart tissue sample. Whereas no papillomavirus DNA could be detected by restriction enzyme digestion of the original sample, multiply primed RCA enabled us to obtain a sufficient amount of papillomavirus DNA for restriction enzyme analysis, cloning, and subsequent sequencing of a novel variant of bovine papillomavirus type 1. The multiply primed RCA method allows the discovery of previously unknown papillomaviruses, and possibly also other circular DNA viruses, without a priori sequence information. PMID:15113879

Comparison of four DNA extraction methods for the detection of Mycobacterium leprae from Ziehl-Neelsen-stained microscopic slides.

PubMed

Ruiz-Fuentes, Jenny Laura; Díaz, Alexis; Entenza, Anayma Elena; Frión, Yahima; Suárez, Odelaisy; Torres, Pedro; de Armas, Yaxsier; Acosta, Lucrecia

2015-12-01

The diagnosis of leprosy has been a challenge due to the low sensibility of the conventional methods and the impossibility of culturing the causative organism. In this study, four methods for Mycobacterium leprae nucleic-acid extraction from Ziehl-Neelsen-stained slides (ZNS slides) were compared: Phenol/chloroform, Chelex 100 resin, and two commercial kits (Wizard Genomic DNA Purification Kit and QIAamp DNA Mini Kit). DNA was extracted from four groups of slides: a high-codification-slide group (bacteriological index [BI]⩾4), a low-codification-slide group (BI=1), a negative-slide group (BI=0), and a negative-control-slide group (BI=0). Quality DNA was evidenced by the amplification of specific repetitive element present in M. leprae genomic DNA (RLEP) using a nested polymerase chain reaction. This is the first report comparing four different extraction methods for obtaining M. leprae DNA from ZNS slides in Cuban patients, and applied in molecular diagnosis. Good-quality DNA and positive amplification were detected in the high-codification-slide group with the four methods, while from the low-codification-slide group only the QIAGEN and phenol-chloroform methods obtained amplification of M. leprae. In the negative-slide group, only the QIAGEN method was able to obtain DNA with sufficient quality for positive amplification of the RLEP region. No amplification was observed in the negative-control-slide group by any method. Patients with ZNS negative slides can still transmit the infection, and molecular methods can help identify and treat them, interrupting the chain of transmission and preventing the onset of disabilities. The ZNS slides can be sent easily to reference laboratories for later molecular analysis that can be useful not only to improve the diagnosis, but also for the application of other molecular techniques. Copyright © 2015 Asian-African Society for Mycobacteriology. Published by Elsevier Ltd. All rights reserved.

Colony-PCR Is a Rapid Method for DNA Amplification of Hyphomycetes

PubMed Central

Walch, Georg; Knapp, Maria; Rainer, Georg; Peintner, Ursula

2016-01-01

Fungal pure cultures identified with both classical morphological methods and through barcoding sequences are a basic requirement for reliable reference sequences in public databases. Improved techniques for an accelerated DNA barcode reference library construction will result in considerably improved sequence databases covering a wider taxonomic range. Fast, cheap, and reliable methods for obtaining DNA sequences from fungal isolates are, therefore, a valuable tool for the scientific community. Direct colony PCR was already successfully established for yeasts, but has not been evaluated for a wide range of anamorphic soil fungi up to now, and a direct amplification protocol for hyphomycetes without tissue pre-treatment has not been published so far. Here, we present a colony PCR technique directly from fungal hyphae without previous DNA extraction or other prior manipulation. Seven hundred eighty-eight fungal strains from 48 genera were tested with a success rate of 86%. PCR success varied considerably: DNA of fungi belonging to the genera Cladosporium, Geomyces, Fusarium, and Mortierella could be amplified with high success. DNA of soil-borne yeasts was always successfully amplified. Absidia, Mucor, Trichoderma, and Penicillium isolates had noticeably lower PCR success. PMID:29376929

Optimization of strand displacement amplification-sensitized G-quadruplex DNAzyme-based sensing system and its application in activity detection of uracil-DNA glycosylase.

PubMed

Du, Yi-Chen; Jiang, Hong-Xin; Huo, Yan-Fang; Han, Gui-Mei; Kong, De-Ming

2016-03-15

As an isothermal nucleic acid amplification technique, strand displacement amplification (SDA) reaction has been introduced in G-quadruplex DNAzyme-based sensing system to improve the sensing performance. To further provide useful information for the design of SDA-amplified G-quadruplex DNAzyme-based sensors, the effects of nicking site number in SDA template DNA were investigated. With the increase of the nicking site number from 1 to 2, enrichment of G-quadruplex DNAzyme by SDA is changed from a linear amplification to an exponential amplification, thus greatly increasing the amplification efficiency and subsequently improving the sensing performance of corresponding sensing system. The nicking site number cannot be further increased because more nicking sites might result in high background signals. However, we demonstrated that G-quadruplex DNAzyme enrichment efficiency could be further improved by introducing a cross-triggered SDA strategy, in which two templates each has two nicking sites are used. To validate the proposed cross-triggered SDA strategy, we used it to develop a sensing platform for the detection of uracil-DNA glycosylase (UDG) activity. The sensor enables sensitive detection of UDG activity in the range of 1 × 10(-4)-1 U/mL with a detection limit of 1 × 10(-4)U/mL. Copyright © 2015 Elsevier B.V. All rights reserved.

Amplified electrochemiluminescence detection of DNA based on novel quantum dots signal probe by multiple cycling amplification strategy.

PubMed

Tan, Lu; Ge, Junjun; Jiao, Meng; Jie, Guifen; Niu, Shuyan

2018-06-01

In the present work, we designed a unique enzyme-aided multiple amplification strategy for sensitive electrochemiluminescence (ECL) detection of DNA by using the amplified gold nanoparticles (GNPS)-polyamidoamine (PAMAM)-CdSe quantum dots (QDs) signal probe. Firstly, the novel GNPS-PAMAM dendrimers nanostructure with good biocompatibility and electroconductibility contains many amino groups, which can load a large number of CdSe QDs to develop amplified ECL signal probe. Then, the presence of target DNA activated the enzyme-assisted polymerization strand-displacement cycling reaction, and a large number of the hairpin template was opened. Subsequently, the opened stem further interacted with the capture hairpin (HP) DNA on the electrode, and the GNPS-PAMAM-CdSe signal probe hybridized with the exposed stem of the HP to trigger the second new polymerization reaction. Meanwhile, the first cycle was generating abundant DNA triggers which could directly open the template. As a result of the cascade amplification technique, a large number of CdSe QDs signal probe could be assembled on the electrode, generating much amplified ECL signal for sensitive detection of target DNA. Thus, this novel QDs-based amplified ECL strategy holds great promise for DNA detection and can be further exploited for sensing applications in clinical diagnostics. Copyright © 2018 Elsevier B.V. All rights reserved.

Direct PCR amplification of forensic touch and other challenging DNA samples: A review.

PubMed

Cavanaugh, Sarah E; Bathrick, Abigail S

2018-01-01

DNA evidence sample processing typically involves DNA extraction, quantification, and STR amplification; however, DNA loss can occur at both the DNA extraction and quantification steps, which is not ideal for forensic evidence containing low levels of DNA. Direct PCR amplification of forensic unknown samples has been suggested as a means to circumvent extraction and quantification, thereby retaining the DNA typically lost during those procedures. Direct PCR amplification is a method in which a sample is added directly to an amplification reaction without being subjected to prior DNA extraction, purification, or quantification. It allows for maximum quantities of DNA to be targeted, minimizes opportunities for error and contamination, and reduces the time and monetary resources required to process samples, although data analysis may take longer as the increased DNA detection sensitivity of direct PCR may lead to more instances of complex mixtures. ISO 17025 accredited laboratories have successfully implemented direct PCR for limited purposes (e.g., high-throughput databanking analysis), and recent studies indicate that direct PCR can be an effective method for processing low-yield evidence samples. Despite its benefits, direct PCR has yet to be widely implemented across laboratories for the processing of evidentiary items. While forensic DNA laboratories are always interested in new methods that will maximize the quantity and quality of genetic information obtained from evidentiary items, there is often a lag between the advent of useful methodologies and their integration into laboratories. Delayed implementation of direct PCR of evidentiary items can be attributed to a variety of factors, including regulatory guidelines that prevent laboratories from omitting the quantification step when processing forensic unknown samples, as is the case in the United States, and, more broadly, a reluctance to validate a technique that is not widely used for evidence samples. The advantages of direct PCR of forensic evidentiary samples justify a re-examination of the factors that have delayed widespread implementation of this method and of the evidence supporting its use. In this review, the current and potential future uses of direct PCR in forensic DNA laboratories are summarized. Copyright © 2017 Elsevier B.V. All rights reserved.

Forensic DNA testing.

PubMed

Butler, John M

2011-12-01

Forensic DNA testing has a number of applications, including parentage testing, identifying human remains from natural or man-made disasters or terrorist attacks, and solving crimes. This article provides background information followed by an overview of the process of forensic DNA testing, including sample collection, DNA extraction, PCR amplification, short tandem repeat (STR) allele separation and sizing, typing and profile interpretation, statistical analysis, and quality assurance. The article concludes with discussions of possible problems with the data and other forensic DNA testing techniques.

Microfluidic electrochemical assay for rapid detection and quantification of Escherichia coli.

PubMed

Safavieh, Mohammadali; Ahmed, Minhaz Uddin; Tolba, Mona; Zourob, Mohammed

2012-01-15

Microfluidic electrochemical biosensor for performing Loop-mediated isothermal amplification (LAMP) was developed for the detection and quantification of Escherichia coli. The electrochemical detection for detecting the DNA amplification was achieved using Hoechst 33258 redox molecule and linear sweep voltametry (LSV). The DNA aggregation and minor groove binding with redox molecule cause a significant drop in the anodic oxidation of LSV. Unlike other electrochemical techniques, this method does not require the probe immobilization and the detection of the bacteria can be accomplished in a single chamber without DNA extraction and purification steps. The isothermal amplification time has a major role in the quantification of the bacteria. We have shown that we could detect and quantify 24 CFU/ml of bacteria and 8.6 fg/μl DNA in 60 min and 48 CFU/ml of bacteria in 35 min in LB media and urine samples. We believe that this microfluidic chip has great potential to be used as a point of care diagnostic (POC) device in the clinical/hospital application. Copyright © 2011 Elsevier B.V. All rights reserved.

Caught in the middle with multiple displacement amplification: the myth of pooling for avoiding multiple displacement amplification bias in a metagenome.

PubMed

Marine, Rachel; McCarren, Coleen; Vorrasane, Vansay; Nasko, Dan; Crowgey, Erin; Polson, Shawn W; Wommack, K Eric

2014-01-30

Shotgun metagenomics has become an important tool for investigating the ecology of microorganisms. Underlying these investigations is the assumption that metagenome sequence data accurately estimates the census of microbial populations. Multiple displacement amplification (MDA) of microbial community DNA is often used in cases where it is difficult to obtain enough DNA for sequencing; however, MDA can result in amplification biases that may impact subsequent estimates of population census from metagenome data. Some have posited that pooling replicate MDA reactions negates these biases and restores the accuracy of population analyses. This assumption has not been empirically tested. Using mock viral communities, we examined the influence of pooling on population-scale analyses. In pooled and single reaction MDA treatments, sequence coverage of viral populations was highly variable and coverage patterns across viral genomes were nearly identical, indicating that initial priming biases were reproducible and that pooling did not alleviate biases. In contrast, control unamplified sequence libraries showed relatively even coverage across phage genomes. MDA should be avoided for metagenomic investigations that require quantitative estimates of microbial taxa and gene functional groups. While MDA is an indispensable technique in applications such as single-cell genomics, amplification biases cannot be overcome by combining replicate MDA reactions. Alternative library preparation techniques should be utilized for quantitative microbial ecology studies utilizing metagenomic sequencing approaches.

Molecular Technique to Understand Deep Microbial Diversity

NASA Technical Reports Server (NTRS)

Vaishampayan, Parag A.; Venkateswaran, Kasthuri J.

2012-01-01

Current sequencing-based and DNA microarray techniques to study microbial diversity are based on an initial PCR (polymerase chain reaction) amplification step. However, a number of factors are known to bias PCR amplification and jeopardize the true representation of bacterial diversity. PCR amplification of the minor template appears to be suppressed by the exponential amplification of the more abundant template. It is widely acknowledged among environmental molecular microbiologists that genetic biosignatures identified from an environment only represent the most dominant populations. The technological bottleneck has overlooked the presence of the less abundant minority population, and underestimated their role in the ecosystem maintenance. To generate PCR amplicons for subsequent diversity analysis, bacterial l6S rRNA genes are amplified by PCR using universal primers. Two distinct PCR regimes are employed in parallel: one using normal and the other using biotinlabeled universal primers. PCR products obtained with biotin-labeled primers are mixed with streptavidin-labeled magnetic beads and selectively captured in the presence of a magnetic field. Less-abundant DNA templates that fail to amplify in this first round of PCR amplification are subjected to a second round of PCR using normal universal primers. These PCR products are then subjected to downstream diversity analyses such as conventional cloning and sequencing. A second round of PCR amplified the minority population and completed the deep diversity picture of the environmental sample.

Dielectrophoretic manipulation of particles for use in microfluidic devices

DOE Office of Scientific and Technical Information (OSTI.GOV)

Belgrader, P; Bettencourt, K; Hamilton, J

1999-06-23

Amplification and hybridization of DNA are commonly used techniques to verify the presence of a specific DNA sequence in a test sample. Automatic sample handling to concentrate and purify sample prior to amplification is desirable both from the cost standpoint and from the standpoint of reducing the possibility of sample contamination. This paper explores the use of the dielectrophoretic force to manipulate DNA, Bacillus globigii spores, and Erwinia herbicola bacteria to provide concentration and purification as part of the sample handling functions in biological monitoring equipment. It was found that for what would be considered a typical microfabricated structure withmore » electrode gaps at 30 {micro}m operating at 5V, that concentration of the particles is very effective.« less

Highly sensitive detection of human IgG using a novel bio-barcode assay combined with DNA chip technology

NASA Astrophysics Data System (ADS)

Liu, Zhenbao; Zhou, Bo; Wang, Haiqing; Lu, Feng; Liu, Tianjun; Song, Cunxian; Leng, Xigang

2013-09-01

A simple and ultrasensitive detection of human IgG based on signal amplification using a novel bio-barcode assay and DNA chip technology was developed. The sensing platform was a sandwich system made up of antibody-modified magnetic microparticles (Ab-MMPs)/human IgG/Cy3-labeled single-stranded DNA and antibody-modified gold nanoparticles (Cy3-ssDNA-Ab-AuNPs). The MMPs (2.5 μm in diameter) modified with mouse anti-human IgG monoclonal-antibodies could capture human IgG and further be separated and enriched via a magnetic field. The AuNPs (13 nm in diameter) conjugated with goat anti-human IgG polyclonal-antibodies and Cy3-ssDNA could further combine with the human IgG/Ab-MMP complex. The Cy3-ssDNA on AuNPs was then released by TCEP to hybridize with the DNA chip, thus generating a detectable signal by the fluorescence intensity of Cy3. In order to improve detection sensitivity, a three-level cascaded signal amplification was developed: (1) The MMP enrichment as the first-level; (2) Large quantities of Cy3-ssDNA on AuNPs as the second-level; (3) The Cy3-ssDNA conjugate with DNA chip as the third-level. The highly sensitive technique showed an increased response of the fluorescence intensity to the increased concentration of human IgG through a detection range from 1 pg mL-1 to 10 ng mL-1. This sensing technique could not only improve the detection sensitivity for the low concentration of human IgG but also present a robust and efficient signal amplification model. The detection method has good stability, specificity, and reproducibility and could be applied in the detection of human IgG in the real samples.

Recombinase polymerase amplification as a promising tool in hepatitis C virus diagnosis.

PubMed

Zaghloul, Hosam; El-Shahat, Mahmoud

2014-12-27

Hepatitis C virus (HCV) infection represents a significant health problem and represents a heavy load on some countries like Egypt in which about 20% of the total population are infected. Initial infection is usually asymptomatic and result in chronic hepatitis that give rise to complications including cirrhosis and hepatocellular carcinoma. The management of HCV infection should not only be focus on therapy, but also to screen carrier individuals in order to prevent transmission. In the present, molecular detection and quantification of HCV genome by real time polymerase chain reaction (PCR) represent the gold standard in HCV diagnosis and plays a crucial role in the management of therapeutic regimens. However, real time PCR is a complicated approach and of limited distribution. On the other hand, isothermal DNA amplification techniques have been developed and offer molecular diagnosis of infectious dieses at point-of-care. In this review we discuss recombinase polymerase amplification technique and illustrate its diagnostic value over both PCR and other isothermal amplification techniques.

Rapid amplification of 5' complementary DNA ends (5' RACE).

PubMed

2005-08-01

This method is used to extend partial cDNA clones by amplifying the 5' sequences of the corresponding mRNAs 1-3. The technique requires knowledge of only a small region of sequence within the partial cDNA clone. During PCR, the thermostable DNA polymerase is directed to the appropriate target RNA by a single primer derived from the region of known sequence; the second primer required for PCR is complementary to a general feature of the target-in the case of 5' RACE, to a homopolymeric tail added (via terminal transferase) to the 3' termini of cDNAs transcribed from a preparation of mRNA. This synthetic tail provides a primer-binding site upstream of the unknown 5' sequence of the target mRNA. The products of the amplification reaction are cloned into a plasmid vector for sequencing and subsequent manipulation.

Development of a Loop Mediated Isothermal Amplification (LAMP) - Surface Enhanced Raman spectroscopy (SERS) Assay for the Detection of Salmonella Enterica Serotype Enteritidis.

PubMed

Draz, Mohamed Shehata; Lu, Xiaonan

2016-01-01

As a major foodborne pathogen, Salmonella enterica serotype Enteritidis is increasingly rising as a global health concern. Here, we developed an integrated assay that combines loop mediated isothermal amplification (LAMP) and surface enhanced Raman spectroscopy (SERS) for DNA detection of S. Enteritidis using specifically designed Raman active Au-nanoprobes. The target DNA was amplified by LAMP and then labeled with Au-nanoprobes comprised of gold nanoparticle-modified with specific cy5/DNA probes to allow the detection by SERS. The sensitivity of the developed LAMP-SERS detection assay (66 CFU/mL) was ~100-fold higher than the conventional polymerase chain reaction (PCR) method. Significantly, this technique allowed highly specific detection of the target DNA of S. Enteritidis and could differentiate it from the DNA of closely related bacterial species or non-specific contamination, making it more accurate and reliable than the standard LAMP technique. The applicability of detection of S. Enteritidis in milk samples using LAMP-SERS assay was validated as well. In sum, the developed LAMP-SERS assay is highly specific and sensitive, and has the potential to be applied for rapid detection of different foodborne pathogens and other microbial contaminants.

Rapid and sensitive detection of Citrus Bacterial Canker by loop-mediated isothermal amplification combined with simple visual evaluation methods

PubMed Central

2010-01-01

Background Citrus Bacterial Canker (CBC) is a major, highly contagious disease of citrus plants present in many countries in Asia, Africa and America, but not in the Mediterranean area. There are three types of Citrus Bacterial Canker, named A, B, and C that have different genotypes and posses variation in host range within citrus species. The causative agent for type A CBC is Xanthomonas citri subsp. citri, while Xanthomonas fuscans subsp. aurantifolii, strain B causes type B CBC and Xanthomonas fuscans subsp. aurantifolii strain C causes CBC type C. The early and accurate identification of those bacteria is essential for the protection of the citrus industry. Detection methods based on bacterial isolation, antibodies or polymerase chain reaction (PCR) have been developed previously; however, these approaches may be time consuming, laborious and, in the case of PCR, it requires expensive laboratory equipment. Loop-mediated isothermal amplification (LAMP), which is a novel isothermal DNA amplification technique, is sensitive, specific, fast and requires no specialized laboratory equipment. Results A loop-mediated isothermal amplification assay for the diagnosis of Citrus Bacterial Canker (CBC-LAMP) was developed and evaluated. DNA samples were obtained from infected plants or cultured bacteria. A typical ladder-like pattern on gel electrophoresis was observed in all positive samples in contrast to the negative controls. In addition, amplification products were detected by visual inspection using SYBRGreen and using a lateral flow dipstick, eliminating the need for gel electrophoresis. The sensitivity and specificity of the assay were evaluated in different conditions and using several sample sources which included purified DNA, bacterium culture and infected plant tissue. The sensitivity of the CBC-LAMP was 10 fg of pure Xcc DNA, 5 CFU in culture samples and 18 CFU in samples of infected plant tissue. No cross reaction was observed with DNA of other phytopathogenic bacteria. The assay was capable of detecting CBC-causing strains from several geographical origins and pathotypes. Conclusions The CBC-LAMP technique is a simple, fast, sensitive and specific method for the diagnosis of Citrus Bacterial Canker. This method can be useful in the phytosanitary programs of the citrus industry worldwide. PMID:20565886

A non-invasive technique for rapid extraction of DNA from fish scales.

PubMed

Kumar, Ravindra; Singh, Poonam Jayant; Nagpure, N S; Kushwaha, Basdeo; Srivastava, S K; Lakra, W S

2007-11-01

DNA markers are being increasingly used in studies related to population genetics and conservation biology of endangered species. DNA isolation for such studies requires a source of biological material that is easy to collect, non-bulky and reliable. Further, the sampling strategies based on non-invasive procedures are desirable, especially for the endangered fish species. In view of above, a rapid DNA extraction method from fish scales has been developed with the use of a modified lysis buffer that require about 2 hr duration. This methodology is non-invasive, less expensive and reproducible with high efficiency of DNA recovery. The DNA extracted by this technique, have been found suitable for performing restriction enzyme digestion and PCR amplification. Therefore, the present DNA extraction procedure can be used as an alternative technique in population genetic studies pertaining to endangered fish species. The technique was also found equally effective for DNA isolation from fresh, dried and ethanol preserved scales.

A cascade signal amplification strategy for sensitive and label-free DNA detection based on Exo III-catalyzed recycling coupled with rolling circle amplification.

PubMed

Liu, Xingti; Xue, Qingwang; Ding, Yongshun; Zhu, Jing; Wang, Lei; Jiang, Wei

2014-06-07

A sensitive and label-free fluorescence assay for DNA detection has been developed based on cascade signal amplification combining exonuclease III (Exo III)-catalyzed recycling with rolling circle amplification. In this assay, probe DNA hybridized with template DNA was coupled onto magnetic nanoparticles to prepare a magnetic bead-probe (MNB-probe)-template complex. The complex could hybridize with the target DNA, which transformed the protruding 3' terminus of template DNA into a blunt end. Exo III could then digest template DNA, liberating the MNB-probe and target DNA. The intact target DNA then hybridized with other templates and released more MNB-probes. The liberated MNB-probe captured the primer, circular DNA and then initiated the rolling circle amplification (RCA) reaction, realizing a cascade signal amplification. Using this cascade amplification strategy, a sensitive DNA detection method was developed which was superior to many existing Exo III-based signal amplification methods. Moreover, N-methyl mesoporphyrin IX, which had a pronounced structural selectivity for the G-quadruplex, was used to combine with the G-quadruplex RCA products and generate a fluorescence signal, avoiding the need for any fluorophore-label probes. The spike and recovery experiments in a human serum sample indicated that our assay also had great potential for DNA detection in real biological samples.

Improved acid tolerance of Lactobacillus pentosus by error-prone whole genome amplification.

PubMed

Ye, Lidan; Zhao, Hua; Li, Zhi; Wu, Jin Chuan

2013-05-01

Acid tolerance of Lactobacillus pentosus ATCC 8041 was improved by error-prone amplification of its genomic DNA using random primers and Taq DNA polymerase. The resulting amplification products were transferred into wild-type L. pentosus by electroporation and the transformants were screened for growth on low-pH agar plates. After only one round of mutation, one mutant (MT3) was identified that was able to completely consume 20 g/L of glucose to produce lactic acid at a yield of 95% in 1L MRS medium at pH 3.8 within 36 h, whereas no growth or lactic acid production was observed for the wild-type strain under the same conditions. The acid tolerance of mutant MT3 remained genetically stable for at least 25 subcultures. Therefore, the error-prone whole genome amplification technique is a very powerful tool for improving phenotypes of this lactic acid bacterium and may also be applicable for other microorganisms. Copyright © 2012 Elsevier Ltd. All rights reserved.

[Evaluation of 3 methods of DNA extraction from paraffin-embedded material for the amplification of genomic DNA using PCR].

PubMed

Mesquita, R A; Anzai, E K; Oliveira, R N; Nunes, F D

2001-01-01

There are several protocols reported in the literature for the extraction of genomic DNA from formalin-fixed paraffin-embedded samples. Genomic DNA is utilized in molecular analyses, including PCR. This study compares three different methods for the extraction of genomic DNA from formalin-fixed paraffin-embedded (inflammatory fibrous hyperplasia) and non-formalin-fixed (normal oral mucosa) samples: phenol with enzymatic digestion, and silica with and without enzymatic digestion. The amplification of DNA by means of the PCR technique was carried out with primers for the exon 7 of human keratin type 14. Amplicons were analyzed by means of electrophoresis in an 8% polyacrylamide gel with 5% glycerol, followed by silver-staining visualization. The phenol/enzymatic digestion and the silica/enzymatic digestion methods provided amplicons from both tissue samples. The method described is a potential aid in the establishment of the histopathologic diagnosis and in retrospective studies with archival paraffin-embedded samples.

Strategy for Extracting DNA from Clay Soil and Detecting a Specific Target Sequence via Selective Enrichment and Real-Time (Quantitative) PCR Amplification ▿

PubMed Central

Yankson, Kweku K.; Steck, Todd R.

2009-01-01

We present a simple strategy for isolating and accurately enumerating target DNA from high-clay-content soils: desorption with buffers, an optional magnetic capture hybridization step, and quantitation via real-time PCR. With the developed technique, μg quantities of DNA were extracted from mg samples of pure kaolinite and a field clay soil. PMID:19633108

A cascade amplification strategy based on rolling circle amplification and hydroxylamine amplified gold nanoparticles enables chemiluminescence detection of adenosine triphosphate.

PubMed

Wang, Ping; Zhang, Tonghuan; Yang, Taoyi; Jin, Nan; Zhao, Yanjun; Fan, Aiping

2014-08-07

A highly sensitive and selective chemiluminescent (CL) biosensor for adenosine triphosphate (ATP) was developed by taking advantage of the ATP-dependent enzymatic reaction (ATP-DER), the powerful signal amplification capability of rolling circle amplification (RCA), and hydroxylamine-amplified gold nanoparticles (Au NPs). The strategy relies on the ability of ATP, a cofactor of T4 DNA ligase, to trigger the ligation-RCA reaction. In the presence of ATP, the T4 DNA ligase catalyzes the ligation reaction between the two ends of the padlock probe, producing a closed circular DNA template that initiates the RCA reaction with phi29 DNA polymerase and dNTP. Therein, many complementary copies of the circular template can be generated. The ATP-DER is eventually converted into a detectable CL signal after a series of processes, including gold probe hybridization, hydroxylamine amplification, and oxidative gold metal dissolution coupled with a simple and sensitive luminol CL reaction. The CL signal is directly proportional to the ATP level. The results showed that the detection limit of the assay is 100 pM of ATP, which compares favorably with those of other ATP detection techniques. In addition, by taking advantage of ATP-DER, the proposed CL sensing system exhibits extraordinary specificity towards ATP and could distinguish the target molecule ATP from its analogues. The proposed method provides a new and versatile platform for the design of novel DNA ligation reaction-based CL sensing systems for other cofactors. This novel ATP-DER based CL sensing system may find wide applications in clinical diagnosis as well as in environmental and biomedical fields.

A Paper-Based Device for Performing Loop-Mediated Isothermal Amplification with Real-Time Simultaneous Detection of Multiple DNA Targets.

PubMed

Seok, Youngung; Joung, Hyou-Arm; Byun, Ju-Young; Jeon, Hyo-Sung; Shin, Su Jeong; Kim, Sanghyo; Shin, Young-Beom; Han, Hyung Soo; Kim, Min-Gon

2017-01-01

Paper-based diagnostic devices have many advantages as a one of the multiple diagnostic test platforms for point-of-care (POC) testing because they have simplicity, portability, and cost-effectiveness. However, despite high sensitivity and specificity of nucleic acid testing (NAT), the development of NAT based on a paper platform has not progressed as much as the others because various specific conditions for nucleic acid amplification reactions such as pH, buffer components, and temperature, inhibitions from technical differences of paper-based device. Here, we propose a paper-based device for performing loop-mediated isothermal amplification (LAMP) with real-time simultaneous detection of multiple DNA targets. We determined the optimal chemical components to enable dry conditions for the LAMP reaction without lyophilization or other techniques. We also devised the simple paper device structure by sequentially stacking functional layers, and employed a newly discovered property of hydroxynaphthol blue fluorescence to analyze real-time LAMP signals in the paper device. This proposed platform allowed analysis of three different meningitis DNA samples in a single device with single-step operation. This LAMP-based multiple diagnostic device has potential for real-time analysis with quantitative detection of 10 2 -10 5 copies of genomic DNA. Furthermore, we propose the transformation of DNA amplification devices to a simple and affordable paper system approach with great potential for realizing a paper-based NAT system for POC testing.

A Paper-Based Device for Performing Loop-Mediated Isothermal Amplification with Real-Time Simultaneous Detection of Multiple DNA Targets

PubMed Central

Seok, Youngung; Joung, Hyou-Arm; Byun, Ju-Young; Jeon, Hyo-Sung; Shin, Su Jeong; Kim, Sanghyo; Shin, Young-Beom; Han, Hyung Soo; Kim, Min-Gon

2017-01-01

Paper-based diagnostic devices have many advantages as a one of the multiple diagnostic test platforms for point-of-care (POC) testing because they have simplicity, portability, and cost-effectiveness. However, despite high sensitivity and specificity of nucleic acid testing (NAT), the development of NAT based on a paper platform has not progressed as much as the others because various specific conditions for nucleic acid amplification reactions such as pH, buffer components, and temperature, inhibitions from technical differences of paper-based device. Here, we propose a paper-based device for performing loop-mediated isothermal amplification (LAMP) with real-time simultaneous detection of multiple DNA targets. We determined the optimal chemical components to enable dry conditions for the LAMP reaction without lyophilization or other techniques. We also devised the simple paper device structure by sequentially stacking functional layers, and employed a newly discovered property of hydroxynaphthol blue fluorescence to analyze real-time LAMP signals in the paper device. This proposed platform allowed analysis of three different meningitis DNA samples in a single device with single-step operation. This LAMP-based multiple diagnostic device has potential for real-time analysis with quantitative detection of 102-105 copies of genomic DNA. Furthermore, we propose the transformation of DNA amplification devices to a simple and affordable paper system approach with great potential for realizing a paper-based NAT system for POC testing. PMID:28740546

[Development of a universal primers PCR-coupled liquid bead array to detect biothreat bacteria].

PubMed

Wen, Hai-yan; Wang, Jing; Liu, Heng-chuan; Sun, Xiao-hong; Yang, Yu; Hu, Kong-xin; Shan, Lin-jun

2009-10-01

To develop a fast, high-throughput screening method with suspension array technique for simultaneous detection of biothreat bacteria. 16 S rDNA universal primers for Bacillus anthracis, Francisella tularensis, Yersinia pestis, Brucella spp.and Burkholderia pseudomallei were selected to amplify corresponding regions and the genus-specific or species-specific probes were designed. After amplification of chromosomal DNA by 16 S rDNA primers 341A and 519B, the PCR products were detected by suspension array technique. The sensitivity, specificity, reproducibility and detection power were also analyzed. After PCR amplification by 16 S rDNA primers and specific probe hybridization, the target microorganisms could be identified at genus level, cross reaction was recognized in the same genus. The detection sensitivity of the assay was 1.5 pg/microl (Burkholderia pseudomallei), 20 pg/microl (Brucella spp.), 7 pg/microl (Bacillus anthracis), 0.1 pg/microl (Francisella tularensis), and 1.1 pg/microl (Yersinia pestis), respectively. The coefficient of variation for 15 test of different probes was ranged from 5.18% to 17.88%, it showed good reproducibility. The assay could correctly identify Bacillus anthracis and Yersinia pestis strains in simulated white powder samples. The suspension array technique could be served as an opening screening method for biothreat bacteria rapid detection.

A Simple Method for Amplifying RNA Targets (SMART)

PubMed Central

McCalla, Stephanie E.; Ong, Carmichael; Sarma, Aartik; Opal, Steven M.; Artenstein, Andrew W.; Tripathi, Anubhav

2012-01-01

We present a novel and simple method for amplifying RNA targets (named by its acronym, SMART), and for detection, using engineered amplification probes that overcome existing limitations of current RNA-based technologies. This system amplifies and detects optimal engineered ssDNA probes that hybridize to target RNA. The amplifiable probe-target RNA complex is captured on magnetic beads using a sequence-specific capture probe and is separated from unbound probe using a novel microfluidic technique. Hybridization sequences are not constrained as they are in conventional target-amplification reactions such as nucleic acid sequence amplification (NASBA). Our engineered ssDNA probe was amplified both off-chip and in a microchip reservoir at the end of the separation microchannel using isothermal NASBA. Optimal solution conditions for ssDNA amplification were investigated. Although KCl and MgCl2 are typically found in NASBA reactions, replacing 70 mmol/L of the 82 mmol/L total chloride ions with acetate resulted in optimal reaction conditions, particularly for low but clinically relevant probe concentrations (≤100 fmol/L). With the optimal probe design and solution conditions, we also successfully removed the initial heating step of NASBA, thus achieving a true isothermal reaction. The SMART assay using a synthetic model influenza DNA target sequence served as a fundamental demonstration of the efficacy of the capture and microfluidic separation system, thus bridging our system to a clinically relevant detection problem. PMID:22691910

Development of a quantitative fluorescence single primer isothermal amplification-based method for the detection of Salmonella.

PubMed

Wang, Jianchang; Li, Rui; Hu, Lianxia; Sun, Xiaoxia; Wang, Jinfeng; Li, Jing

2016-02-16

Food-borne disease caused by Salmonella has long been, and continues to be, an important global public health problem, necessitating rapid and accurate detection of Salmonella in food. Real time PCR is the most recently developed approach for Salmonella detection. Single primer isothermal amplification (SPIA), a novel gene amplification technique, has emerged as an attractive microbiological testing method. SPIA is performed under a constant temperature, eliminating the need for an expensive thermo-cycler. In addition, SPIA reactions can be accomplished in 30 min, faster than real time PCR that usually takes over 2h. We developed a quantitative fluorescence SPIA-based method for the detection of Salmonella. Using Salmonella Typhimurium genomic DNA as template and a primer targeting Salmonella invA gene, we showed the detection limit of SPIA was 2.0 × 10(1)fg DNA. Its successful amplification of different serotypic Salmonella genomic DNA but not non-Salmonella bacterial DNA demonstrated the specificity of SPIA. Furthermore, this method was validated with artificially contaminated beef. In conclusion, we showed high sensitivity and specificity of SPIA in the detection of Salmonella, comparable to real time PCR. In addition, SPIA is faster and more cost-effective (non-use of expensive cyclers), making it a potential alternative for field detection of Salmonella in resource-limited settings that are commonly encountered in developing countries. Copyright © 2015 Elsevier B.V. All rights reserved.

Integrated microfluidic systems for cell lysis, mixing/pumping and DNA amplification

NASA Astrophysics Data System (ADS)

Lee, Chia-Yen; Lee, Gwo-Bin; Lin, Jr-Lung; Huang, Fu-Chun; Liao, Chia-Sheng

2005-06-01

The present paper reports a fully automated microfluidic system for the DNA amplification process by integrating an electroosmotic pump, an active micromixer and an on-chip temperature control system. In this DNA amplification process, the cell lysis is initially performed in a micro cell lysis reactor. Extracted DNA samples, primers and reagents are then driven electroosmotically into a mixing region where they are mixed by the active micromixer. The homogeneous mixture is then thermally cycled in a micro-PCR (polymerase chain reaction) chamber to perform DNA amplification. Experimental results show that the proposed device can successfully automate the sample pretreatment operation for DNA amplification, thereby delivering significant time and effort savings. The new microfluidic system, which facilitates cell lysis, sample driving/mixing and DNA amplification, could provide a significant contribution to ongoing efforts to miniaturize bio-analysis systems by utilizing a simple fabrication process and cheap materials.

Differential impact of ionic and coordinate covalent chromium (Cr)-DNA binding on DNA replication.

PubMed

Fornsaglio, Jamie L; O'Brien, Travis J; Patierno, Steven R

2005-11-01

The reactive species produced by the reduction of Cr(VI), particularly Cr(III), can form both ionic and coordinate covalent complexes with DNA. These Cr(III)-DNA interactions consist of Cr-DNA monoadducts, Cr-DNA ternary adducts, and Cr-DNA interstrand cross-links (Cr-ICLs), the latter of which are DNA polymerase arresting lesions (PALs). We sought to determine the impact of Cr-DNA interactions on the formation of replication blocking lesions in S. cerevisiae using a PCR-based method. We found that target sequence (TS) amplification using DNA isolated from Cr(VI)-treated yeast actually increased as a function of Cr(VI) concentration. Moreover, the enhanced TS amplification was reproduced in vitro using Cr(III)-treated DNA. In contrast, PCR amplification of TS from DNA isolated from yeast exposed to equitoxic doses of the inorganic DNA cross-linking agent cisplatin (CDDP), was decreased in a concentration-dependent manner. This paradox suggested that a specific Cr-DNA interaction, such as an ionic Cr-DNA complex, was responsible for the enhanced TS amplification, thereby masking the replication-blocking effect of certain ternary Cr-DNA adducts (i.e. interstrand cross-links). To test this possibility, we removed ionically associated Cr from the DNA using salt extraction prior to PCR analysis. This procedure obviated the increased amplification and revealed a dose-dependent decrease in TS amplification and an increase in Cr-PALs. These data from DNA analyzed ex vivo after treatment of intact cells indicate that ionic interactions of Cr with DNA result in increased DNA amplification whereas coordinate-covalent Cr-DNA complexes lead to formation of Cr-PALs. Thus, these results suggest that treatment of living cells with Cr(VI) leads to two modes of Cr-binding, which may have conflicting effects on DNA replication.

Detection of Hepatitis A Virus by the Nucleic Acid Sequence-Based Amplification Technique and Comparison with Reverse Transcription-PCR

PubMed Central

Jean, Julie; Blais, Burton; Darveau, André; Fliss, Ismaïl

2001-01-01

A nucleic acid sequence-based amplification (NASBA) technique for the detection of hepatitis A virus (HAV) in foods was developed and compared to the traditional reverse transcription (RT)-PCR technique. Oligonucleotide primers targeting the VP1 and VP2 genes encoding the major HAV capsid proteins were used for the amplification of viral RNA in an isothermal process resulting in the accumulation of RNA amplicons. Amplicons were detected by hybridization with a digoxigenin-labeled oligonucleotide probe in a dot blot assay format. Using the NASBA, as little as 0.4 ng of target RNA/ml was detected per comparison to 4 ng/ml for RT-PCR. When crude HAV viral lysate was used, a detection limit of 2 PFU (4 × 102 PFU/ml) was obtained with NASBA, compared to 50 PFU (1 × 104 PFU/ml) obtained with RT-PCR. No interference was encountered in the amplification of HAV RNA in the presence of excess nontarget RNA or DNA. The NASBA system successfully detected HAV recovered from experimentally inoculated samples of waste water, lettuce, and blueberries. Compared to RT-PCR and other amplification techniques, the NASBA system offers several advantages in terms of sensitivity, rapidity, and simplicity. This technique should be readily adaptable for detection of other RNA viruses in both foods and clinical samples. PMID:11722911

Detection of hepatitis A virus by the nucleic acid sequence-based amplification technique and comparison with reverse transcription-PCR.

PubMed

Jean, J; Blais, B; Darveau, A; Fliss, I

2001-12-01

A nucleic acid sequence-based amplification (NASBA) technique for the detection of hepatitis A virus (HAV) in foods was developed and compared to the traditional reverse transcription (RT)-PCR technique. Oligonucleotide primers targeting the VP1 and VP2 genes encoding the major HAV capsid proteins were used for the amplification of viral RNA in an isothermal process resulting in the accumulation of RNA amplicons. Amplicons were detected by hybridization with a digoxigenin-labeled oligonucleotide probe in a dot blot assay format. Using the NASBA, as little as 0.4 ng of target RNA/ml was detected per comparison to 4 ng/ml for RT-PCR. When crude HAV viral lysate was used, a detection limit of 2 PFU (4 x 10(2) PFU/ml) was obtained with NASBA, compared to 50 PFU (1 x 10(4) PFU/ml) obtained with RT-PCR. No interference was encountered in the amplification of HAV RNA in the presence of excess nontarget RNA or DNA. The NASBA system successfully detected HAV recovered from experimentally inoculated samples of waste water, lettuce, and blueberries. Compared to RT-PCR and other amplification techniques, the NASBA system offers several advantages in terms of sensitivity, rapidity, and simplicity. This technique should be readily adaptable for detection of other RNA viruses in both foods and clinical samples.

The influence of diet on faecal DNA amplification and sex identification in brown bears (Ursus arctos)

USGS Publications Warehouse

Murphy, M.A.; Waits, L.P.; Kendall, K.C.

2003-01-01

To evaluate the influence of diet on faecal DNA amplification, 11 captive brown bears (Ursus arctos) were placed on six restricted diets: grass (Trifolium spp., Haplopappus hirtus and Poa pratensis), alfalfa (Lupinus spp.), carrots (Daucus spp.), white-tailed deer (Odocoileus virginianus), blueberries (Vaccinium spp.) and salmon (Salmo spp.). DNA was extracted from 50 faecal samples of each restricted diet, and amplification of brown bear DNA was attempted for a mitochondrial DNA (mtDNA) locus and nuclear DNA (nDNA) locus. For mtDNA, no significant differences were observed in amplification success rates across diets. For nDNA, amplification success rates for salmon diet extracts were significantly lower than all other diet extracts (P < 0.001). To evaluate the accuracy of faecal DNA sex identification when female carnivores consume male mammalian prey, female bears were fed male white-tailed deer. Four of 10 extracts amplified, and all extracts were incorrectly scored as male due to amplification of X and Y-chromosome fragments. The potential biases highlighted in this study have broad implications for researchers using faecal DNA for individual and sex identification, and should be evaluated in other species.

Amplification of bovine papillomavirus DNA by N-methyl-N'-nitro-N-nitrosoguanidine, ultraviolet irradiation, or infection with herpes simplex virus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schmitt, J.; Schlehofer, J.R.; Mergener, K.

1989-09-01

Treatment with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) or irradiation with ultraviolet light (uv254 nm) induces amplification of integrated as well as episomal sequences of bovine papillomavirus (BPV) type 1 DNA in BPV-1-transformed mouse C127 cells (i.e., ID13 cells). This is shown by filter in situ hybridization and Southern blot analysis of cellular DNA. Similarly, infection of ID13 cells with herpes simplex virus (HSV) type 1 which has been shown to be mutagenic for host cell DNA leads to amplification of BPV DNA sequences. In contrast to this induction of DNA amplification by initiators, treatment of ID13 cells with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA)more » does not result in increased synthesis of BPV DNA nor does TPA treatment modulate the initiator-induced DNA amplification. Similar to other cell systems infection with adeno-associated virus (AAV) type 2 inhibits BPV-1 DNA amplification irrespective of the inducing agent. In contrast to initiator-induced DNA amplification, treatment with carcinogen (MNNG) or tumor promoters or combination of MNNG and promoter of C127 cells prior to transformation by BPV-1 does not lead to an increase in the number of transformed foci. The induction of amplification of papillomavirus DNA by initiating agents possibly represents one of the mechanisms by which the observed synergism between papillomavirus infection and initiators in tumorigenesis might occur.« less

An extended sequence specificity for UV-induced DNA damage.

PubMed

Chung, Long H; Murray, Vincent

2018-01-01

The sequence specificity of UV-induced DNA damage was determined with a higher precision and accuracy than previously reported. UV light induces two major damage adducts: cyclobutane pyrimidine dimers (CPDs) and pyrimidine(6-4)pyrimidone photoproducts (6-4PPs). Employing capillary electrophoresis with laser-induced fluorescence and taking advantages of the distinct properties of the CPDs and 6-4PPs, we studied the sequence specificity of UV-induced DNA damage in a purified DNA sequence using two approaches: end-labelling and a polymerase stop/linear amplification assay. A mitochondrial DNA sequence that contained a random nucleotide composition was employed as the target DNA sequence. With previous methodology, the UV sequence specificity was determined at a dinucleotide or trinucleotide level; however, in this paper, we have extended the UV sequence specificity to a hexanucleotide level. With the end-labelling technique (for 6-4PPs), the consensus sequence was found to be 5'-GCTC*AC (where C* is the breakage site); while with the linear amplification procedure, it was 5'-TCTT*AC. With end-labelling, the dinucleotide frequency of occurrence was highest for 5'-TC*, 5'-TT* and 5'-CC*; whereas it was 5'-TT* for linear amplification. The influence of neighbouring nucleotides on the degree of UV-induced DNA damage was also examined. The core sequences consisted of pyrimidine nucleotides 5'-CTC* and 5'-CTT* while an A at position "1" and C at position "2" enhanced UV-induced DNA damage. Crown Copyright © 2017. Published by Elsevier B.V. All rights reserved.

Ligation-mediated PCR with a back-to-back adapter reduces amplification bias resulting from variations in GC content.

PubMed

Ishihara, Satoru; Kotomura, Naoe; Yamamoto, Naoki; Ochiai, Hiroshi

2017-08-15

Ligation-mediated polymerase chain reaction (LM-PCR) is a common technique for amplification of a pool of DNA fragments. Here, a double-stranded oligonucleotide consisting of two primer sequences in back-to-back orientation was designed as an adapter for LM-PCR. When DNA fragments were ligated with this adapter, the fragments were sandwiched between two adapters in random orientations. In the ensuing PCR, ligation products linked at each end to an opposite side of the adapter, i.e. to a distinct primer sequence, were preferentially amplified compared with products linked at each end to an identical primer sequence. The use of this adapter in LM-PCR reduced the impairment of PCR by substrate DNA with a high GC content, compared with the use of traditional LM-PCR adapters. This result suggested that our method has the potential to contribute to reduction of the amplification bias that is caused by an intrinsic property of the sequence context in substrate DNA. A DNA preparation obtained from a chromatin immunoprecipitation assay using pulldown of a specific form of histone H3 was successfully amplified using the modified LM-PCR, and the amplified products could be used as probes in a fluorescence in situ hybridization analysis. Copyright © 2017 Elsevier Inc. All rights reserved.

Extraction of High Quality DNA from Seized Moroccan Cannabis Resin (Hashish)

PubMed Central

El Alaoui, Moulay Abdelaziz; Melloul, Marouane; Alaoui Amine, Sanaâ; Stambouli, Hamid; El Bouri, Aziz; Soulaymani, Abdelmajid; El Fahime, Elmostafa

2013-01-01

The extraction and purification of nucleic acids is the first step in most molecular biology analysis techniques. The objective of this work is to obtain highly purified nucleic acids derived from Cannabis sativa resin seizure in order to conduct a DNA typing method for the individualization of cannabis resin samples. To obtain highly purified nucleic acids from cannabis resin (Hashish) free from contaminants that cause inhibition of PCR reaction, we have tested two protocols: the CTAB protocol of Wagner and a CTAB protocol described by Somma (2004) adapted for difficult matrix. We obtained high quality genomic DNA from 8 cannabis resin seizures using the adapted protocol. DNA extracted by the Wagner CTAB protocol failed to give polymerase chain reaction (PCR) amplification of tetrahydrocannabinolic acid (THCA) synthase coding gene. However, the extracted DNA by the second protocol permits amplification of THCA synthase coding gene using different sets of primers as assessed by PCR. We describe here for the first time the possibility of DNA extraction from (Hashish) resin derived from Cannabis sativa. This allows the use of DNA molecular tests under special forensic circumstances. PMID:24124454

New, Improved Version of the mCOP-PCR Screening System for Detection of Spinal Muscular Atrophy Gene (SMN1) Deletion.

PubMed

Shinohara, Masakazu; Ar Rochmah, Mawaddah; Nakanishi, Kenta; Harahap, Nur Imma Fatimah; Niba, Emma Tabe Eko; Saito, Toshio; Saito, Kayoko; Takeuchi, Atsuko; Bouike, Yoshihiro; Nishio, Hisahide

2017-09-07

Spinal muscular atrophy (SMA) is a frequent autosomal recessive disorder, characterized by lower motor neuron loss in the spinal cord. More than 95% of SMA patients show homozygous survival motor neuron 1 (SMN1) deletion. We previously developed a screening system for SMN1 deletion based on a modified competitive oligonucleotide priming-PCR (mCOP-PCR) technique. However, non-specific amplification products were observed with mCOP-PCR, which might lead to erroneous interpretation of the screening results. To establish an improved version of the mCOP-PCR screening system without non-specific amplification. DNA samples were assayed using a new version of the mCOP-PCR screening system. DNA samples had already been genotyped by PCR-restriction fragment length polymorphism (PCR-RFLP), showing the presence or absence of SMN1 exon 7. The new mCOP-PCR method contained a targeted pre-amplification step of the region, including an SMN1-specific nucleotide, prior to the mCOP-PCR step. mCOP-PCR products were electrophoresed on agarose gels. No non-specific amplification products were detected in electrophoresis gels with the new mCOP-PCR screening system. An additional targeted pre-amplification step eliminated non-specific amplification from mCOP-PCR screening.

Relative quantification of 40 nucleic acid sequences by multiplex ligation-dependent probe amplification

PubMed Central

Schouten, Jan P.; McElgunn, Cathal J.; Waaijer, Raymond; Zwijnenburg, Danny; Diepvens, Filip; Pals, Gerard

2002-01-01

We describe a new method for relative quantification of 40 different DNA sequences in an easy to perform reaction requiring only 20 ng of human DNA. Applications shown of this multiplex ligation-dependent probe amplification (MLPA) technique include the detection of exon deletions and duplications in the human BRCA1, MSH2 and MLH1 genes, detection of trisomies such as Down’s syndrome, characterisation of chromosomal aberrations in cell lines and tumour samples and SNP/mutation detection. Relative quantification of mRNAs by MLPA will be described elsewhere. In MLPA, not sample nucleic acids but probes added to the samples are amplified and quantified. Amplification of probes by PCR depends on the presence of probe target sequences in the sample. Each probe consists of two oligonucleotides, one synthetic and one M13 derived, that hybridise to adjacent sites of the target sequence. Such hybridised probe oligonucleotides are ligated, permitting subsequent amplification. All ligated probes have identical end sequences, permitting simultaneous PCR amplification using only one primer pair. Each probe gives rise to an amplification product of unique size between 130 and 480 bp. Probe target sequences are small (50–70 nt). The prerequisite of a ligation reaction provides the opportunity to discriminate single nucleotide differences. PMID:12060695

Relative quantification of 40 nucleic acid sequences by multiplex ligation-dependent probe amplification.

PubMed

Schouten, Jan P; McElgunn, Cathal J; Waaijer, Raymond; Zwijnenburg, Danny; Diepvens, Filip; Pals, Gerard

2002-06-15

We describe a new method for relative quantification of 40 different DNA sequences in an easy to perform reaction requiring only 20 ng of human DNA. Applications shown of this multiplex ligation-dependent probe amplification (MLPA) technique include the detection of exon deletions and duplications in the human BRCA1, MSH2 and MLH1 genes, detection of trisomies such as Down's syndrome, characterisation of chromosomal aberrations in cell lines and tumour samples and SNP/mutation detection. Relative quantification of mRNAs by MLPA will be described elsewhere. In MLPA, not sample nucleic acids but probes added to the samples are amplified and quantified. Amplification of probes by PCR depends on the presence of probe target sequences in the sample. Each probe consists of two oligonucleotides, one synthetic and one M13 derived, that hybridise to adjacent sites of the target sequence. Such hybridised probe oligonucleotides are ligated, permitting subsequent amplification. All ligated probes have identical end sequences, permitting simultaneous PCR amplification using only one primer pair. Each probe gives rise to an amplification product of unique size between 130 and 480 bp. Probe target sequences are small (50-70 nt). The prerequisite of a ligation reaction provides the opportunity to discriminate single nucleotide differences.

Amplification of a specific repetitive DNA sequence for Trypanosoma rangeli identification and its potential application in epidemiological investigations.

PubMed

Vargas, N; Souto, R P; Carranza, J C; Vallejo, G A; Zingales, B

2000-11-01

Trypanosoma rangeli can infect humans as well as the same domestic and wild animals and triatomine vectors infected by Trypanosoma cruzi in Central and South America. This overlapping distribution complicates the epidemiology of American trypanosomiasis due to the cross-reactivity between T. rangeli and T. cruzi antigens and the presence of conserved DNA sequences in these parasites. We have isolated a T. rangeli-specific DNA repetitive element which is represented in approximately 103 copies per parasite genome and is distributed in several chromosomal bands. The 542-bp nucleotide sequence of this element, named P542, was determined and a PCR assay was standardized for its amplification. The sensitivity of the assay is high, allowing the detection of one tenth of the DNA content of a single parasite. The presence of the P542 element was confirmed in 11 T. rangeli isolates from mammalian hosts and insect vectors originating from several countries in Latin America. Negative amplification was observed with different T. cruzi strains and other trypanosomatids. The potential field application of the P542 PCR assay was investigated in simulated samples containing T. rangeli and/or T. cruzi and intestinal tract and feces of Rhodnius prolixus. Epidemiological studies were conducted in DNA preparations obtained from the digestive tracts of 12 Rhodnius colombiensis insects collected in a sylvatic area in Colombia. Positive amplification of the P542 element was obtained in 9/12 insects. We have also compared in the same samples the diagnostic performance of two PCR assays for the amplification of the variable domain of minicircle kinetoplast DNA (kDNA) and of the large subunit (LSU) of the ribosomal RNA gene of T. cruzi and T. rangeli. Data indicate that the kDNA PCR assay does not allow diagnosis of mixed infections in most insects. On the other hand, the PCR assay of the LSU RNA gene showed lower sensitivity in the detection of T. rangeli than the PCR assay of the P542 element. It is predicted that the use of sensitive detection techniques will indicate that the actual distribution of T. rangeli in America is wider than presumed. Copyright 2000 Academic Press.

Evaluation of fluorescence in situ hybridization techniques to study long non-coding RNA expression in cultured cells

PubMed Central

Soares, Ricardo J; Maglieri, Giulia; Gutschner, Tony; Lund, Anders H; Nielsen, Boye S

2018-01-01

Abstract Deciphering the functions of long non-coding RNAs (lncRNAs) is facilitated by visualization of their subcellular localization using in situ hybridization (ISH) techniques. We evaluated four different ISH methods for detection of MALAT1 and CYTOR in cultured cells: a multiple probe detection approach with or without enzymatic signal amplification, a branched-DNA (bDNA) probe and an LNA-modified probe with enzymatic signal amplification. All four methods adequately stained MALAT1 in the nucleus in all of three cell lines investigated, HeLa, NHDF and T47D, and three of the methods detected the less expressed CYTOR. The sensitivity of the four ISH methods was evaluated by image analysis. In all three cell lines, the two methods involving enzymatic amplification gave the most intense MALAT1 signal, but the signal-to-background ratios were not different. CYTOR was best detected using the bDNA method. All four ISH methods showed significantly reduced MALAT1 signal in knock-out cells, and siRNA-induced knock-down of CYTOR resulted in significantly reduced CYTOR ISH signal, indicating good specificity of the probe designs and detection systems. Our data suggest that the ISH methods allow detection of both abundant and less abundantly expressed lncRNAs, although the latter required the use of the most specific and sensitive probe detection system. PMID:29059327

Amplification of chromosomal DNA in situ

DOEpatents

Christian, Allen T.; Coleman, Matthew A.; Tucker, James D.

2002-01-01

Amplification of chromosomal DNA in situ to increase the amount of DNA associated with a chromosome or chromosome region is described. The amplification of chromosomal DNA in situ provides for the synthesis of Fluorescence in situ Hybridization (FISH) painting probes from single dissected chromosome fragments, the production of cDNA libraries from low copy mRNAs and improved in Comparative Genomic Hybridization (CGH) procedures.

Recombinase-based isothermal amplification of nucleic acids with self-avoiding molecular recognition systems (SAMRS).

PubMed

Sharma, Nidhi; Hoshika, Shuichi; Hutter, Daniel; Bradley, Kevin M; Benner, Steven A

2014-10-13

Recombinase polymerase amplification (RPA) is an isothermal method to amplify nucleic acid sequences without the temperature cycling that classical PCR uses. Instead of using heat to denature the DNA duplex, RPA uses recombination enzymes to swap single-stranded primers into the duplex DNA product; these are then extended using a strand-displacing polymerase to complete the cycle. Because RPA runs at low temperatures, it never forces the system to recreate base-pairs following Watson-Crick rules, and therefore it produces undesired products that impede the amplification of the desired product, complicating downstream analysis. Herein, we show that most of these undesired side products can be avoided if the primers contain components of a self-avoiding molecular recognition system (SAMRS). Given the precision that is necessary in the recombination systems for them to function biologically, it is surprising that they accept SAMRS. SAMRS-RPA is expected to be a powerful tool within the range of amplification techniques available to scientists. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

GMO detection using a bioluminescent real time reporter (BART) of loop mediated isothermal amplification (LAMP) suitable for field use.

PubMed

Kiddle, Guy; Hardinge, Patrick; Buttigieg, Neil; Gandelman, Olga; Pereira, Clint; McElgunn, Cathal J; Rizzoli, Manuela; Jackson, Rebecca; Appleton, Nigel; Moore, Cathy; Tisi, Laurence C; Murray, James A H

2012-04-30

There is an increasing need for quantitative technologies suitable for molecular detection in a variety of settings for applications including food traceability and monitoring of genetically modified (GM) crops and their products through the food processing chain. Conventional molecular diagnostics utilising real-time polymerase chain reaction (RT-PCR) and fluorescence-based determination of amplification require temperature cycling and relatively complex optics. In contrast, isothermal amplification coupled to a bioluminescent output produced in real-time (BART) occurs at a constant temperature and only requires a simple light detection and integration device. Loop mediated isothermal amplification (LAMP) shows robustness to sample-derived inhibitors. Here we show the applicability of coupled LAMP and BART reactions (LAMP-BART) for determination of genetically modified (GM) maize target DNA at low levels of contamination (0.1-5.0% GM) using certified reference material, and compare this to RT-PCR. Results show that conventional DNA extraction methods developed for PCR may not be optimal for LAMP-BART quantification. Additionally, we demonstrate that LAMP is more tolerant to plant sample-derived inhibitors, and show this can be exploited to develop rapid extraction techniques suitable for simple field-based qualitative tests for GM status determination. We also assess the effect of total DNA assay load on LAMP-BART quantitation. LAMP-BART is an effective and sensitive technique for GM detection with significant potential for quantification even at low levels of contamination and in samples derived from crops such as maize with a large genome size. The resilience of LAMP-BART to acidic polysaccharides makes it well suited to rapid sample preparation techniques and hence to both high throughput laboratory settings and to portable GM detection applications. The impact of the plant sample matrix and genome loading within a reaction must be controlled to ensure quantification at low target concentrations.

Rapid authentication of the precious herb saffron by loop-mediated isothermal amplification (LAMP) based on internal transcribed spacer 2 (ITS2) sequence

PubMed Central

Zhao, Mingming; Shi, Yuhua; Wu, Lan; Guo, Licheng; Liu, Wei; Xiong, Chao; Yan, Song; Sun, Wei; Chen, Shilin

2016-01-01

Saffron is one of the most expensive species of Chinese herbs and has been subjected to various types of adulteration because of its high price and limited production. The present study introduces a loop-mediated isothermal amplification (LAMP) technique for the differentiation of saffron from its adulterants. This novel technique is sensitive, efficient and simple. Six specific LAMP primers were designed on the basis of the nucleotide sequence of the internal transcribed spacer 2 (ITS2) nuclear ribosomal DNA of Crocus sativus. All LAMP amplifications were performed successfully, and visual detection occurred within 60 min at isothermal conditions of 65 °C. The results indicated that the LAMP primers are accurate and highly specific for the discrimination of saffron from its adulterants. In particular, 10 fg of genomic DNA was determined to be the limit for template accuracy of LAMP in saffron. Thus, the proposed novel, simple, and sensitive LAMP assay is well suited for immediate on-site discrimination of herbal materials. Based on the study, a practical standard operating procedure (SOP) for utilizing the LAMP protocol for herbal authentication is provided. PMID:27146605

Rapid authentication of the precious herb saffron by loop-mediated isothermal amplification (LAMP) based on internal transcribed spacer 2 (ITS2) sequence.

PubMed

Zhao, Mingming; Shi, Yuhua; Wu, Lan; Guo, Licheng; Liu, Wei; Xiong, Chao; Yan, Song; Sun, Wei; Chen, Shilin

2016-05-05

Saffron is one of the most expensive species of Chinese herbs and has been subjected to various types of adulteration because of its high price and limited production. The present study introduces a loop-mediated isothermal amplification (LAMP) technique for the differentiation of saffron from its adulterants. This novel technique is sensitive, efficient and simple. Six specific LAMP primers were designed on the basis of the nucleotide sequence of the internal transcribed spacer 2 (ITS2) nuclear ribosomal DNA of Crocus sativus. All LAMP amplifications were performed successfully, and visual detection occurred within 60 min at isothermal conditions of 65 °C. The results indicated that the LAMP primers are accurate and highly specific for the discrimination of saffron from its adulterants. In particular, 10 fg of genomic DNA was determined to be the limit for template accuracy of LAMP in saffron. Thus, the proposed novel, simple, and sensitive LAMP assay is well suited for immediate on-site discrimination of herbal materials. Based on the study, a practical standard operating procedure (SOP) for utilizing the LAMP protocol for herbal authentication is provided.

Real-time polymerase chain reaction assay for rapid and sensitive detection of anthrax spores in spiked soil and talcum powder.

PubMed

Jain, Neha; Merwyn, S; Rai, G P; Agarwal, G S

2012-05-01

Real-time polymerase chain reaction (real-time PCR) is a laboratory technique based on PCR. This technique is able to detect sequence-specific PCR products as they accumulate in "real time" during the PCR amplification, and also to quantify the number of substrates present in the initial PCR mixture before amplification begins. In the present study, real-time PCR assay was employed for rapid and real-time detection of Bacillus anthracis spores spiked in 0.1 g of soil and talcum powder ranging from 5 to 10(7) spores. DNA was isolated from spiked soil and talcum powder, using PBS containing 1 % Triton-X-100, followed by heat treatment. The isolated DNA was used as template for real-time PCR and PCR. Real-time PCR amplification was obtained in 60 min under the annealing condition at 60°C by employing primers targeting the pag gene of B. anthracis. In the present study, the detection limit of real-time PCR assay in soil was 10(3) spores and 10(2) spores in talcum powder, respectively, whereas PCR could detect 10(4) spores in soil and 10(3) spores in talcum powder, respectively.

Preparation of DNA-containing extract for PCR amplification

DOEpatents

Dunbar, John M.; Kuske, Cheryl R.

2006-07-11

Environmental samples typically include impurities that interfere with PCR amplification and DNA quantitation. Samples of soil, river water, and aerosol were taken from the environment and added to an aqueous buffer (with or without detergent). Cells from the sample are lysed, releasing their DNA into the buffer. After removing insoluble cell components, the remaining soluble DNA-containing extract is treated with N-phenacylthiazolium bromide, which causes rapid precipitation of impurities. Centrifugation provides a supernatant that can be used or diluted for PCR amplification of DNA, or further purified. The method may provide a DNA-containing extract sufficiently pure for PCR amplification within 5–10 minutes.

The successes and future prospects of the linear antisense RNA amplification methodology.

PubMed

Li, Jifen; Eberwine, James

2018-05-01

It has been over a quarter of a century since the introduction of the linear RNA amplification methodology known as antisense RNA (aRNA) amplification. Whereas most molecular biology techniques are rapidly replaced owing to the fast-moving nature of development in the field, the aRNA procedure has become a base that can be built upon through varied uses of the technology. The technique was originally developed to assess RNA populations from small amounts of starting material, including single cells, but over time its use has evolved to include the detection of various cellular entities such as proteins, RNA-binding-protein-associated cargoes, and genomic DNA. In this Perspective we detail the linear aRNA amplification procedure and its use in assessing various components of a cell's chemical phenotype. This procedure is particularly useful in efforts to multiplex the simultaneous detection of various cellular processes. These efforts are necessary to identify the quantitative chemical phenotype of cells that underlies cellular function.

Direct LAMP Assay without Prior DNA Purification for Sex Determination of Papaya.

PubMed

Tsai, Chi-Chu; Shih, Huei-Chuan; Ko, Ya-Zhu; Wang, Ren-Huang; Li, Shu-Ju; Chiang, Yu-Chung

2016-09-24

Papaya (Carica papaya L.) is an economically important tropical fruit tree with hermaphrodite, male and female sex types. Hermaphroditic plants are the major type used for papaya production because their fruits have more commercial advantages than those of female plants. Sex determination of the seedlings, or during the early growth stages, is very important for the papaya seedling industry. Thus far, the only method for determining the sex type of a papaya at the seedling stage has been DNA analysis. In this study, a molecular technique-based on DNA analysis-was developed for detecting male-hermaphrodite-specific markers to examine the papaya's sex type. This method is based on the loop-mediated isothermal amplification (LAMP) and does not require prior DNA purification. The results show that the method is an easy, efficient, and inexpensive way to determine a papaya's sex. This is the first report on the LAMP assay, using intact plant materials-without DNA purification-as samples for the analysis of sex determination of papaya. We found that using high-efficiency DNA polymerase was essential for successful DNA amplification, using trace intact plant material as a template DNA source.

Micropreparative capillary gel electrophoresis of DNA: rapid expressed sequence tag library construction.

PubMed

Shi, Liang; Khandurina, Julia; Ronai, Zsolt; Li, Bi-Yu; Kwan, Wai King; Wang, Xun; Guttman, András

2003-01-01

A capillary gel electrophoresis based automated DNA fraction collection technique was developed to support a novel DNA fragment-pooling strategy for expressed sequence tag (EST) library construction. The cDNA population is first cleaved by BsaJ I and EcoR I restriction enzymes, and then subpooled by selective ligation with specific adapters followed by polymerase chain reaction (PCR) amplification and labeling. Combination of this cDNA fingerprinting method with high-resolution capillary gel electrophoresis separation and precise fractionation of individual cDNA transcript representatives avoids redundant fragment selection and concomitant repetitive sequencing of abundant transcripts. Using a computer-controlled capillary electrophoresis device the transcript representatives were separated by their size and fractions were automatically collected in every 30 s into 96-well plates. The high resolving power of the sieving matrix ensured sequencing grade separation of the DNA fragments (i.e., single-base resolution) and successful fraction collection. Performance and precision of the fraction collection procedure was validated by PCR amplification of the collected DNA fragments followed by capillary electrophoresis analysis for size and purity verification. The collected and PCR-amplified transcript representatives, ranging up to several hundred base pairs, were then sequenced to create an EST library.

Enhanced genetic analysis of single human bioparticles recovered by simplified micromanipulation from forensic 'touch DNA' evidence.

PubMed

Farash, Katherine; Hanson, Erin K; Ballantyne, Jack

2015-03-09

DNA profiles can be obtained from 'touch DNA' evidence, which comprises microscopic traces of human biological material. Current methods for the recovery of trace DNA employ cotton swabs or adhesive tape to sample an area of interest. However, such a 'blind-swabbing' approach will co-sample cellular material from the different individuals, even if the individuals' cells are located in geographically distinct locations on the item. Thus, some of the DNA mixtures encountered in touch DNA samples are artificially created by the swabbing itself. In some instances, a victim's DNA may be found in significant excess thus masking any potential perpetrator's DNA. In order to circumvent the challenges with standard recovery and analysis methods, we have developed a lower cost, 'smart analysis' method that results in enhanced genetic analysis of touch DNA evidence. We describe an optimized and efficient micromanipulation recovery strategy for the collection of bio-particles present in touch DNA samples, as well as an enhanced amplification strategy involving a one-step 5 µl microvolume lysis/STR amplification to permit the recovery of STR profiles from the bio-particle donor(s). The use of individual or few (i.e., "clumps") bioparticles results in the ability to obtain single source profiles. These procedures represent alternative enhanced techniques for the isolation and analysis of single bioparticles from forensic touch DNA evidence. While not necessary in every forensic investigation, the method could be highly beneficial for the recovery of a single source perpetrator DNA profile in cases involving physical assault (e.g., strangulation) that may not be possible using standard analysis techniques. Additionally, the strategies developed here offer an opportunity to obtain genetic information at the single cell level from a variety of other non-forensic trace biological material.

The Interplay Between Estrogen and Replication Origins in Breast Cancer DNA Amplification

DTIC Science & Technology

2013-09-01

Replication Origins in Breast Cancer DNA Amplification PRINCIPAL INVESTIGATOR: Cinzia Casella CONTRACTING ORGANIZATION: Brown...Interplay Between Estrogen and Replication Origins in Breast Cancer DNA Amplification 5b. GRANT NUMBER W81XWH-11-1-0599 5c. PROGRAM ELEMENT NUMBER 6... amplification and oncogenes activation in breast cancer cells? This project aims to understand the role of estrogen in inducing re-replication, thus

Isothermal amplification of environmental DNA (eDNA) for direct field-based monitoring and laboratory confirmation of Dreissena sp.

PubMed

Williams, Maggie R; Stedtfeld, Robert D; Engle, Cathrine; Salach, Paul; Fakher, Umama; Stedtfeld, Tiffany; Dreelin, Erin; Stevenson, R Jan; Latimore, Jo; Hashsham, Syed A

2017-01-01

Loop-mediated isothermal amplification (LAMP) of aquatic invasive species environmental DNA (AIS eDNA) was used for rapid, sensitive, and specific detection of Dreissena sp. relevant to the Great Lakes (USA) basin. The method was validated for two uses including i) direct amplification of eDNA using a hand filtration system and ii) confirmation of the results after DNA extraction using a conventional thermal cycler run at isothermal temperatures. Direct amplification eliminated the need for DNA extraction and purification and allowed detection of target invasive species in grab or concentrated surface water samples, containing both free DNA as well as larger cells and particulates, such as veligers, eggs, or seeds. The direct amplification method validation was conducted using Dreissena polymorpha and Dreissena bugensis and uses up to 1 L grab water samples for high target abundance (e.g., greater than 10 veligers (larval mussels) per L for Dreissena sp.) or 20 L samples concentrated through 35 μm nylon screens for low target abundance, at less than 10 veligers per liter water. Surface water concentrate samples were collected over a period of three years, mostly from inland lakes in Michigan with the help of a network of volunteers. Field samples collected from 318 surface water locations included i) filtered concentrate for direct amplification validation and ii) 1 L grab water sample for eDNA extraction and confirmation. Though the extraction-based protocol was more sensitive (resulting in more positive detections than direct amplification), direct amplification could be used for rapid screening, allowing for quicker action times. For samples collected between May and August, results of eDNA direct amplification were consistent with known presence/absence of selected invasive species. A cross-platform smartphone application was also developed to disseminate the analyzed results to volunteers. Field tests of the direct amplification protocol using a portable device (Gene-Z) showed the method could be used in the field to obtain results within one hr (from sample to result). Overall, the direct amplification has the potential to simplify the eDNA-based monitoring of multiple aquatic invasive species. Additional studies are warranted to establish quantitative correlation between eDNA copy number, veliger, biomass or organismal abundance in the field.

Isothermal amplification of environmental DNA (eDNA) for direct field-based monitoring and laboratory confirmation of Dreissena sp.

PubMed Central

Stedtfeld, Robert D.; Engle, Cathrine; Salach, Paul; Fakher, Umama; Stedtfeld, Tiffany; Dreelin, Erin; Stevenson, R. Jan; Latimore, Jo; Hashsham, Syed A.

2017-01-01

Loop-mediated isothermal amplification (LAMP) of aquatic invasive species environmental DNA (AIS eDNA) was used for rapid, sensitive, and specific detection of Dreissena sp. relevant to the Great Lakes (USA) basin. The method was validated for two uses including i) direct amplification of eDNA using a hand filtration system and ii) confirmation of the results after DNA extraction using a conventional thermal cycler run at isothermal temperatures. Direct amplification eliminated the need for DNA extraction and purification and allowed detection of target invasive species in grab or concentrated surface water samples, containing both free DNA as well as larger cells and particulates, such as veligers, eggs, or seeds. The direct amplification method validation was conducted using Dreissena polymorpha and Dreissena bugensis and uses up to 1 L grab water samples for high target abundance (e.g., greater than 10 veligers (larval mussels) per L for Dreissena sp.) or 20 L samples concentrated through 35 μm nylon screens for low target abundance, at less than 10 veligers per liter water. Surface water concentrate samples were collected over a period of three years, mostly from inland lakes in Michigan with the help of a network of volunteers. Field samples collected from 318 surface water locations included i) filtered concentrate for direct amplification validation and ii) 1 L grab water sample for eDNA extraction and confirmation. Though the extraction-based protocol was more sensitive (resulting in more positive detections than direct amplification), direct amplification could be used for rapid screening, allowing for quicker action times. For samples collected between May and August, results of eDNA direct amplification were consistent with known presence/absence of selected invasive species. A cross-platform smartphone application was also developed to disseminate the analyzed results to volunteers. Field tests of the direct amplification protocol using a portable device (Gene-Z) showed the method could be used in the field to obtain results within one hr (from sample to result). Overall, the direct amplification has the potential to simplify the eDNA-based monitoring of multiple aquatic invasive species. Additional studies are warranted to establish quantitative correlation between eDNA copy number, veliger, biomass or organismal abundance in the field. PMID:29036210

fM to aM nucleic acid amplification for molecular diagnostics in a non-stick-coated metal microfluidic bioreactor

PubMed Central

Huang, Guoliang; Huang, Qin; Ma, Li; Luo, Xianbo; Pang, Biao; Zhang, Zhixin; Wang, Ruliang; Zhang, Junqi; Li, Qi; Fu, Rongxin; Ye, Jiancheng

2014-01-01

A sensitive DNA isothermal amplification method for the detection of DNA at fM to aM concentrations for pathogen identification was developed using a non-stick-coated metal microfluidic bioreactor. A portable confocal optical detector was utilized to monitor the DNA amplification in micro- to nanoliter reaction assays in real-time, with fluorescence collection near the optical diffraction limit. The non-stick-coated metal microfluidic bioreactor, with a surface contact angle of 103°, was largely inert to bio-molecules, and DNA amplification could be performed in a minimum reaction volume of 40 nL. The isothermal nucleic acid amplification for Mycoplasma pneumoniae identification in the non-stick-coated microfluidic bioreactor could be performed at a minimum DNA template concentration of 1.3 aM, and a detection limit of three copies of genomic DNA was obtained. This microfluidic bioreactor offers a promising clinically relevant pathogen molecular diagnostic method via the amplification of targets from only a few copies of genomic DNA from a single bacterium. PMID:25475544

Indel analysis by droplet digital PCR: a sensitive method for DNA mixture detection and chimerism analysis.

PubMed

Santurtún, Ana; Riancho, José A; Arozamena, Jana; López-Duarte, Mónica; Zarrabeitia, María T

2017-01-01

Several methods have been developed to determinate genetic profiles from a mixed samples and chimerism analysis in transplanted patients. The aim of this study was to explore the effectiveness of using the droplet digital PCR (ddPCR) for mixed chimerism detection (a mixture of genetic profiles resulting after allogeneic hematopoietic stem cell transplantation (HSCT)). We analyzed 25 DNA samples from patients who had undergone HSCT and compared the performance of ddPCR and two established methods for chimerism detection, based upon the Indel and STRs analysis, respectively. Additionally, eight artificial mixture DNA samples were created to evaluate the sensibility of ddPCR. Our results show that the chimerism percentages estimated by the analysis of a single Indel using ddPCR were very similar to those calculated by the amplification of 15 STRs (r 2  = 0.970) and with the results obtained by the amplification of 38 Indels (r 2  = 0.975). Moreover, the amplification of a single Indel by ddPCR was sensitive enough to detect a minor DNA contributor comprising down to 0.5 % of the sample. We conclude that ddPCR can be a powerful tool for the determination of a genetic profile of forensic mixtures and clinical chimerism analysis when traditional techniques are not sensitive enough.

Construction and Evaluation of Internal Control DNA for PCR Amplification of Chlamydia trachomatis DNA from Urine Samples

PubMed Central

Betsou, Fotini; Beaumont, Katy; Sueur, Jean Marie; Orfila, Jeanne

2003-01-01

An internal control DNA (ICD) with the same primer binding sequences as the target Chlamydia trachomatis DNA was constructed and evaluated in a PCR assay with immunoenzymatic detection. One hundred urine specimens were tested, and 23 were found to contain inhibitors of the PCR, if not subjected to DNA extraction prior to amplification. Coamplification and detection of the ICD appeared to be a useful method for estimating the effects of inhibitors on C. trachomatis DNA amplification. PMID:12624066

System for portable nucleic acid testing in low resource settings

NASA Astrophysics Data System (ADS)

Lu, Hsiang-Wei; Roskos, Kristina; Hickerson, Anna I.; Carey, Thomas; Niemz, Angelika

2013-03-01

Our overall goal is to enable timely diagnosis of infectious diseases through nucleic acid testing at the point-of-care and in low resource settings, via a compact system that integrates nucleic acid sample preparation, isothermal DNA amplification, and nucleic acid lateral flow (NALF) detection. We herein present an interim milestone, the design of the amplification and detection subsystem, and the characterization of thermal and fluidic control and assay execution within this system. Using an earlier prototype of the amplification and detection unit, comprised of a disposable cartridge containing flexible pouches, passive valves, and electrolysis-driven pumps, in conjunction with a small heater, we have demonstrated successful execution of an established and clinically validated isothermal loop-mediated amplification (LAMP) reaction targeting Mycobacterium tuberculosis (M.tb) DNA, coupled to NALF detection. The refined design presented herein incorporates miniaturized and integrated electrolytic pumps, novel passive valves, overall design changes to facilitate integration with an upstream sample preparation unit, and a refined instrument design that automates pumping, heating, and timing. Nucleic acid amplification occurs in a two-layer pouch that facilitates fluid handling and appropriate thermal control. The disposable cartridge is manufactured using low-cost and scalable techniques and forms a closed system to prevent workplace contamination by amplicons. In a parallel effort, we are developing a sample preparation unit based on similar design principles, which performs mechanical lysis of mycobacteria and DNA extraction from liquefied and disinfected sputum. Our next step is to combine sample preparation, amplification, and detection in a final integrated cartridge and device, to enable fully automated sample-in to answer-out diagnosis of active tuberculosis in primary care facilities of low-resource and high-burden countries.

Development of internal amplification controls for DNA profiling with the AmpFℓSTR(®) SGM Plus(®) kit.

PubMed

Zahra, Nathalie; Hadi, Sibte; Smith, Judith A; Iyengar, Arati; Goodwin, William

2011-06-01

DNA extracted from forensic samples can be degraded and also contain co-extracted contaminants that inhibit PCR. The effects of DNA degradation and PCR inhibition are often indistinguishable when examining a DNA profile. Two internal amplification controls (IACs) were developed to improve quality control of PCR using the AmpFℓSTR® SGM Plus® kit. The co-amplification of these controls with DNA samples was used to monitor amplification efficiency and detect PCR inhibitors. IAC fragments of 90 and 410 bp (IAC₉₀ and IAC₄₁₀) were generated from the plasmid pBR322 using tailed primers and then amplified with ROX-labelled primers. Co-amplification of IAC₉₀ and IAC₄₁₀ was performed with varying amounts of template DNA, degraded DNA and DNA contaminated with humic acid, heme and indigo dye. Both IAC₉₀ and IAC₄₁₀ were successfully amplified with human DNA without significantly affecting the quality of the DNA profile, even with DNA amounts lower than 0.5 ng. In the presence of inhibitors, the IAC₉₀ signal was still present after all human DNA loci fail to amplify; in contrast, the IAC₄₁₀ signal was reduced or absent at low levels of inhibition. Amplification of the two IACs provided an internal PCR control and allowed partial profiles caused by inhibition to be distinguished from degraded DNA profiles. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Whole-genome multiple displacement amplification from single cells.

PubMed

Spits, Claudia; Le Caignec, Cédric; De Rycke, Martine; Van Haute, Lindsey; Van Steirteghem, André; Liebaers, Inge; Sermon, Karen

2006-01-01

Multiple displacement amplification (MDA) is a recently described method of whole-genome amplification (WGA) that has proven efficient in the amplification of small amounts of DNA, including DNA from single cells. Compared with PCR-based WGA methods, MDA generates DNA with a higher molecular weight and shows better genome coverage. This protocol was developed for preimplantation genetic diagnosis, and details a method for performing single-cell MDA using the phi29 DNA polymerase. It can also be useful for the amplification of other minute quantities of DNA, such as from forensic material or microdissected tissue. The protocol includes the collection and lysis of single cells, and all materials and steps involved in the MDA reaction. The whole procedure takes 3 h and generates 1-2 microg of DNA from a single cell, which is suitable for multiple downstream applications, such as sequencing, short tandem repeat analysis or array comparative genomic hybridization.

Adaptation of a visualized loop-mediated isothermal amplification technique for field detection of Plasmodium vivax infection.

PubMed

Tao, Zhi-Yong; Zhou, Hua-Yun; Xia, Hui; Xu, Sui; Zhu, Han-Wu; Culleton, Richard L; Han, Eun-Taek; Lu, Feng; Fang, Qiang; Gu, Ya-Ping; Liu, Yao-Bao; Zhu, Guo-Ding; Wang, Wei-Ming; Li, Ju-Lin; Cao, Jun; Gao, Qi

2011-06-21

Loop-mediated isothermal amplification (LAMP) is a high performance method for detecting DNA and holds promise for use in the molecular detection of infectious pathogens, including Plasmodium spp. However, in most malaria-endemic areas, which are often resource-limited, current LAMP methods are not feasible for diagnosis due to difficulties in accurately interpreting results with problems of sensitive visualization of amplified products, and the risk of contamination resulting from the high quantity of amplified DNA produced. In this study, we establish a novel visualized LAMP method in a closed-tube system, and validate it for the diagnosis of malaria under simulated field conditions. A visualized LAMP method was established by the addition of a microcrystalline wax-dye capsule containing the highly sensitive DNA fluorescence dye SYBR Green I to a normal LAMP reaction prior to the initiation of the reaction. A total of 89 blood samples were collected on filter paper and processed using a simple boiling method for DNA extraction, and then tested by the visualized LAMP method for Plasmodium vivax infection. The wax capsule remained intact during isothermal amplification, and released the DNA dye to the reaction mixture only when the temperature was raised to the melting point following amplification. Soon after cooling down, the solidified wax sealed the reaction mix at the bottom of the tube, thus minimizing the risk of aerosol contamination. Compared to microscopy, the sensitivity and specificity of LAMP were 98.3% (95% confidence interval (CI): 91.1-99.7%) and 100% (95% CI: 88.3-100%), and were in close agreement with a nested polymerase chain reaction method. This novel, cheap and quick visualized LAMP method is feasible for malaria diagnosis in resource-limited field settings.

Diagnostic application of polymerase chain reaction for detection of Ehrlichia risticii in equine monocytic ehrlichiosis (Potomac horse fever).

PubMed

Biswas, B; Mukherjee, D; Mattingly-Napier, B L; Dutta, S K

1991-10-01

Genomic amplification by the polymerase chain reaction (PCR) was used to identify a unique genomic sequence of Ehrlichia risticii directly in DNA isolated from peripheral-blood buffy coat cells of E. risticii-infected horses (Potomac horse fever) and from infected cell cultures. A specific primer pair, selected from a cloned, species-specific, 1-kb DNA fragment of the E. risticii genome as a template, was used for the amplification of the target DNA of 247 bp. The optimal number of 40 PCR cycles, determined by analyzing an amplification profile obtained with a constant Taq polymerase concentration, was used to achieve maximum amplification of the E. risticii DNA segment. Efficient amplification of target DNA was achieved with specimens processed by either the phenol extraction or rapid lysis method. The specificity of the amplified DNA product was confirmed by the proper size (247 bp) and appropriate restriction enzyme cleavage pattern of the amplified target DNA, as well as by the specific hybridization signal obtained by using a PCR-amplified 185-bp internal DNA probe. A 10(5)- to 10(6)-fold amplification of target DNA, which allowed detection of E. risticii from as few as two to three infected cells in culture and from a very small volume of buffy coat cells from infected horses, was achieved. This PCR amplification procedure was found to be highly specific and sensitive for the detection of E. risticii for the study of Potomac horse fever.

Rapid and Sensitive Isothermal Detection of Nucleic-acid Sequence by Multiple Cross Displacement Amplification.

PubMed

Wang, Yi; Wang, Yan; Ma, Ai-Jing; Li, Dong-Xun; Luo, Li-Juan; Liu, Dong-Xin; Jin, Dong; Liu, Kai; Ye, Chang-Yun

2015-07-08

We have devised a novel amplification strategy based on isothermal strand-displacement polymerization reaction, which was termed multiple cross displacement amplification (MCDA). The approach employed a set of ten specially designed primers spanning ten distinct regions of target sequence and was preceded at a constant temperature (61-65 °C). At the assay temperature, the double-stranded DNAs were at dynamic reaction environment of primer-template hybrid, thus the high concentration of primers annealed to the template strands without a denaturing step to initiate the synthesis. For the subsequent isothermal amplification step, a series of primer binding and extension events yielded several single-stranded DNAs and single-stranded single stem-loop DNA structures. Then, these DNA products enabled the strand-displacement reaction to enter into the exponential amplification. Three mainstream methods, including colorimetric indicators, agarose gel electrophoresis and real-time turbidity, were selected for monitoring the MCDA reaction. Moreover, the practical application of the MCDA assay was successfully evaluated by detecting the target pathogen nucleic acid in pork samples, which offered advantages on quick results, modest equipment requirements, easiness in operation, and high specificity and sensitivity. Here we expounded the basic MCDA mechanism and also provided details on an alternative (Single-MCDA assay, S-MCDA) to MCDA technique.

A novel loop-mediated isothermal amplification-based test for detecting Neospora caninum DNA.

PubMed

Ramos, Andrea Estefanía; Muñoz, Marina; Cortés-Vecino, Jesús Alfredo; Barato, Paola; Patarroyo, Manuel Alfonso

2017-11-29

Neospora caninum is a cyst-forming, coccidian parasite which is known to cause neurological disorders in dogs and abortion and neonatal mortality in cows and other livestock. This study reports the development of a loop-mediated isothermal amplification (LAMP) assay based on the Neospora caninum Nc-5 gene and compares its efficacy for detecting DNA to that of a semi-nested PCR test. Six primers were designed based on the Nc-5 repeat region of N. caninum. Specific LAMP primers led to successful amplification of N. caninum DNA at 63 °C in 30 min. The LAMP assay was highly specific (i.e. it did not reveal cross-reactivity with other parasite species) and had a low N. caninum plasmid DNA limit of detection (1 fg), which is ten times higher than that for the semi-nested PCR. LAMP applicability was evaluated using a set of naturally-infected samples (59 from canine faeces and five from bovine abortions). Thirty-nine percent (25/64) of the naturally-infected samples were positive for N. caninum DNA by LAMP and 36% (23/64) by semi-nested PCR. However, the LAMP assay is much faster to perform than semi-nested PCR and provides results in 30 min. The optimized reaction conditions described in this study resulted in a sensitive, specific and rapid technique for detecting N. caninum DNA. Considering the advantages of LAMP for detecting N. caninum DNA, further assays aimed at testing its usefulness on a wider range of field samples are recommended.

Molecular contributions to conservation

USGS Publications Warehouse

Haig, Susan M.

1998-01-01

Recent advances in molecular technology have opened a new chapter in species conservation efforts, as well as population biology. DNA sequencing, MHC (major histocompatibility complex), minisatellite, microsatellite, and RAPD (random amplified polymorphic DNA) procedures allow for identification of parentage, more distant relatives, founders to new populations, unidentified individuals, population structure, effective population size, population-specific markers, etc. PCR (polymerase chain reaction) amplification of mitochondrial DNA, nuclear DNA, ribosomal DNA, chloroplast DNA, and other systems provide for more sophisticated analyses of metapopulation structure, hybridization events, and delineation of species, subspecies, and races, all of which aid in setting species recovery priorities. Each technique can be powerful in its own right but is most credible when used in conjunction with other molecular techniques and, most importantly, with ecological and demographic data collected from the field. Surprisingly few taxa of concern have been assayed with any molecular technique. Thus, rather than showcasing exhaustive details from a few well-known examples, this paper attempts to present a broad range of cases in which molecular techniques have been used to provide insight into conservation efforts.

[Synthesis of Circular DNA Templates with T4 RNA Ligase for Rolling Circle Amplification].

PubMed

Sakhabutdinova, A R; Maksimova, M A; Garafutdinov, R R

2017-01-01

Currently, isothermal methods of nucleic acid amplification have been well established; in particular, rolling circle amplification is of great interest. In this approach, circular ssDNA molecules have been used as a target that can be obtained by the intramolecular template-dependent ligation of an oligonucleotide C-probe. Here, a new method of synthesizing small circular DNA molecules via the cyclization of ssDNA based on T4 RNA ligase has been proposed. Circular ssDNA is further used as the template for the rolling circle amplification. The maximum yield of the cyclization products was observed in the presence of 5-10% polyethylene glycol 4000, and the optimum DNA length for the cyclization constituted 50 nucleotides. This highly sensitive method was shown to detect less than 10^(2) circular DNA molecules. The method reliability was proved based on artificially destroyed dsDNA, which suggests its implementation for analyzing any significantly fragmented dsDNA.

Deep Diversity: Novel Approach to Overcoming the PCR Bias Encountered During Environmental Analysis of Microbial Populations for Alpha-Diversity

NASA Technical Reports Server (NTRS)

Ramirez, Gustavo A; Vaishampayan, Parag A.

2011-01-01

Alpha-diversity studies are of crucial importance to environmental microbiologists. The polymerase chain reaction (PCR) method has been paramount for studies interrogating microbial environmental samples for taxon richness. Phylogenetic studies using this technique are based on the amplification and comparison of the 16S rRNA coding regions. PCR, due disproportionate distribution of microbial species in the environment, increasingly favors the amplification of the most predominant phylotypes with every subsequent reaction cycle. The genetic and chemical complexity of environmental samples are intrinsic factors that exacerbate an inherit bias in PCR-based quantitative and qualitative studies of microbial communities. We report that treatment of a genetically complex total genomic environmental DNA extract with Propidium Monoazide (PMA), a DNA intercalating molecule capable of forming a covalent cross-linkage to organic moieties upon light exposure, disproportionally inactivates predominant phylotypes and results in the exponential amplification of previously shadowed microbial ?-diversity quantified as a 19.5% increase in OUTs reported via phylogenetic screening using PhyloChip.

SMA Diagnosis: Detection of SMN1 Deletion with Real-Time mCOP-PCR System Using Fresh Blood DNA.

PubMed

Niba, Emma Tabe Eko; Ar Rochmah, Mawaddah; Harahap, Nur Imma Fatimah; Awano, Hiroyuki; Morioka, Ichiro; Iijima, Kazumoto; Saito, Toshio; Saito, Kayoko; Takeuchi, Atsuko; Lai, Poh San; Bouike, Yoshihiro; Nishio, Hisahide; Shinohara, Masakazu

2017-12-18

Spinal muscular atrophy (SMA) is one of the most common autosomal recessive disorders. The symptoms are caused by defects of lower motor neurons in the spinal cord. More than 95% of SMA patients are homozygous for survival motor neuron 1 (SMN1) deletion. We previously developed a screening system for SMN1 deletion based on a modified competitive oligonucleotide priming-PCR (mCOP-PCR) technique using dried blood spot (DBS) on filter paper. This system is convenient for mass screening in the large population and/or first-tier diagnostic method of the patients in the remote areas. However, this system was still time-consuming and effort-taking, because it required pre-amplification procedure to avoid non-specific amplification and gel-electrophoresis to detect the presence or absence of SMN1 deletion. When the fresh blood samples are used instead of DBS, or when the gel-electrophoresis is replaced by real-time PCR, we may have a simpler and more rapid diagnostic method for SMA. To establish a simpler and more rapid diagnostic method of SMN1 deletion using fresh blood DNA. DNA samples extracted from fresh blood and stored at 4 ℃ for 1 month. The samples were assayed using a real-time mCOP-PCR system without pre-amplification procedures. DNA samples had already been genotyped by PCR-restriction fragment length polymorphism (PCR-RFLP), showing the presence or absence of SMN1 exon 7. The DNA samples were directly subjected to the mCOP-PCR step. The amplification of mCOP-PCR was monitored in a real-time PCR apparatus. The genotyping results of the real-time mCOP-PCR system using fresh blood DNA were completely matched with those of PCR-RFLP. In this real-time mCOP-PCR system using fresh blood-DNA, it took only four hours from extraction of DNA to detection of the presence or absence of SMN1 deletion, while it took more than 12 hours in PCR-RFLP. Our real-time mCOP-PCR system using fresh blood DNA was rapid and accurate, suggesting it may be useful for the first-tier diagnostic method of SMA.

Simplified Real-Time Multiplex Detection of Loop-Mediated Isothermal Amplification Using Novel Mediator Displacement Probes with Universal Reporters.

PubMed

Becherer, Lisa; Bakheit, Mohammed; Frischmann, Sieghard; Stinco, Silvina; Borst, Nadine; Zengerle, Roland; von Stetten, Felix

2018-04-03

A variety of real-time detection techniques for loop-mediated isothermal amplification (LAMP) based on the change in fluorescence intensity during DNA amplification enable simultaneous detection of multiple targets. However, these techniques depend on fluorogenic probes containing target-specific sequences. That complicates the adaption to different targets leading to time-consuming assay optimization. Here, we present the first universal real-time detection technique for multiplex LAMP. The novel approach allows simple assay design and is easy to implement for various targets. The innovation features a mediator displacement probe and a universal reporter. During amplification of target DNA the mediator is displaced from the mediator displacement probe. Then it hybridizes to the reporter generating a fluorescence signal. The novel mediator displacement (MD) detection was validated against state-of-the-art molecular beacon (MB) detection by means of a HIV-1 RT-LAMP: MD surpassed MB detection by accelerated probe design (MD: 10 min, MB: 3-4 h), shorter times to positive (MD 4.1 ± 0.1 min shorter than MB, n = 36), improved signal-to-noise fluorescence ratio (MD: 5.9 ± 0.4, MB: 2.7 ± 0.4; n = 15), and showed equally good or better analytical performance parameters. The usability of one universal mediator-reporter set in different multiplex assays was successfully demonstrated for a biplex RT-LAMP of HIV-1 and HTLV-1 and a biplex LAMP of Haemophilus ducreyi and Treponema pallidum, both showing good correlation between target concentration and time to positive. Due to its simple implementation it is suggested to extend the use of the universal mediator-reporter sets to the detection of various other diagnostic panels.

Computational method and system for modeling, analyzing, and optimizing DNA amplification and synthesis

DOEpatents

Vandersall, Jennifer A.; Gardner, Shea N.; Clague, David S.

2010-05-04

A computational method and computer-based system of modeling DNA synthesis for the design and interpretation of PCR amplification, parallel DNA synthesis, and microarray chip analysis. The method and system include modules that address the bioinformatics, kinetics, and thermodynamics of DNA amplification and synthesis. Specifically, the steps of DNA selection, as well as the kinetics and thermodynamics of DNA hybridization and extensions, are addressed, which enable the optimization of the processing and the prediction of the products as a function of DNA sequence, mixing protocol, time, temperature and concentration of species.

Nondestructive DNA extraction from blackflies (Diptera: Simuliidae): retaining voucher specimens for DNA barcoding projects.

PubMed

Hunter, Stephanie J; Goodall, Tim I; Walsh, Kerry A; Owen, Richard; Day, John C

2008-01-01

A nondestructive, chemical-free method is presented for the extraction of DNA from small insects. Blackflies were submerged in sterile, distilled water and sonicated for varying lengths of time to provide DNA which was assessed in terms of quantity, purity and amplification efficiency. A verified DNA barcode was produced from DNA extracted from blackfly larvae, pupae and adult specimens. A 60-second sonication period was found to release the highest quality and quantity of DNA although the amplification efficiency was found to be similar regardless of sonication time. Overall, a 66% amplification efficiency was observed. Examination of post-sonicated material confirmed retention of morphological characters. Sonication was found to be a reliable DNA extraction approach for barcoding, providing sufficient quality template for polymerase chain reaction amplification as well as retaining the voucher specimen for post-barcoding morphological evaluation. © 2007 The Authors.

Sequencing of the large dsDNA genome of Oryctes rhinoceros nudivirus using multiple displacement amplification of nanogram amounts of virus DNA.

PubMed

Wang, Yongjie; Kleespies, Regina G; Ramle, Moslim B; Jehle, Johannes A

2008-09-01

The genomic sequence analysis of many large dsDNA viruses is hampered by the lack of enough sample materials. Here, we report a whole genome amplification of the Oryctes rhinoceros nudivirus (OrNV) isolate Ma07 starting from as few as about 10 ng of purified viral DNA by application of phi29 DNA polymerase- and exonuclease-resistant random hexamer-based multiple displacement amplification (MDA) method. About 60 microg of high molecular weight DNA with fragment sizes of up to 25 kbp was amplified. A genomic DNA clone library was generated using the product DNA. After 8-fold sequencing coverage, the 127,615 bp of OrNV whole genome was sequenced successfully. The results demonstrate that the MDA-based whole genome amplification enables rapid access to genomic information from exiguous virus samples.

Rapid Amplification of Plasmid and Phage DNA Using Phi29 DNA Polymerase and Multiply-Primed Rolling Circle Amplification

PubMed Central

Dean, Frank B.; Nelson, John R.; Giesler, Theresa L.; Lasken, Roger S.

2001-01-01

We describe a simple method of using rolling circle amplification to amplify vector DNA such as M13 or plasmid DNA from single colonies or plaques. Using random primers and φ29 DNA polymerase, circular DNA templates can be amplified 10,000-fold in a few hours. This procedure removes the need for lengthy growth periods and traditional DNA isolation methods. Reaction products can be used directly for DNA sequencing after phosphatase treatment to inactivate unincorporated nucleotides. Amplified products can also be used for in vitro cloning, library construction, and other molecular biology applications. PMID:11381035

Maintenance of host DNA integrity in field-preserved mosquito (Diptera: Culicidae) blood meals for identification by DNA barcoding.

PubMed

Reeves, Lawrence E; Holderman, Chris J; Gillett-Kaufman, Jennifer L; Kawahara, Akito Y; Kaufman, Phillip E

2016-09-15

Determination of the interactions between hematophagous arthropods and their hosts is a necessary component to understanding the transmission dynamics of arthropod-vectored pathogens. Current molecular methods to identify hosts of blood-fed arthropods require the preservation of host DNA to serve as an amplification template. During transportation to the laboratory and storage prior to molecular analysis, genetic samples need to be protected from nucleases, and the degradation effects of hydrolysis, oxidation and radiation. Preservation of host DNA contained in field-collected blood-fed specimens has an additional caveat: suspension of the degradative effects of arthropod digestion on host DNA. Unless effective preservation methods are implemented promptly after blood-fed specimens are collected, host DNA will continue to degrade. Preservation methods vary in their efficacy, and need to be selected based on the logistical constraints of the research program. We compared four preservation methods (cold storage at -20 °C, desiccation, ethanol storage of intact mosquito specimens and crushed specimens on filter paper) for field storage of host DNA from blood-fed mosquitoes across a range of storage and post-feeding time periods. The efficacy of these techniques in maintaining host DNA integrity was evaluated using a polymerase chain reaction (PCR) to detect the presence of a sufficient concentration of intact host DNA templates for blood meal analysis. We applied a logistic regression model to assess the effects of preservation method, storage time and post-feeding time on the binomial response variable, amplification success. Preservation method, storage time and post-feeding time all significantly impacted PCR amplification success. Filter papers and, to a lesser extent, 95 % ethanol, were the most effective methods for the maintenance of host DNA templates. Amplification success of host DNA preserved in cold storage at -20 °C and desiccation was poor. Our data suggest that, of the methods tested, host DNA template integrity was most stable when blood meals were preserved using filter papers. Filter paper preservation is effective over short- and long-term storage, while ethanol preservation is only suitable for short-term storage. Cold storage at -20 °C, and desiccation of blood meal specimens, even for short time periods, should be avoided.

Label-free electrical detection of pyrophosphate generated from DNA polymerase reactions on field-effect devices.

PubMed

Credo, Grace M; Su, Xing; Wu, Kai; Elibol, Oguz H; Liu, David J; Reddy, Bobby; Tsai, Ta-Wei; Dorvel, Brian R; Daniels, Jonathan S; Bashir, Rashid; Varma, Madoo

2012-03-21

We introduce a label-free approach for sensing polymerase reactions on deoxyribonucleic acid (DNA) using a chelator-modified silicon-on-insulator field-effect transistor (SOI-FET) that exhibits selective and reversible electrical response to pyrophosphate anions. The chemical modification of the sensor surface was designed to include rolling-circle amplification (RCA) DNA colonies for locally enhanced pyrophosphate (PPi) signal generation and sensors with immobilized chelators for capture and surface-sensitive detection of diffusible reaction by-products. While detecting arrays of enzymatic base incorporation reactions is typically accomplished using optical fluorescence or chemiluminescence techniques, our results suggest that it is possible to develop scalable and portable PPi-specific sensors and platforms for broad biomedical applications such as DNA sequencing and microbe detection using surface-sensitive electrical readout techniques.

Optimization and evaluation of T7 based RNA linear amplification protocols for cDNA microarray analysis

PubMed Central

Zhao, Hongjuan; Hastie, Trevor; Whitfield, Michael L; Børresen-Dale, Anne-Lise; Jeffrey, Stefanie S

2002-01-01

Background T7 based linear amplification of RNA is used to obtain sufficient antisense RNA for microarray expression profiling. We optimized and systematically evaluated the fidelity and reproducibility of different amplification protocols using total RNA obtained from primary human breast carcinomas and high-density cDNA microarrays. Results Using an optimized protocol, the average correlation coefficient of gene expression of 11,123 cDNA clones between amplified and unamplified samples is 0.82 (0.85 when a virtual array was created using repeatedly amplified samples to minimize experimental variation). Less than 4% of genes show changes in expression level by 2-fold or greater after amplification compared to unamplified samples. Most changes due to amplification are not systematic both within one tumor sample and between different tumors. Amplification appears to dampen the variation of gene expression for some genes when compared to unamplified poly(A)+ RNA. The reproducibility between repeatedly amplified samples is 0.97 when performed on the same day, but drops to 0.90 when performed weeks apart. The fidelity and reproducibility of amplification is not affected by decreasing the amount of input total RNA in the 0.3–3 micrograms range. Adding template-switching primer, DNA ligase, or column purification of double-stranded cDNA does not improve the fidelity of amplification. The correlation coefficient between amplified and unamplified samples is higher when total RNA is used as template for both experimental and reference RNA amplification. Conclusion T7 based linear amplification reproducibly generates amplified RNA that closely approximates original sample for gene expression profiling using cDNA microarrays. PMID:12445333

Evaluation of whole genome amplified DNA to decrease material expenditure and increase quality.

PubMed

Bækvad-Hansen, Marie; Bybjerg-Grauholm, Jonas; Poulsen, Jesper B; Hansen, Christine S; Hougaard, David M; Hollegaard, Mads V

2017-06-01

The overall aim of this study is to evaluate whole genome amplification of DNA extracted from dried blood spot samples. We wish to explore ways of optimizing the amplification process, while decreasing the amount of input material and inherently the cost. Our primary focus of optimization is on the amount of input material, the amplification reaction volume, the number of replicates and amplification time and temperature. Increasing the quality of the amplified DNA and the subsequent results of array genotyping is a secondary aim of this project. This study is based on DNA extracted from dried blood spot samples. The extracted DNA was subsequently whole genome amplified using the REPLIg kit and genotyped on the PsychArray BeadChip (assessing > 570,000 SNPs genome wide). We used Genome Studio to evaluate the quality of the genotype data by call rates and log R ratios. The whole genome amplification process is robust and does not vary between replicates. Altering amplification time, temperature or number of replicates did not affect our results. We found that spot size i.e. amount of input material could be reduced without compromising the quality of the array genotyping data. We also showed that whole genome amplification reaction volumes can be reduced by a factor of 4, without compromising the DNA quality. Whole genome amplified DNA samples from dried blood spots is well suited for array genotyping and produces robust and reliable genotype data. However, the amplification process introduces additional noise to the data, making detection of structural variants such as copy number variants difficult. With this study, we explore ways of optimizing the amplification protocol in order to reduce noise and increase data quality. We found, that the amplification process was very robust, and that changes in amplification time or temperature did not alter the genotyping calls or quality of the array data. Adding additional replicates of each sample also lead to insignificant changes in the array data. Thus, the amount of noise introduced by the amplification process was consistent regardless of changes made to the amplification protocol. We also explored ways of decreasing material expenditure by reducing the spot size or the amplification reaction volume. The reduction did not affect the quality of the genotyping data.

Amplification of Mitochondrial DNA for detection of Plasmodiumvivax in Balochistan.

PubMed

Shahwani, Muhammad Naeem; Nisar, Samia; Aleem, Abdul; Panezai, Marina; Afridi, Sarwat; Malik, Shaukat Iqbal

2017-05-01

To access a new step using PCR to amplify the targeted mtDNA sequence for detecting specifically Plasmodium vivax and its co-infections, false positive and false negative results with Plasmodium falciparum. In this study we have standardized a new technical approach in which the target mitochondrial DNA sequence (mtDNA) was amplified by using a PCR technique as a tool to detect Plasmodium spp. Species specific primers were designed to hybridize with cytochrome c oxidase gene of P. vivax (cox I) and P. falciparum (cox III). Two hundred blood samples were collected on the basis of clinical symptoms which were initially examined through microscopic analysis after preparing Giemsa stained thick and thin blood smears. Afterwards genomic DNA was extracted from all samples and was then subjected to PCR amplification by using species specific primers and amplified segments were sequenced for confirmation of results. One-hundred and thirty-two blood samples were detected as positive for malaria by PCR, out of which 64 were found to be positive by PCR and 53 by both microscopy and PCR for P.vivax infection. Nine samples were found to be false negative, one P.vivax mono infection was declared as co infection by PCR and 3 samples identified as having P.falciparum gametes were confirmed as P.vivax by PCR amplification. Sensitivity and specificity were found to be 85% and 92% respectively. Results obtained through PCR method were comparatively better and reliable than microscopy.

Development of a real-time fluorescence loop-mediated isothermal amplification assay for rapid and quantitative detection of Fusarium oxysporum f. sp. cubense tropical race 4 in soil.

PubMed

Zhang, Xin; Zhang, He; Pu, Jinji; Qi, Yanxiang; Yu, Qunfang; Xie, Yixian; Peng, Jun

2013-01-01

Fusarium oxysporum f. sp. cubense (Foc), the causal agent of Fusarium wilt (Panama disease), is one of the most devastating diseases of banana (Musa spp.). The Foc tropical race 4 (TR4) is currently known as a major concern in global banana production. No effective resistance is known in Musa to Foc, and no effective measures for controlling Foc once banana plants have been infected in place. Early and accurate detection of Foc TR4 is essential to protect banana industry and guide banana planting. A real-time fluorescence loop-mediated isothermal amplification assay (RealAmp) was developed for the rapid and quantitative detection of Foc TR4 in soil. The detection limit of the RealAmp assay was approximately 0.4 pg/µl plasmid DNA when mixed with extracted soil DNA or 10(3) spores/g of artificial infested soil, and no cross-reaction with other relative pathogens were observed. The RealAmp assay for quantifying genomic DNA of TR4 was confirmed by testing both artificially and naturally infested samples. Quantification of the soil-borne pathogen DNA of Foc TR4 in naturally infested samples was no significant difference compared to classic real-time PCR (P>0.05). Additionally, RealAmp assay was visual with an improved closed-tube visual detection system by adding SYBR Green I fluorescent dye to the inside of the lid prior to amplification, which avoided the inhibitory effects of the stain on DNA amplification and makes the assay more convenient in the field and could thus become a simple, rapid and effective technique that has potential as an alternative tool for the detection and monitoring of Foc TR4 in field, which would be a routine DNA-based testing service for the soil-borne pathogen in South China.

Recombinase Polymerase Amplification Combined with Lateral Flow Strip for Listeria monocytogenes Detection in Food.

PubMed

Du, Xin-Jun; Zang, Yu-Xuan; Liu, Hai-Bin; Li, Ping; Wang, Shuo

2018-04-01

Listeria monocytogenes is an important food-borne pathogenic bacterium that causes human disease, resulting in economic losses worldwide. The current detection methods for L. monocytogenes are not well suited for direct field testing because they involve complicated, time-consuming operations. A simple, efficient method is vital for L. monocytogenes detection. In this study, we combined isothermal recombinase polymerase amplification (RPA) with a lateral flow (LF) strip to rapidly and reliably detect L. monocytogenes. In the presence of biotin- and digoxin-modified primers, RPA produced numerous digoxin- and biotin-attached duplex DNA products. These products were detected on an LF strip via dual immunoreactions (digoxin on the duplex DNA reacted with the anti-digoxin antibody on the gold nanoparticle (Au-NP) and the biotin on the duplex DNA captured by the streptavidin on the LF test zone). The accumulation of Au-NPs produced characteristic bands, enabling the visual detection of L. monocytogenes without instrumentation. This assay could be used to detect L. monocytogenes within 15 min, including DNA amplification with RPA for 10 min at 39 °C and visualization of the amplicons by LF strips for 5 min. Experiments confirmed a detection limit as low as 300 fg of DNA and 1.5 × 10 1 CFU in pure cultures. Furthermore, RPA-LF exhibited no cross-reactions with pathogens. Evaluation of the method with food samples indicated that the detection limit was substantially improved to 1.5 × 10° CFU for the original bacterial content in 25 g/mL samples after enrichment for 6 hr. RPA-LF can be used as a sensitive and rapid detection technique for L. monocytogenes. Recombinase polymerase amplification (RPA) can amplify target DNA at 37 to 42 °C without a thermal cycler. Lateral flow (LF) strips are portable, cheap and easy to operate. RPA combined with LF strips to detect Listeria monocytogenes can be widely used in remote areas. © 2018 Institute of Food Technologists®.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Vladimir Larionov, Ph D

A special interest in the organization of human centromeric DNA was stimulated a few years ago when two independent groups succeeded in reconstituting a functional human centromere, using constructs carrying centromere-specific alphoid DNA arrays. This work demonstrated the importance of DNA components in mammalian centromeres and opened a way for studying the structural requirements for de novo kinetochore formation and for construction of human artificial chromosomes (HACs) with therapeutic potential. To elucidate the structural requirements for formation of HACs with a functional kinetochore, we developed a new method for cloning of large DNA fragments for human centromeric regions that canmore » be used as a substrate for HAC formation. This method exploits in vivo recombination in yeast (TAR cloning). In addition, a new strategy for the construction of alphoid DNA arrays was developed in our lab. The strategy involves the construction of uniform or hybrid synthetic alphoid DNA arrays by the RCA-TAR technique. This technique comprises two steps: rolling circle amplification of an alphoid DNA dimer and subsequent assembling of the amplified fragments by in vivo homologous recombination in yeast (Figure 1). Using this system, we constructed a set of different synthetic alphoid DNA arrays with a predetermined sequence varying in size from 30 to 140 kb and demonstrated that some of the arrays are competent in HAC formation. Because any nucleotide can be changed in a dimer before its amplification, this new technique is optimal for identifying the structural requirements for de novo kinetochore formation in HACs. Moreover, the technique makes possible to introduce into alphoid DNA arrays recognition sites for DNA-binding proteins. We have made the following progress on the studying of human centromeric regions using transformation-associated recombination cloning technology: i) minimal size of alphoid DNA array required for de novo kinetochore formation was estimated; ii) critical role of CENP-B binding site in do novo kinetochore formation was demonstrated; iii) role of gamma-satellite DNA in functional centromere was elucidated; iv) new generation of HAC with a conditional centromere was constructed for the study of epigenetic control of kinetochore function and for gene expression studies. These studies de novo kinetochore formation may thus provide both a fundamental knowledge and new points of intervention for therapy.« less

A loop-mediated isothermal amplification assay for rapid and sensitive detection of bovine papular stomatitis virus.

PubMed

Kurosaki, Yohei; Okada, Sayaka; Nakamae, Sayuri; Yasuda, Jiro

2016-12-01

Bovine papular stomatitis virus (BPSV) causes pustular cutaneous disease in cattle worldwide. This paper describes the development of a specific loop-mediated isothermal amplification (LAMP) assay to detect BPSV which did not cross-react with other parapoxviruses. To assess analytical sensitivity of this LAMP assay, DNA was extracted from serially diluted BPSV from which the infectious titer was determined by a novel assay based on calf kidney epithelial cells. The LAMP assay had equivalent analytical sensitivity to quantitative PCR, and could detect as few as 86 copies of viral DNA per reaction. These results suggest that the assay is a specific and sensitive technique to rapidly diagnose bovine papular stomatitis in domestic animals. Copyright © 2016 Elsevier B.V. All rights reserved.

Photocleavable DNA Barcoding Antibodies for Multiplexed Protein Analysis in Single Cells.

PubMed

Ullal, Adeeti V; Weissleder, Ralph

2015-01-01

We describe a DNA-barcoded antibody sensing technique for single cell protein analysis in which the barcodes are photocleaved and digitally detected without amplification steps (Ullal et al., Sci Transl Med 6:219, 2014). After photocleaving the unique ~70 mer DNA barcodes we use a fluorescent hybridization technology for detection, similar to what is commonly done for nucleic acid readouts. This protocol offers a simple method for multiplexed protein detection using 100+ antibodies and can be performed on clinical samples as well as single cells.

Graphene Nanoprobes for Real-Time Monitoring of Isothermal Nucleic Acid Amplification.

PubMed

Li, Fan; Liu, Xiaoguo; Zhao, Bin; Yan, Juan; Li, Qian; Aldalbahi, Ali; Shi, Jiye; Song, Shiping; Fan, Chunhai; Wang, Lihua

2017-05-10

Isothermal amplification is an efficient way to amplify DNA with high accuracy; however, the real-time monitoring for quantification analysis mostly relied on expensive and precisely designed probes. In the present study, a graphene oxide (GO)-based nanoprobe was used to real-time monitor the isothermal amplification process. The interaction between GO and different DNA structures was systematically investigated, including single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), DNA 3-helix, and long rolling circle amplification (RCA) and hybridization chain reaction (HCR) products, which existed in one-, two-, and three-dimensional structures. It was found that the high rigid structures exhibited much lower affinity with GO than soft ssDNA, and generally the rigidity was dependent on the length of targets and the hybridization position with probe DNA. On the basis of these results, we successfully monitored HCR amplification process, RCA process, and the enzyme restriction of RCA products with GO nanoprobe; other applications including the detection of the assembly/disassembly of DNA 3-helix structures were also performed. Compared to the widely used end-point detection methods, the GO-based sensing platform is simple, sensitive, cost-effective, and especially in a real-time monitoring mode. We believe such studies can provide comprehensive understandings and evocation on design of GO-based biosensors for broad application in various fields.

Highly sensitive detection of DNA methylation levels by using a quantum dot-based FRET method

NASA Astrophysics Data System (ADS)

Ma, Yunfei; Zhang, Honglian; Liu, Fangming; Wu, Zhenhua; Lu, Shaohua; Jin, Qinghui; Zhao, Jianlong; Zhong, Xinhua; Mao, Hongju

2015-10-01

DNA methylation is the most frequently studied epigenetic modification that is strongly involved in genomic stability and cellular plasticity. Aberrant changes in DNA methylation status are ubiquitous in human cancer and the detection of these changes can be informative for cancer diagnosis. Herein, we reported a facile quantum dot-based (QD-based) fluorescence resonance energy transfer (FRET) technique for the detection of DNA methylation. The method relies on methylation-sensitive restriction enzymes for the differential digestion of genomic DNA based on its methylation status. Digested DNA is then subjected to PCR amplification for the incorporation of Alexa Fluor-647 (A647) fluorophores. DNA methylation levels can be detected qualitatively through gel analysis and quantitatively by the signal amplification from QDs to A647 during FRET. Furthermore, the methylation levels of three tumor suppressor genes, PCDHGB6, HOXA9 and RASSF1A, in 20 lung adenocarcinoma and 20 corresponding adjacent nontumorous tissue (NT) samples were measured to verify the feasibility of the QD-based FRET method and a high sensitivity for cancer detection (up to 90%) was achieved. Our QD-based FRET method is a convenient, continuous and high-throughput method, and is expected to be an alternative for detecting DNA methylation as a biomarker for certain human cancers.DNA methylation is the most frequently studied epigenetic modification that is strongly involved in genomic stability and cellular plasticity. Aberrant changes in DNA methylation status are ubiquitous in human cancer and the detection of these changes can be informative for cancer diagnosis. Herein, we reported a facile quantum dot-based (QD-based) fluorescence resonance energy transfer (FRET) technique for the detection of DNA methylation. The method relies on methylation-sensitive restriction enzymes for the differential digestion of genomic DNA based on its methylation status. Digested DNA is then subjected to PCR amplification for the incorporation of Alexa Fluor-647 (A647) fluorophores. DNA methylation levels can be detected qualitatively through gel analysis and quantitatively by the signal amplification from QDs to A647 during FRET. Furthermore, the methylation levels of three tumor suppressor genes, PCDHGB6, HOXA9 and RASSF1A, in 20 lung adenocarcinoma and 20 corresponding adjacent nontumorous tissue (NT) samples were measured to verify the feasibility of the QD-based FRET method and a high sensitivity for cancer detection (up to 90%) was achieved. Our QD-based FRET method is a convenient, continuous and high-throughput method, and is expected to be an alternative for detecting DNA methylation as a biomarker for certain human cancers. Electronic supplementary information (ESI) available: Synthesis of CdSe/CdS/ZnS core/shell/shell QDs. Sequences of primers used for amplifying the promoter regions in bisulfate-modified DNA. Comparison of detected methylation levels in different gene promoters using the QD-based FRET method versus bisulfite pyrosequencing. Methylation levels of the RASSF1A gene in one pair of NT and cancer samples as indicated by pyrosequencing. Theoretical calculation of the Förster distance R0. See DOI: 10.1039/c5nr04956c

The principle and application of new PCR Technologies

NASA Astrophysics Data System (ADS)

Yu, Miao; Cao, Yue; Ji, Yubin

2017-12-01

Polymerase chain reaction (PCR) is essentially a selective DNA amplification technique commonlyapplied for genetic testing and molecular diagnosis because of its high specificity and sensitivity.PCR technologies as the key of molecular biology, has realized that the qualitative detection of absolute quantitative has been changed. It has produced a variety of new PCR technologies, such as extreme PCR, photonic PCR, o-amplification at lower denaturation temperature PCR, nanoparticle PCR and so on. In this paper, the principle and application of PCR technologies are reviewed, and its development is prospected too.

Methylation Sensitive Amplification Polymorphism Sequencing (MSAP-Seq)-A Method for High-Throughput Analysis of Differentially Methylated CCGG Sites in Plants with Large Genomes.

PubMed

Chwialkowska, Karolina; Korotko, Urszula; Kosinska, Joanna; Szarejko, Iwona; Kwasniewski, Miroslaw

2017-01-01

Epigenetic mechanisms, including histone modifications and DNA methylation, mutually regulate chromatin structure, maintain genome integrity, and affect gene expression and transposon mobility. Variations in DNA methylation within plant populations, as well as methylation in response to internal and external factors, are of increasing interest, especially in the crop research field. Methylation Sensitive Amplification Polymorphism (MSAP) is one of the most commonly used methods for assessing DNA methylation changes in plants. This method involves gel-based visualization of PCR fragments from selectively amplified DNA that are cleaved using methylation-sensitive restriction enzymes. In this study, we developed and validated a new method based on the conventional MSAP approach called Methylation Sensitive Amplification Polymorphism Sequencing (MSAP-Seq). We improved the MSAP-based approach by replacing the conventional separation of amplicons on polyacrylamide gels with direct, high-throughput sequencing using Next Generation Sequencing (NGS) and automated data analysis. MSAP-Seq allows for global sequence-based identification of changes in DNA methylation. This technique was validated in Hordeum vulgare . However, MSAP-Seq can be straightforwardly implemented in different plant species, including crops with large, complex and highly repetitive genomes. The incorporation of high-throughput sequencing into MSAP-Seq enables parallel and direct analysis of DNA methylation in hundreds of thousands of sites across the genome. MSAP-Seq provides direct genomic localization of changes and enables quantitative evaluation. We have shown that the MSAP-Seq method specifically targets gene-containing regions and that a single analysis can cover three-quarters of all genes in large genomes. Moreover, MSAP-Seq's simplicity, cost effectiveness, and high-multiplexing capability make this method highly affordable. Therefore, MSAP-Seq can be used for DNA methylation analysis in crop plants with large and complex genomes.

Methylation Sensitive Amplification Polymorphism Sequencing (MSAP-Seq)—A Method for High-Throughput Analysis of Differentially Methylated CCGG Sites in Plants with Large Genomes

PubMed Central

Chwialkowska, Karolina; Korotko, Urszula; Kosinska, Joanna; Szarejko, Iwona; Kwasniewski, Miroslaw

2017-01-01

Epigenetic mechanisms, including histone modifications and DNA methylation, mutually regulate chromatin structure, maintain genome integrity, and affect gene expression and transposon mobility. Variations in DNA methylation within plant populations, as well as methylation in response to internal and external factors, are of increasing interest, especially in the crop research field. Methylation Sensitive Amplification Polymorphism (MSAP) is one of the most commonly used methods for assessing DNA methylation changes in plants. This method involves gel-based visualization of PCR fragments from selectively amplified DNA that are cleaved using methylation-sensitive restriction enzymes. In this study, we developed and validated a new method based on the conventional MSAP approach called Methylation Sensitive Amplification Polymorphism Sequencing (MSAP-Seq). We improved the MSAP-based approach by replacing the conventional separation of amplicons on polyacrylamide gels with direct, high-throughput sequencing using Next Generation Sequencing (NGS) and automated data analysis. MSAP-Seq allows for global sequence-based identification of changes in DNA methylation. This technique was validated in Hordeum vulgare. However, MSAP-Seq can be straightforwardly implemented in different plant species, including crops with large, complex and highly repetitive genomes. The incorporation of high-throughput sequencing into MSAP-Seq enables parallel and direct analysis of DNA methylation in hundreds of thousands of sites across the genome. MSAP-Seq provides direct genomic localization of changes and enables quantitative evaluation. We have shown that the MSAP-Seq method specifically targets gene-containing regions and that a single analysis can cover three-quarters of all genes in large genomes. Moreover, MSAP-Seq's simplicity, cost effectiveness, and high-multiplexing capability make this method highly affordable. Therefore, MSAP-Seq can be used for DNA methylation analysis in crop plants with large and complex genomes. PMID:29250096

Comparison of STR profiling from low template DNA extracts with and without the consensus profiling method

PubMed Central

2012-01-01

Background The consensus profiling method was introduced to overcome the exaggerated stochastic effects associated with low copy number DNA typing. However, little empirical evidence has been provided which shows that a consensus profile, derived from dividing a sample into separate aliquots and including only alleles seen at least twice, gives the most informative profile, compared to a profile obtained by amplifying the entire low template DNA extract in one reaction. Therefore, this study aimed to investigate the quality of consensus profiles compared to profiles obtained using the whole low template extract for amplification. Methods A total of 100 pg and 25 pg DNA samples were amplified with the PowerPlex® ESI 16 Kits using 30 or 34 PCR cycles. A total of 100 pg and 25 pg DNA samples were then divided into three aliquots for a 34-cycle PCR and a consensus profile derived that included alleles that appeared in at least two of the replicates. Profiles from the non-split samples were compared to the consensus profiles focusing on peak heights, allele drop out, locus drop out and allele drop in. Results Performing DNA profiling on non-split extracts produced profiles with a higher percentage of correct loci compared to the consensus profiling technique. Consensus profiling did eliminate any spurious alleles from the final profile. However, there was a notable increase in allele and locus drop out when a LTDNA sample was divided prior to amplification. Conclusions The loss of information that occurs when a sample is split for amplification indicates that consensus profiling may not be producing the most informative DNA profile for samples where the template amount is limited. PMID:22748106

Improved multiple displacement amplification (iMDA) and ultraclean reagents.

PubMed

Motley, S Timothy; Picuri, John M; Crowder, Chris D; Minich, Jeremiah J; Hofstadler, Steven A; Eshoo, Mark W

2014-06-06

Next-generation sequencing sample preparation requires nanogram to microgram quantities of DNA; however, many relevant samples are comprised of only a few cells. Genomic analysis of these samples requires a whole genome amplification method that is unbiased and free of exogenous DNA contamination. To address these challenges we have developed protocols for the production of DNA-free consumables including reagents and have improved upon multiple displacement amplification (iMDA). A specialized ethylene oxide treatment was developed that renders free DNA and DNA present within Gram positive bacterial cells undetectable by qPCR. To reduce DNA contamination in amplification reagents, a combination of ion exchange chromatography, filtration, and lot testing protocols were developed. Our multiple displacement amplification protocol employs a second strand-displacing DNA polymerase, improved buffers, improved reaction conditions and DNA free reagents. The iMDA protocol, when used in combination with DNA-free laboratory consumables and reagents, significantly improved efficiency and accuracy of amplification and sequencing of specimens with moderate to low levels of DNA. The sensitivity and specificity of sequencing of amplified DNA prepared using iMDA was compared to that of DNA obtained with two commercial whole genome amplification kits using 10 fg (~1-2 bacterial cells worth) of bacterial genomic DNA as a template. Analysis showed >99% of the iMDA reads mapped to the template organism whereas only 0.02% of the reads from the commercial kits mapped to the template. To assess the ability of iMDA to achieve balanced genomic coverage, a non-stochastic amount of bacterial genomic DNA (1 pg) was amplified and sequenced, and data obtained were compared to sequencing data obtained directly from genomic DNA. The iMDA DNA and genomic DNA sequencing had comparable coverage 99.98% of the reference genome at ≥1X coverage and 99.9% at ≥5X coverage while maintaining both balance and representation of the genome. The iMDA protocol in combination with DNA-free laboratory consumables, significantly improved the ability to sequence specimens with low levels of DNA. iMDA has broad utility in metagenomics, diagnostics, ancient DNA analysis, pre-implantation embryo screening, single-cell genomics, whole genome sequencing of unculturable organisms, and forensic applications for both human and microbial targets.

A surface-confined DNA assembly amplification strategy on DNA nanostructural scaffold for electrochemiluminescence biosensing.

PubMed

Feng, Qiu-Mei; Guo, Yue-Hua; Xu, Jing-Juan; Chen, Hong-Yuan

2018-02-15

A critical challenge in surface-based DNA assembly amplification is the reduced accessibility of DNA strands arranged on a heterogeneous surface compared to that in homogeneous solution. Here, a novel in situ surface-confined DNA assembly amplification electrochemiluminescence (ECL) biosensor based on DNA nanostructural scaffold was presented. In this design, a stem-loop structural DNA segment (Hairpin 1) was constructed on the vertex of DNA nanostructural scaffold as recognition probe. In the present of target DNA, the hairpin structure changed to rod-like through complementary hybridization with target DNA, resulting in the formation of Hairpin 1:target DNA. When the obtained Hairpin 1:target DNA met Hairpin 2 labeled with glucose oxidase (GOD), the DNA cyclic amplification was activated, releasing target DNA into homogeneous solution for the next recycling. Thus, the ECL signal of Ru(bpy) 3 2+ -TPrA system was quenched by H 2 O 2 , the product of GOD catalyzing glucose. As a result, this proposed method achieved a linear range response from 50 aM to 10 pM with lower detection limit of 20 aM. Copyright © 2017 Elsevier B.V. All rights reserved.

Recombinase polymerase amplification applied to plant virus detection and potential implications.

PubMed

Babu, Binoy; Ochoa-Corona, Francisco M; Paret, Mathews L

2018-04-01

Several isothermal techniques for the detection of plant pathogens have been developed with the advent of molecular techniques. Among them, Recombinase Polymerase Amplification (RPA) is becoming an important technique for the rapid, sensitive and cost-effective detection of plant viruses. The RPA technology has the advantage to be implemented in field-based scenarios because the method requires a minimal sample preparation, and is performed at constant low temperature (37-42 °C). The RPA technique is rapidly becoming a promising tool for use in rapid detection and further diagnostics in plant clinics and monitoring quarantine services. This paper presents a review of studies conducted using RPA for detection/diagnosis of plant viruses with either DNA genomes (Banana bunchy top virus, Bean golden yellow mosaic virus, Tomato mottle virus, Tomato yellow leaf curl virus) or RNA genomes (Little Cherry virus 2, Plum pox virus and Rose rosette virus). Copyright © 2018 Elsevier Inc. All rights reserved.

An isothermal based recombinase polymerase amplification assay for rapid, sensitive and robust indexing of citrus yellow mosaic virus.

PubMed

Kumar, P V; Sharma, S K; Rishi, N; Ghosh, D K; Baranwal, V K

Management of viral diseases relies on definite and sensitive detection methods. Citrus yellow mosaic virus (CYMV), a double stranded DNA virus of the genus Badnavirus, causes yellow mosaic disease in citrus plants. CYMV is transmitted through budwood and requires a robust and simplified indexing protocol for budwood certification programme. The present study reports development and standardization of an isothermal based recombinase polymerase amplification (RPA) assay for a sensitive, rapid, easy, and cost-effective method for detection and diagnosis of CYMV. Two different oligonucleotide primer sets were designed from ORF III (coding for polyprotein) and ORF II (coding for virion associated protein) regions of CYMV to perform amplification assays. Comparative evaluation of RPA, PCR and immuno-capture recombinase polymerase amplification (IC-RPA) based assays were done using purified DNA and plant crude sap. CYMV infection was efficiently detected from the crude sap in RPA and IC-RPA assays. The primer set used in RPA was specific and did not show any cross-amplification with banana streak MY virus (BSMYV), another Badnavirus species. The results from the present study indicated that RPA assay can be used easily in routine indexing of citrus planting material. To the best of our knowledge, this is the first report on development of a rapid and simplified isothermal detection assay for CYMV and can be utilized as an effective technique in quarantine and budwood certification process.

Monodisperse Picoliter Droplets for Low-Bias and Contamination-Free Reactions in Single-Cell Whole Genome Amplification

PubMed Central

Maruyama, Toru; Yamagishi, Keisuke; Mori, Tetsushi; Takeyama, Haruko

2015-01-01

Whole genome amplification (WGA) is essential for obtaining genome sequences from single bacterial cells because the quantity of template DNA contained in a single cell is very low. Multiple displacement amplification (MDA), using Phi29 DNA polymerase and random primers, is the most widely used method for single-cell WGA. However, single-cell MDA usually results in uneven genome coverage because of amplification bias, background amplification of contaminating DNA, and formation of chimeras by linking of non-contiguous chromosomal regions. Here, we present a novel MDA method, termed droplet MDA, that minimizes amplification bias and amplification of contaminants by using picoliter-sized droplets for compartmentalized WGA reactions. Extracted DNA fragments from a lysed cell in MDA mixture are divided into 105 droplets (67 pL) within minutes via flow through simple microfluidic channels. Compartmentalized genome fragments can be individually amplified in these droplets without the risk of encounter with reagent-borne or environmental contaminants. Following quality assessment of WGA products from single Escherichia coli cells, we showed that droplet MDA minimized unexpected amplification and improved the percentage of genome recovery from 59% to 89%. Our results demonstrate that microfluidic-generated droplets show potential as an efficient tool for effective amplification of low-input DNA for single-cell genomics and greatly reduce the cost and labor investment required for determination of nearly complete genome sequences of uncultured bacteria from environmental samples. PMID:26389587

Evaluation of two molecular techniques for rapid detection of the main dermatophytic agents of tinea capitis.

PubMed

Deng, S; Zhou, Z; de Hoog, G S; Wang, X; Abliz, P; Sun, J; Najafzadeh, M J; Pan, W; Lei, W; Zhu, S; Hasimu, H; Zhang, P; Guo, Y; Deng, D; Liao, W

2015-12-01

Tinea capitis is very common in Western China, with the most widespread aetiological agent being Trichophyton violaceum, while Microsporum canis is prevalent in the remainder of China. Conventional diagnostics and internal transcribed spacer (ITS) sequencing analyses have proven relatively limited due to the close phylogenetic relationship of anthropophilic dermatophytes. Therefore, alternative molecular tools with sufficient specificity, reproducibility and sensitivity are necessary. To evaluate two molecular techniques [multiplex ligation-dependent probe amplification (MLPA) and rolling circle amplification (RCA)] for rapid detection of the aetiological agents of tinea capitis, T. violaceum and M. canis. Probes of RCA and MLPA were designed with target sequences in the rDNA ITS gene region. Strains tested consist of 31 T. violaceum, 22 M. canis and 24 reference strains of species that are taxonomically close to the target species. The specificity and reproducibility of RCA and MLPA in detection of T. violaceum and M. canis were both 100% in both species. Sensitivity testing showed that RCA was positive at concentrations down to 1·68 × 10(6) copies of DNA in the TvioRCA probe, and 2·7 × 10(8) copies of DNA in McRCA. MLPA yielded positive results at concentrations of DNA down to 1·68 × 10(1) copies in the TvioMLPA probe and 2·7 × 10(2) in McMLPA. The two techniques were sufficiently specific and sensitive for discriminating the target DNA of T. violaceum and M. canis from that of closely related dermatophytes. RCA and MLPA are advantageous in their reliability and ease of operation compared with standard polymerase chain reaction and conventional methods. © 2015 British Association of Dermatologists.

Formation of template-switching artifacts by linear amplification.

PubMed

Chakravarti, Dhrubajyoti; Mailander, Paula C

2008-07-01

Linear amplification is a method of synthesizing single-stranded DNA from either a single-stranded DNA or one strand of a double-stranded DNA. In this protocol, molecules of a single primer DNA are extended by multiple rounds of DNA synthesis at high temperature using thermostable DNA polymerases. Although linear amplification generates the intended full-length single-stranded product, it is more efficient over single-stranded templates than double-stranded templates. We analyzed linear amplification over single- or double-stranded mouse H-ras DNA (exon 1-2 region). The single-stranded H-ras template yielded only the intended product. However, when the double-stranded template was used, additional artifact products were observed. Increasing the concentration of the double-stranded template produced relatively higher amounts of these artifact products. One of the artifact DNA bands could be mapped and analyzed by sequencing. It contained three template-switching products. These DNAs were formed by incomplete DNA strand extension over the template strand, followed by switching to the complementary strand at a specific Ade nucleotide within a putative hairpin sequence, from which DNA synthesis continued over the complementary strand.

[Multilocus genotyping of polymorphous STR-loci of chromosomal DNA in individual cells: technical difficulties].

PubMed

Ivanov, P L; Leonov, S N; Zemskova, E Iu; Kobylianskiĭ, A G; Dziubenko, E V

2013-01-01

This study was designed to estimate the effectiveness of special technical procedures for the enhancement of sensitivity of multiplex analysis of DNA, such as the use of low-plexity PCR systems and the whole genome preamplification technology, and the possibility of their application for the purpose of forensic medical genotyping of polymorphous STR-loci of chromosomal DNA in individual cells. The authors refused to use the imitation model (equivalent DNA dilutions) for the sake of obtaining the maximally informative data and chose to work with real preparations of solitary buccal epithelial cells isolated by the laser microdissection technique. It was shown that neither the use of the low-plexity multilocus PCR systems nor the whole genome pre-amplification technology makes possible reliable genotyping of STR-loci of chromosomal DNA in individual cells. The proposed techniques allow for DNA genotyping in preparations consisting of 10 diploid cells whereas the methods for reliable genotyping of STR-loci of chromosomal DNA in individual cells remains to be developed.

Population diversity of ammonium oxidizers investigated by specific PCR amplification

USGS Publications Warehouse

Ward, B.B.; Voytek, M.A.; Witzel, K.-P.

1997-01-01

The species composition of ammonia-oxidizing bacteria in aquatic environments was investigated using PCR primers for 16S rRNA genes to amplify specific subsets of the total ammonia-oxidizer population. The specificity of the amplification reactions was determined using total genomic DNA from known nitrifying strains and non-nitrifying strains identified as having similar rDNA sequences. Specificity of amplification was determined both for direct amplification, using the nitrifier specific primers, and with nested amplification, in which the nitrifier primers were used to reamplify a fragment obtained from direct amplification with Eubacterial universal primers. The present level of specificity allows the distinction between Nitrosomonas europaea, Nitrosomonas sp. (marine) and the other known ammonia-oxidizers in the beta subclass of the Proteobacteria. Using total DNA extracted from natural samples, we used direct amplification to determine presence/absence of different species groups. Species composition was found to differ among depths in vertical profiles of lake samples and among samples and enrichments from various other aquatic environments. Nested PCR yielded several more positive reactions, which implies that nitrifier DNA was present in most samples, but often at very low levels.

Genetic diversity analysis of Jatropha curcas L. (Euphorbiaceae) based on methylation-sensitive amplification polymorphism.

PubMed

Kanchanaketu, T; Sangduen, N; Toojinda, T; Hongtrakul, V

2012-04-13

Genetic analysis of 56 samples of Jatropha curcas L. collected from Thailand and other countries was performed using the methylation-sensitive amplification polymorphism (MSAP) technique. Nine primer combinations were used to generate MSAP fingerprints. When the data were interpreted as amplified fragment length polymorphism (AFLP) markers, 471 markers were scored. All 56 samples were classified into three major groups: γ-irradiated, non-toxic and toxic accessions. Genetic similarity among the samples was extremely high, ranging from 0.95 to 1.00, which indicated very low genetic diversity in this species. The MSAP fingerprint was further analyzed for DNA methylation polymorphisms. The results revealed differences in the DNA methylation level among the samples. However, the samples collected from saline areas and some species hybrids showed specific DNA methylation patterns. AFLP data were used, together with methylation-sensitive AFLP (MS-AFLP) data, to construct a phylogenetic tree, resulting in higher efficiency to distinguish the samples. This combined analysis separated samples previously grouped in the AFLP analysis. This analysis also distinguished some hybrids. Principal component analysis was also performed; the results confirmed the separation in the phylogenetic tree. Some polymorphic bands, involving both nucleotide and DNA methylation polymorphism, that differed between toxic and non-toxic samples were identified, cloned and sequenced. BLAST analysis of these fragments revealed differences in DNA methylation in some known genes and nucleotide polymorphism in chloroplast DNA. We conclude that MSAP is a powerful technique for the study of genetic diversity for organisms that have a narrow genetic base.

An evaluation of direct PCR amplification

PubMed Central

Hall, Daniel E.; Roy, Reena

2014-01-01

Aim To generate complete DNA profiles from blood and saliva samples deposited on FTA® and non-FTA® paper substrates following a direct amplification protocol. Methods Saliva samples from living donors and blood samples from deceased individuals were deposited on ten different FTA® and non-FTA® substrates. These ten paper substrates containing body fluids were kept at room temperature for varying lengths of time ranging from one day to approximately one year. For all assays in this research, 1.2 mm punches were collected from each substrate containing one type of body fluid and amplified with reagents provided in the nine commercial polymerase chain reaction (PCR) amplification kits. The substrates were not subjected to purification reagent or extraction buffer prior to amplification. Results Success rates were calculated for all nine amplification kits and all ten substrates based on their ability to yield complete DNA profiles following a direct amplification protocol. Six out of the nine amplification kits, and four out of the ten paper substrates had the highest success rates overall. Conclusion The data show that it is possible to generate complete DNA profiles following a direct amplification protocol using both standard (non-direct) and direct PCR amplification kits. The generation of complete DNA profiles appears to depend more on the success of the amplification kit rather than the than the FTA®- or non-FTA®-based substrates. PMID:25559837

Droplet Microfluidic Device Fabrication and Use for Isothermal Amplification and Detection of MicroRNA.

PubMed

Giuffrida, Maria Chiara; D'Agata, Roberta; Spoto, Giuseppe

2017-01-01

Droplet microfluidics combined with the isothermal circular strand displacement polymerization (ICSDP) represents a powerful new technique to detect both single-stranded DNA and microRNA sequences. The method here described helps in overcoming some drawbacks of the lately introduced droplet polymerase chain reaction (PCR) amplification when implemented in microfluidic devices. The method also allows the detection of nanoliter droplets of nucleic acids sequences solutions, with a particular attention to microRNA sequences that are detected at the picomolar level. The integration of the ICSDP amplification protocol in droplet microfluidic devices reduces the time of analysis and the amount of sample required. In addition, there is also the possibility to design parallel analyses to be integrated in portable devices.

Profiling DNA methylome landscapes of mammalian cells with single-cell reduced-representation bisulfite sequencing.

PubMed

Guo, Hongshan; Zhu, Ping; Guo, Fan; Li, Xianlong; Wu, Xinglong; Fan, Xiaoying; Wen, Lu; Tang, Fuchou

2015-05-01

The heterogeneity of DNA methylation within a population of cells necessitates DNA methylome profiling at single-cell resolution. Recently, we developed a single-cell reduced-representation bisulfite sequencing (scRRBS) technique in which we modified the original RRBS method by integrating all the experimental steps before PCR amplification into a single-tube reaction. These modifications enable scRRBS to provide digitized methylation information on ∼1 million CpG sites within an individual diploid mouse or human cell at single-base resolution. Compared with the single-cell bisulfite sequencing (scBS) technique, scRRBS covers fewer CpG sites, but it provides better coverage for CpG islands (CGIs), which are likely to be the most informative elements for DNA methylation. The entire procedure takes ∼3 weeks, and it requires strong molecular biology skills.

Unusual isothermal multimerization and amplification by the strand-displacing DNA polymerases with reverse transcription activities.

PubMed

Wang, Guoping; Ding, Xiong; Hu, Jiumei; Wu, Wenshuai; Sun, Jingjing; Mu, Ying

2017-10-24

Existing isothermal nucleic acid amplification (INAA) relying on the strand displacement activity of DNA polymerase usually requires at least two primers. However, in this paper, we report an unusual isothermal multimerization and amplification (UIMA) which only needs one primer and is efficiently initiated by the strand-displacing DNA polymerases with reverse transcription activities. On electrophoresis, the products of UIMA present a cascade-shape band and they are confirmed to be multimeric DNAs with repeated target sequences. In contrast to current methods, UIMA is simple to product multimeric DNA, due to the independent of multiple primers and rolling circle structures. Through assaying the synthesized single-stranded DNA targets, UIMA performs high sensitivity and specificity, as well as the universality. In addition, a plausible mechanism of UIMA is proposed, involving short DNA bending, mismatch extension, and template slippage. UIMA is a good explanation for why nonspecific amplification easily happens in existing INAAs. As the simplest INAA till now, UIMA provides a new insight for deeply understanding INAA and opens a new avenue for thoroughly addressing nonspecific amplification.

Rolling-circle amplification under topological constraints

PubMed Central

Kuhn, Heiko; Demidov, Vadim V.; Frank-Kamenetskii, Maxim D.

2002-01-01

We have performed rolling-circle amplification (RCA) reactions on three DNA templates that differ distinctly in their topology: an unlinked DNA circle, a linked DNA circle within a pseudorotaxane-type structure and a linked DNA circle within a catenane. In the linked templates, the single-stranded circle (dubbed earring probe) is threaded, with the aid of two peptide nucleic acid openers, between the two strands of double-stranded DNA (dsDNA). We have found that the RCA efficiency of amplification was essentially unaffected when the linked templates were employed. By showing that the DNA catenane remains intact after RCA reactions, we prove that certain DNA polymerases can carry out the replicative synthesis under topological constraints allowing detection of several hundred copies of a dsDNA marker without DNA denaturation. Our finding may have practical implications in the area of DNA diagnostics. PMID:11788721

The development of a loop-mediated isothermal amplification assay for rapid and sensitive detection of abalone herpesvirus DNA.

PubMed

Chen, M H; Kuo, S T; Renault, T; Chang, P H

2014-02-01

A loop-mediated isothermal amplification (LAMP) assay was developed for the detection of abalone herpesvirus DNA. Two pairs of primers were designed, based on the sequence of the DNA polymerase gene of abalone herpesvirus. The reaction temperature and time were optimized to 63°C and 60min, respectively. LAMP amplicons were analyzed by 2% agarose gel electrophoresis or by visual inspection of a colour change emitted by fluorescent dye. The method developed was specific for the detection of abalone herpesvirus, without cross-reactions with other tested herpesviruses including ostreid herpesvirus 1 (OsHV-1), European eel herpesvirus, koi herpesvirus (KHV) and an avian herpesvirus. The LAMP assay was 100 folds more sensitive than a conventional PCR and 10 folds less sensitive than a SYBR Green PCR. These results indicate that the developed LAMP assay is a simple, rapid, sensitive, specific and reliable technique for the detection of abalone herpesvirus. Copyright © 2013 Elsevier B.V. All rights reserved.

Miniaturized thermocycler based on thermoelectric heating for diagnosis of sexually transmitted disease by DNA amplification

NASA Astrophysics Data System (ADS)

Lim, Hyunjung; Jo, Ga Eun; Kim, Kyong Soo; Back, Seung Min; Choi, Hyuk

2017-05-01

Sexually transmitted disease (STD) is among the most common infectious diseases; therefore, it is necessary to develop sensitive early diagnostic techniques. As the gold standard, polymerase chain reaction (PCR) has been most widely employed for STD diagnosis; however, PCR requires large and expensive instruments. In this study, miniaturized thermal cycler using Peltier modules was developed for the PCR analysis. In comparison with the conventional PCR instrument, the Peltier-based micro-PCR (P-mPCR) device developed in this study enables one to amplify and successfully distinguish between DNA of different sizes. Furthermore, by using the clinical vaginal sample collected with the vaginal swab and tampon, different kinds of STD bacteria could be detected with high accuracy (˜94.19%) and high sensitivity (˜95.6%). Therefore, the P-mPCR device will be applicable in STD diagnosis as well as the detection of other bacteria/viruses using DNA amplification in regions including those with limited resources.

Vaccinia virus, herpes simplex virus, and carcinogens induce DNA amplification in a human cell line and support replication of a helpervirus dependent parvovirus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schlehofer, J.R.; Ehrbar, M.; zur Hausen, H.

1986-07-15

The SV40-transformed human kidney cell line, NB-E, amplifies integrated as well as episomal SV40 DNA upon treatment with chemical (DMBA) or physical (uv irradiation) carcinogens (initiators) as well as after infection with herpes simplex virus (HSV) type 1 or with vaccinia virus. In addition it is shown that vaccinia virus induces SV40 DNA amplification also in the SV40-transformed Chinese hamster embryo cell line, CO631. These findings demonstrate that human cells similar to Chinese hamster cells amplify integrated DNA sequences after treatment with carcinogens or infection with specific viruses. Furthermore, a poxvirus--vaccinia virus--similar to herpes group viruses induces DNA amplification. Asmore » reported for other systems, the vaccinia virus-induced DNA amplification in NB-E cells is inhibited by coinfection with adeno-associated virus (AAV) type 5. This is in line with previous studies on inhibition of carcinogen- or HSV-induced DNA amplification in CO631 cells. The experiments also demonstrate that vaccinia virus, in addition to herpes and adenoviruses acts as a helper virus for replication and structural antigen synthesis of AAV-5 in NB-E cells.« less

Expanded Genetic Codes in Next Generation Sequencing Enable Decontamination and Mitochondrial Enrichment

PubMed Central

McKernan, Kevin J.; Spangler, Jessica; Zhang, Lei; Tadigotla, Vasisht; McLaughlin, Stephen; Warner, Jason; Zare, Amir; Boles, Richard G.

2014-01-01

We have developed a PCR method, coined Déjà vu PCR, that utilizes six nucleotides in PCR with two methyl specific restriction enzymes that respectively digest these additional nucleotides. Use of this enzyme-and-nucleotide combination enables what we term a “DNA diode”, where DNA can advance in a laboratory in only one direction and cannot feedback into upstream assays. Here we describe aspects of this method that enable consecutive amplification with the introduction of a 5th and 6th base while simultaneously providing methylation dependent mitochondrial DNA enrichment. These additional nucleotides enable a novel DNA decontamination technique that generates ephemeral and easy to decontaminate DNA. PMID:24788618

Novel selection methods for DNA-encoded chemical libraries

PubMed Central

Chan, Alix I.; McGregor, Lynn M.; Liu, David R.

2015-01-01

Driven by the need for new compounds to serve as biological probes and leads for therapeutic development and the growing accessibility of DNA technologies including high-throughput sequencing, many academic and industrial groups have begun to use DNA-encoded chemical libraries as a source of bioactive small molecules. In this review, we describe the technologies that have enabled the selection of compounds with desired activities from these libraries. These methods exploit the sensitivity of in vitro selection coupled with DNA amplification to overcome some of the limitations and costs associated with conventional screening methods. In addition, we highlight newer techniques with the potential to be applied to the high-throughput evaluation of DNA-encoded chemical libraries. PMID:25723146

Study of DNA extraction methods for use in loop-mediated isothermal amplification detection of single resting cysts in the toxic dinoflagellates Alexandrium tamarense and A. catenella.

PubMed

Nagai, Satoshi; Yamamoto, Keigo; Hata, Naotugu; Itakura, Shigeru

2012-09-01

In a previous study, we experienced instable amplification and a low amplification success in loop-mediated isothermal amplification (LAMP) reactions from naturally occurring vegetative cells or resting cysts of the toxic dinoflagellates Alexandrium tamarense and Alexandrium catenella. In this study, we examined 4 methods for extracting DNA from single resting cysts of A. tamarense and A. catenella to obtain more stable and better amplification success and to facilitate unambiguous detection using the LAMP method. Apart from comparing the 4 different DNA extraction methods, namely, (1) boiling in Tris-EDTA (TE) buffer, (2) heating at 65 °C in hexadecyltrimethylammonium bromide buffer, (3) boiling in 0.5% Chelex buffer, and (4) boiling in 5% Chelex buffer, we also examined the need for homogenization to crush the resting cysts before DNA extraction in each method. Homogenization of resting cysts was found to be essential for DNA extraction in all 4 methods. The detection time was significantly shorter in 5% Chelex buffer than in the other buffers and the amplification success was 100% (65/65), indicating the importance of DNA extraction and the effectiveness of 5% Chelex buffer in the Alexandrium LAMP. Copyright © 2012 Elsevier B.V. All rights reserved.

DNA sequence responsible for the amplification of adjacent genes.

PubMed

Pasion, S G; Hartigan, J A; Kumar, V; Biswas, D K

1987-10-01

A 10.3-kb DNA fragment in the 5'-flanking region of the rat prolactin (rPRL) gene was isolated from F1BGH(1)2C1, a strain of rat pituitary tumor cells (GH cells) that produces prolactin in response to 5-bromodeoxyuridine (BrdU). Following transfection and integration into genomic DNA of recipient mouse L cells, this DNA induced amplification of the adjacent thymidine kinase gene from Herpes simplex virus type 1 (HSV1TK). We confirmed the ability of this "Amplicon" sequence to induce amplification of other linked or unlinked genes in DNA-mediated gene transfer studies. When transferred into the mouse L cells with the 10.3-5'rPRL gene sequence of BrdU-responsive cells, both the human growth hormone and the HSV1TK genes are amplified in response to 5-bromodeoxyuridine. This observation is substantiated by BrdU-induced amplification of the cotransferred bacterial Neo gene. Cotransfection studies reveal that the BrdU-induced amplification capability is associated with a 4-kb DNA sequence in the 5'-flanking region of the rPRL gene of BrdU-responsive cells. These results demonstrate that genes of heterologous origin, linked or unlinked, and selected or unselected, can be coamplified when located within the amplification boundary of the Amplicon sequence.

Detection and Characterization of Viral Species/Subspecies Using Isothermal Recombinase Polymerase Amplification (RPA) Assays.

PubMed

Glais, Laurent; Jacquot, Emmanuel

2015-01-01

Numerous molecular-based detection protocols include an amplification step of the targeted nucleic acids. This step is important to reach the expected sensitive detection of pathogens in diagnostic procedures. Amplifications of nucleic acid sequences are generally performed, in the presence of appropriate primers, using thermocyclers. However, the time requested to amplify molecular targets and the cost of the thermocycler machines could impair the use of these methods in routine diagnostics. Recombinase polymerase amplification (RPA) technique allows rapid (short-term incubation of sample and primers in an enzymatic mixture) and simple (isothermal) amplification of molecular targets. RPA protocol requires only basic molecular steps such as extraction procedures and agarose gel electrophoresis. Thus, RPA can be considered as an interesting alternative to standard molecular-based diagnostic tools. In this paper, the complete procedures to set up an RPA assay, applied to detection of RNA (Potato virus Y, Potyvirus) and DNA (Wheat dwarf virus, Mastrevirus) viruses, are described. The proposed procedure allows developing species- or subspecies-specific detection assay.

Detection of DNA Sequences Refractory to PCR Amplification Using a Biophysical SERRS Assay (Surface Enhanced Resonant Raman Spectroscopy)

PubMed Central

Feuillie, Cécile; Merheb, Maxime M.; Gillet, Benjamin; Montagnac, Gilles; Daniel, Isabelle; Hänni, Catherine

2014-01-01

The analysis of ancient or processed DNA samples is often a great challenge, because traditional Polymerase Chain Reaction – based amplification is impeded by DNA damage. Blocking lesions such as abasic sites are known to block the bypass of DNA polymerases, thus stopping primer elongation. In the present work, we applied the SERRS-hybridization assay, a fully non-enzymatic method, to the detection of DNA refractory to PCR amplification. This method combines specific hybridization with detection by Surface Enhanced Resonant Raman Scattering (SERRS). It allows the detection of a series of double-stranded DNA molecules containing a varying number of abasic sites on both strands, when PCR failed to detect the most degraded sequences. Our SERRS approach can quickly detect DNA molecules without any need for DNA repair. This assay could be applied as a pre-requisite analysis prior to enzymatic reparation or amplification. A whole new set of samples, both forensic and archaeological, could then deliver information that was not yet available due to a high degree of DNA damage. PMID:25502338

Detection of DNA sequences refractory to PCR amplification using a biophysical SERRS assay (Surface Enhanced Resonant Raman Spectroscopy).

PubMed

Feuillie, Cécile; Merheb, Maxime M; Gillet, Benjamin; Montagnac, Gilles; Daniel, Isabelle; Hänni, Catherine

2014-01-01

The analysis of ancient or processed DNA samples is often a great challenge, because traditional Polymerase Chain Reaction - based amplification is impeded by DNA damage. Blocking lesions such as abasic sites are known to block the bypass of DNA polymerases, thus stopping primer elongation. In the present work, we applied the SERRS-hybridization assay, a fully non-enzymatic method, to the detection of DNA refractory to PCR amplification. This method combines specific hybridization with detection by Surface Enhanced Resonant Raman Scattering (SERRS). It allows the detection of a series of double-stranded DNA molecules containing a varying number of abasic sites on both strands, when PCR failed to detect the most degraded sequences. Our SERRS approach can quickly detect DNA molecules without any need for DNA repair. This assay could be applied as a pre-requisite analysis prior to enzymatic reparation or amplification. A whole new set of samples, both forensic and archaeological, could then deliver information that was not yet available due to a high degree of DNA damage.

Novel rolling circle amplification and DNA origami-based DNA belt-involved signal amplification assay for highly sensitive detection of prostate-specific antigen (PSA).

PubMed

Yan, Juan; Hu, Chongya; Wang, Ping; Liu, Rui; Zuo, Xiaolei; Liu, Xunwei; Song, Shiping; Fan, Chunhai; He, Dannong; Sun, Gang

2014-11-26

Prostate-specific antigen (PSA) is one of the most important biomarkers for the early diagnosis and prognosis of prostate cancer. Although many efforts have been made to achieve significant progress for the detection of PSA, challenges including relative low sensitivity, complicated operation, sophisticated instruments, and high cost remain unsolved. Here, we have developed a strategy combining rolling circle amplification (RCA)-based DNA belts and magnetic bead-based enzyme-linked immunosorbent assay (ELISA) for the highly sensitive and specific detection of PSA. At first, a 96-base circular DNA template was designed and prepared for the following RCA. Single stranded DNA (ssDNA) products from RCA were used as scaffold strand for DNA origami, which was hybridized with three staple strands of DNA. The resulting DNA belts were conjugated with multiple enzymes for signal amplification and then employed to magnetic bead based ELISA for PSA detection. Through our strategy, as low as 50 aM of PSA can be detected with excellent specificity.

Improved Method for Direct Detection of Environmental Microorganisms Using an Amplification of 16S rDNA Region

NASA Astrophysics Data System (ADS)

Tsujimura, M.; Akutsu, J.; Zhang, Z.; Sasaki, M.; Tajima, H.; Kawarabayasi, Y.

2004-12-01

The thermostable proteins or enzymes were expected to be capable to be utilized in many areas of industries. Many thermophilic microorganisms, which possess the thermostable proteins or enzymes, were identified from the extreme environment. However, many unidentified and uncultivable microorganisms are still remaining in the environment on the earth. It is generally said that the cultivable microorganisms are less than 1% of entire microorganisms living in the earth, remaining over 99% are still uncultivable. As an approach to the uncultivable microorganisms, the PCR amplification of 16S rDNA region using primer sets designed from the conserved region has been generally utilized for detection and community analysis of microorganism in the environment. However, the facts, that PCR amplification introduces the mutation in the amplified DNA fragment and efficiency of PCR amplification is depend on the sequences of primer sets, indicated that the improving of PCR analysis was necessary for more correct detection of microorganisms. As the result of evaluation for the quality of DNA polymerases, sequences of primers used for amplification and conditions of PCR amplification, the DNA polymerase, the primer set and the conditions for amplification, which did not amplify the DNA fragment from the DNA contaminated within the DNA polymerase itself, were successfully selected. Also the rate of mutation in the DNA fragment amplified was evaluated using this conditions and the genomic DNA from cultivable microbes as a template. The result indicated the rate of mutation introduced by PCR was approximately 0.1% to 0.125%. The improved method using these conditions and error rate calculated was applied for the analysis of microorganisms in the geothermal environment. The result indicated that four kinds of dominant microorganisms, including both of bacteria and archaea, were alive within soil in the hot spring in Tohoku Area. We would like to apply this improved method to detection of microorganisms with important genes from more other environments.

Novel Degenerate PCR Method for Whole-Genome Amplification Applied to Peru Margin (ODP Leg 201) Subsurface Samples

PubMed Central

Martino, Amanda J.; Rhodes, Matthew E.; Biddle, Jennifer F.; Brandt, Leah D.; Tomsho, Lynn P.; House, Christopher H.

2011-01-01

A degenerate polymerase chain reaction (PCR)-based method of whole-genome amplification, designed to work fluidly with 454 sequencing technology, was developed and tested for use on deep marine subsurface DNA samples. While optimized here for use with Roche 454 technology, the general framework presented may be applicable to other next generation sequencing systems as well (e.g., Illumina, Ion Torrent). The method, which we have called random amplification metagenomic PCR (RAMP), involves the use of specific primers from Roche 454 amplicon sequencing, modified by the addition of a degenerate region at the 3′ end. It utilizes a PCR reaction, which resulted in no amplification from blanks, even after 50 cycles of PCR. After efforts to optimize experimental conditions, the method was tested with DNA extracted from cultured E. coli cells, and genome coverage was estimated after sequencing on three different occasions. Coverage did not vary greatly with the different experimental conditions tested, and was around 62% with a sequencing effort equivalent to a theoretical genome coverage of 14.10×. The GC content of the sequenced amplification product was within 2% of the predicted values for this strain of E. coli. The method was also applied to DNA extracted from marine subsurface samples from ODP Leg 201 site 1229 (Peru Margin), and results of a taxonomic analysis revealed microbial communities dominated by Proteobacteria, Chloroflexi, Firmicutes, Euryarchaeota, and Crenarchaeota, among others. These results were similar to those obtained previously for those samples; however, variations in the proportions of taxa identified illustrates well the generally accepted view that community analysis is sensitive to both the amplification technique used and the method of assigning sequences to taxonomic groups. Overall, we find that RAMP represents a valid methodology for amplifying metagenomes from low-biomass samples. PMID:22319519

Development of a Rapid Point-of-Use DNA Test for the Screening of Genuity® Roundup Ready 2 Yield® Soybean in Seed Samples.

PubMed

Chandu, Dilip; Paul, Sudakshina; Parker, Mathew; Dudin, Yelena; King-Sitzes, Jennifer; Perez, Tim; Mittanck, Don W; Shah, Manali; Glenn, Kevin C; Piepenburg, Olaf

2016-01-01

Testing for the presence of genetically modified material in seed samples is of critical importance for all stakeholders in the agricultural industry, including growers, seed manufacturers, and regulatory bodies. While rapid antibody-based testing for the transgenic protein has fulfilled this need in the past, the introduction of new variants of a given transgene demands new diagnostic regimen that allows distinguishing different traits at the nucleic acid level. Although such molecular tests can be performed by PCR in the laboratory, their requirement for expensive equipment and sophisticated operation have prevented its uptake in point-of-use applications. A recently developed isothermal DNA amplification technique, recombinase polymerase amplification (RPA), combines simple sample preparation and amplification work-flow procedures with the use of minimal detection equipment in real time. Here, we report the development of a highly sensitive and specific RPA-based detection system for Genuity Roundup Ready 2 Yield (RR2Y) material in soybean (Glycine max) seed samples and present the results of studies applying the method in both laboratory and field-type settings.

Self-Assembled DNA Tetrahedral Scaffolds for the Construction of Electrochemiluminescence Biosensor with Programmable DNA Cyclic Amplification.

PubMed

Feng, Qiu-Mei; Guo, Yue-Hua; Xu, Jing-Juan; Chen, Hong-Yuan

2017-05-24

A novel DNA tetrahedron-structured electrochemiluminescence (ECL) platform for bioanalysis with programmable DNA cyclic amplification was developed. In this work, glucose oxidase (GOD) was labeled to a DNA sequence (S) as functional conjugation (GOD-S), which could hybridize with other DNA sequences (L and P) to form GOD-S:L:P probe. In the presence of target DNA and a help DNA (A), the programmable DNA cyclic amplification was activated and released GOD-S via toehold-mediated strand displacement. Then, the obtained GOD-S was further immobilized on the DNA tetrahedral scaffolds with a pendant capture DNA and Ru(bpy) 3 2+ -conjugated silica nanoparticles (RuSi NPs) decorated on the electrode surface. Thus, the amount of GOD-S assembled on the electrode surface depended on the concentration of target DNA and GOD could catalyze glucose to generate H 2 O 2 in situ. The ECL signal of Ru(bpy) 3 2+ -TPrA system was quenched by the presence of H 2 O 2 . By integrating the programmable DNA cyclic amplification and in situ generating H 2 O 2 as Ru(bpy) 3 2+ ECL quencher, a sensitive DNA tetrahedron-structured ECL sensing platform was proposed for DNA detection. Under optimized conditions, this biosensor showed a wide linear range from 100 aM to 10 pM with a detection limit of 40 aM, indicating a promising application in DNA analysis. Furthermore, by labeling GOD to different recognition elements, the proposed strategy could be used for the detection of various targets. Thus, this programmable cascade amplification strategy not only retains the high selectivity and good capturing efficiency of tetrahedral-decorated electrode surface but also provides potential applications in the construction of ECL biosensor.

Generation of DNA profiles from fingerprints developed with columnar thin film technique.

PubMed

Plazibat, Stephanie L; Roy, Reena; Swiontek, Stephen E; Lakhtakia, Akhlesh

2015-12-01

Partial-bloody fingerprints and partial fingerprints with saliva are often encountered at crime scenes, potentially enabling the combination of fingerprint and DNA analyses for absolute identification, provided that the development technique for fingerprint analysis does not inhibit DNA analysis. 36 partial-bloody fingerprints and 30 fingerprints wetted with saliva, all deposited on brass, were first developed using the columnar-thin-film (CTF) technique and then subjected to short tandem repeat (STR) DNA analysis. Equal numbers of samples were subjected to the same DNA analysis without development. Tris (8-hydroxyquinolinato) aluminum, or Alq3, was evaporated to deposit CTFs for development of the prints. DNA was extracted from all 132 samples, quantified, and amplified with AmpFlSTR(®) Identifiler Plus Amplification Kit. Additionally, DNA analyses were conducted on four blood smears on un-fingerprinted brass that had been subjected to CTF deposition and four blood smears on un-fingerprinted brass that had not been subjected to CTF deposition. Complete and concordant autosomal STR profiles of the same quality were obtained from both undeveloped and CTF-developed fingerprints, indicating that CTF development of fingerprints preserves DNA and does not inhibit subsequent DNA analysis. Even when there were no fingerprints, CTF deposition did not lead to inhibition of DNA analysis. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

The Microbial Detection Array Combined with Random Phi29-Amplification Used as a Diagnostic Tool for Virus Detection in Clinical Samples

PubMed Central

Erlandsson, Lena; Rosenstierne, Maiken W.; McLoughlin, Kevin; Jaing, Crystal; Fomsgaard, Anders

2011-01-01

A common technique used for sensitive and specific diagnostic virus detection in clinical samples is PCR that can identify one or several viruses in one assay. However, a diagnostic microarray containing probes for all human pathogens could replace hundreds of individual PCR-reactions and remove the need for a clear clinical hypothesis regarding a suspected pathogen. We have established such a diagnostic platform for random amplification and subsequent microarray identification of viral pathogens in clinical samples. We show that Phi29 polymerase-amplification of a diverse set of clinical samples generates enough viral material for successful identification by the Microbial Detection Array, demonstrating the potential of the microarray technique for broad-spectrum pathogen detection. We conclude that this method detects both DNA and RNA virus, present in the same sample, as well as differentiates between different virus subtypes. We propose this assay for diagnostic analysis of viruses in clinical samples. PMID:21853040

Electrochemical sensor for glutathione detection based on mercury ion triggered hybridization chain reaction signal amplification.

PubMed

Wang, Yonghong; Jiang, Lun; Leng, Qinggang; Wu, Yaohui; He, Xiaoxiao; Wang, Kemin

2016-03-15

In this work, we design a new simple and highly sensitive strategy for electrochemical detection of glutathione (GSH) via mercury ion (Hg(2+)) triggered hybridization chain reaction (HCR) signal amplification. It is observed that in the absence of GSH, a specific thymine-Hg(2+)-thymine (T-Hg(2+)-T) coordination can fold into hairpin structures. While in the presence of GSH, it thus can be chelated with Hg(2+), resulting in Hg(2+) released from the T-Hg(2+)-T hairpin complex which then forms into ssDNA structure to further hybridize with the surface-immobilized capture DNA probe on the gold electrode with a sticky tail left. The presence of two hairpin helper probes through HCR leads to the formation of extended dsDNA superstructure on the electrode surface, which therefore causes the intercalation of numerous electroactive species ([Ru(NH3)6](3+)) into the dsDNA grooves, followed by a significantly amplified signal output whose intensity is related to the concentration of the GSH. Taking advantage of merits of enzyme-free amplification power of the HCR, the inherent high sensitivity of the electrochemical technique, and label-free detection which utilizes an electroactive species as a signaling molecule that binds to the anionic phosphate backbone of DNA strands via electrostatic force, not only does the proposed strategy enable sensitive detection of GSH, but show high selectivity against other amino acid, making our method a simple and sensitive addition to the amplified GSH detection. Copyright © 2015 Elsevier B.V. All rights reserved.

Isothermal amplification detection of nucleic acids by a double-nicked beacon.

PubMed

Shi, Chao; Zhou, Meiling; Pan, Mei; Zhong, Guilin; Ma, Cuiping

2016-03-01

Isothermal and rapid amplification detection of nucleic acids is an important technology in environmental monitoring, foodborne pathogen detection, and point-of-care clinical diagnostics. Here we have developed a novel method of isothermal signal amplification for single-stranded DNA (ssDNA) detection. The ssDNA target could be used as an initiator, coupled with a double-nicked molecular beacon, to originate amplification cycles, achieving cascade signal amplification. In addition, the method showed good specificity and strong anti-jamming capability. Overall, it is a one-pot and isothermal strand displacement amplification method without the requirement of a stepwise procedure, which greatly simplifies the experimental procedure and decreases the probability of contamination of samples. With its advantages, the method would be very useful to detect nucleic acids in point-of-care or field use. Copyright © 2015 Elsevier Inc. All rights reserved.

Comparison of whole-genome bisulfite sequencing library preparation strategies identifies sources of biases affecting DNA methylation data.

PubMed

Olova, Nelly; Krueger, Felix; Andrews, Simon; Oxley, David; Berrens, Rebecca V; Branco, Miguel R; Reik, Wolf

2018-03-15

Whole-genome bisulfite sequencing (WGBS) is becoming an increasingly accessible technique, used widely for both fundamental and disease-oriented research. Library preparation methods benefit from a variety of available kits, polymerases and bisulfite conversion protocols. Although some steps in the procedure, such as PCR amplification, are known to introduce biases, a systematic evaluation of biases in WGBS strategies is missing. We perform a comparative analysis of several commonly used pre- and post-bisulfite WGBS library preparation protocols for their performance and quality of sequencing outputs. Our results show that bisulfite conversion per se is the main trigger of pronounced sequencing biases, and PCR amplification builds on these underlying artefacts. The majority of standard library preparation methods yield a significantly biased sequence output and overestimate global methylation. Importantly, both absolute and relative methylation levels at specific genomic regions vary substantially between methods, with clear implications for DNA methylation studies. We show that amplification-free library preparation is the least biased approach for WGBS. In protocols with amplification, the choice of bisulfite conversion protocol or polymerase can significantly minimize artefacts. To aid with the quality assessment of existing WGBS datasets, we have integrated a bias diagnostic tool in the Bismark package and offer several approaches for consideration during the preparation and analysis of WGBS datasets.

Sequence independent amplification of DNA

DOEpatents

Bohlander, S.K.

1998-03-24

The present invention is a rapid sequence-independent amplification procedure (SIA). Even minute amounts of DNA from various sources can be amplified independent of any sequence requirements of the DNA or any a priori knowledge of any sequence characteristics of the DNA to be amplified. This method allows, for example, the sequence independent amplification of microdissected chromosomal material and the reliable construction of high quality fluorescent in situ hybridization (FISH) probes from YACs or from other sources. These probes can be used to localize YACs on metaphase chromosomes but also--with high efficiency--in interphase nuclei. 25 figs.

Sequence independent amplification of DNA

DOEpatents

Bohlander, Stefan K.

1998-01-01

The present invention is a rapid sequence-independent amplification procedure (SIA). Even minute amounts of DNA from various sources can be amplified independent of any sequence requirements of the DNA or any a priori knowledge of any sequence characteristics of the DNA to be amplified. This method allows, for example the sequence independent amplification of microdissected chromosomal material and the reliable construction of high quality fluorescent in situ hybridization (FISH) probes from YACs or from other sources. These probes can be used to localize YACs on metaphase chromosomes but also--with high efficiency--in interphase nuclei.

Comparison of Three Different Sensitive Assays for Hepatitis B Virus DNA in Monitoring of Responses to Antiviral Therapy

PubMed Central

Chan, Henry L. Y.; Leung, Nancy W. Y.; Lau, Tracy C. M.; Wong, May L.; Sung, Joseph J. Y.

2000-01-01

The aim of our study was to compare the performances of two new hepatitis B virus (HBV) DNA assays, a cross-linking assay (NAXCOR) and a hybrid-capture amplification assay (Digene), versus the widely used branched-DNA (bDNA) assay (Chiron) in the monitoring of HBV DNA levels during antiviral treatment. Serial serum samples from 12 chronically HBV infected patients undergoing a phase II trial of an antiviral drug, 2′,3′-dideoxy-5-fluoro-3′-thiacytidine (FTC), were studied. A total of 96 serum samples were tested for HBV DNA using the cross-linking, hybrid-capture amplification, and bDNA assays. In the comparison of the cross-linking and bDNA assays, concordant results were found in 77 (80.3%) samples, no significant difference was found between the median log10 HBV DNA levels (6.66 versus 7.17 meq/ml), and the results of the two assays were closely correlated (r = 0.95). In the comparison of the hybrid-capture amplification and bDNA assays, concordant results were found in 79 (82.3%) samples, no significant difference was found between the median log10 HBV DNA levels (6.98 versus 6.99 meq/ml), and the results of the two assays were closely correlated (r = 0.99). Six (6.3%) samples by the cross-linking assay and 10 (10.4%) samples by the bDNA assay required retesting because of unacceptably high within-run coefficients of variance. No sample required retesting in the hybrid-capture amplification assay according to the internal validation. In conclusion, the cross-linking and hybrid-capture amplification assays were as sensitive as the bDNA assay for HBV DNA detection and can be recommended for monitoring of HBV DNA levels during antiviral treatment. PMID:10970358

Identification of Prostate Cancer-Specific microDNAs

DTIC Science & Technology

2014-12-01

displacement amplification (MDA). 2 adopted multiple displacement amplification (MDA) with random primers for enriched circular DNA by rolling circle ... amplification (RCA) (Fig. 1) and then amplified DNA fragments were subject to deep sequencing. Sequence NO of Reads seq 1 184 seq 2 133 seq 3 2407 seq...prostate cancer cells through multiple displacement amplification .  Clone #7 is the top candidate which has been cloned in an expression vector and it

Sensitive and inexpensive digital DNA analysis by microfluidic enrichment of rolling circle amplified single-molecules

PubMed Central

Kühnemund, Malte; Hernández-Neuta, Iván; Sharif, Mohd Istiaq; Cornaglia, Matteo; Gijs, Martin A.M.

2017-01-01

Abstract Single molecule quantification assays provide the ultimate sensitivity and precision for molecular analysis. However, most digital analysis techniques, i.e. droplet PCR, require sophisticated and expensive instrumentation for molecule compartmentalization, amplification and analysis. Rolling circle amplification (RCA) provides a simpler means for digital analysis. Nevertheless, the sensitivity of RCA assays has until now been limited by inefficient detection methods. We have developed a simple microfluidic strategy for enrichment of RCA products into a single field of view of a low magnification fluorescent sensor, enabling ultra-sensitive digital quantification of nucleic acids over a dynamic range from 1.2 aM to 190 fM. We prove the broad applicability of our analysis platform by demonstrating 5-plex detection of as little as ∼1 pg (∼300 genome copies) of pathogenic DNA with simultaneous antibiotic resistance marker detection, and the analysis of rare oncogene mutations. Our method is simpler, more cost-effective and faster than other digital analysis techniques and provides the means to implement digital analysis in any laboratory equipped with a standard fluorescent microscope. PMID:28077562

Enhanced Genetic Analysis of Single Human Bioparticles Recovered by Simplified Micromanipulation from Forensic ‘Touch DNA’ Evidence

PubMed Central

Farash, Katherine; Hanson, Erin K.; Ballantyne, Jack

2015-01-01

DNA profiles can be obtained from ‘touch DNA’ evidence, which comprises microscopic traces of human biological material. Current methods for the recovery of trace DNA employ cotton swabs or adhesive tape to sample an area of interest. However, such a ‘blind-swabbing’ approach will co-sample cellular material from the different individuals, even if the individuals’ cells are located in geographically distinct locations on the item. Thus, some of the DNA mixtures encountered in touch DNA samples are artificially created by the swabbing itself. In some instances, a victim’s DNA may be found in significant excess thus masking any potential perpetrator’s DNA. In order to circumvent the challenges with standard recovery and analysis methods, we have developed a lower cost, ‘smart analysis’ method that results in enhanced genetic analysis of touch DNA evidence. We describe an optimized and efficient micromanipulation recovery strategy for the collection of bio-particles present in touch DNA samples, as well as an enhanced amplification strategy involving a one-step 5 µl microvolume lysis/STR amplification to permit the recovery of STR profiles from the bio-particle donor(s). The use of individual or few (i.e., “clumps”) bioparticles results in the ability to obtain single source profiles. These procedures represent alternative enhanced techniques for the isolation and analysis of single bioparticles from forensic touch DNA evidence. While not necessary in every forensic investigation, the method could be highly beneficial for the recovery of a single source perpetrator DNA profile in cases involving physical assault (e.g., strangulation) that may not be possible using standard analysis techniques. Additionally, the strategies developed here offer an opportunity to obtain genetic information at the single cell level from a variety of other non-forensic trace biological material. PMID:25867046

A novel SERRS sandwich-hybridization assay to detect specific DNA target.

PubMed

Feuillie, Cécile; Merheb, Maxime Mohamad; Gillet, Benjamin; Montagnac, Gilles; Daniel, Isabelle; Hänni, Catherine

2011-01-01

In this study, we have applied Surface Enhanced Resonance Raman Scattering (SERRS) technology to the specific detection of DNA. We present an innovative SERRS sandwich-hybridization assay that allows specific DNA detection without any enzymatic amplification, such as is the case with Polymerase Chain Reaction (PCR). In some substrates, such as ancient or processed remains, enzymatic amplification fails due to DNA alteration (degradation, chemical modification) or to the presence of inhibitors. Consequently, the development of a non-enzymatic method, allowing specific DNA detection, could avoid long, expensive and inconclusive amplification trials. Here, we report the proof of concept of a SERRS sandwich-hybridization assay that leads to the detection of a specific chamois DNA. This SERRS assay reveals its potential as a non-enzymatic alternative technology to DNA amplification methods (particularly the PCR method) with several applications for species detection. As the amount and type of damage highly depend on the preservation conditions, the present SERRS assay would enlarge the range of samples suitable for DNA analysis and ultimately would provide exciting new opportunities for the investigation of ancient DNA in the fields of evolutionary biology and molecular ecology, and of altered DNA in food frauds detection and forensics.

A Novel SERRS Sandwich-Hybridization Assay to Detect Specific DNA Target

PubMed Central

Gillet, Benjamin; Montagnac, Gilles; Daniel, Isabelle; Hänni, Catherine

2011-01-01

In this study, we have applied Surface Enhanced Resonance Raman Scattering (SERRS) technology to the specific detection of DNA. We present an innovative SERRS sandwich-hybridization assay that allows specific DNA detection without any enzymatic amplification, such as is the case with Polymerase Chain Reaction (PCR). In some substrates, such as ancient or processed remains, enzymatic amplification fails due to DNA alteration (degradation, chemical modification) or to the presence of inhibitors. Consequently, the development of a non-enzymatic method, allowing specific DNA detection, could avoid long, expensive and inconclusive amplification trials. Here, we report the proof of concept of a SERRS sandwich-hybridization assay that leads to the detection of a specific chamois DNA. This SERRS assay reveals its potential as a non-enzymatic alternative technology to DNA amplification methods (particularly the PCR method) with several applications for species detection. As the amount and type of damage highly depend on the preservation conditions, the present SERRS assay would enlarge the range of samples suitable for DNA analysis and ultimately would provide exciting new opportunities for the investigation of ancient DNA in the fields of evolutionary biology and molecular ecology, and of altered DNA in food frauds detection and forensics. PMID:21655320

The thermal history of human fossils and the likelihood of successful DNA amplification.

PubMed

Smith, Colin I; Chamberlain, Andrew T; Riley, Michael S; Stringer, Chris; Collins, Matthew J

2003-09-01

Recent success in the amplification of ancient DNA (aDNA) from fossil humans has led to calls for further tests to be carried out on similar material. However, there has been little systematic research on the survival of DNA in the fossil record, even though the environment of the fossil is known to be of paramount importance for the survival of biomolecules over archaeological and geological timescales. A better understanding of aDNA survival would enable research to focus on material with greater chances of successful amplification, thus preventing the unnecessary loss of material and valuable researcher time. We argue that the thermal history of a fossil is a key parameter for the survival of biomolecules. The thermal history of a number of northwest European Neanderthal cave sites is reconstructed here and they are ranked in terms of the relative likelihood of aDNA survival at the sites, under the assumption that DNA depurination is the principal mechanism of degradation. The claims of aDNA amplification from material found at Lake Mungo, Australia, are also considered in the light of the thermal history of this site.

In Silico PCR Tools for a Fast Primer, Probe, and Advanced Searching.

PubMed

Kalendar, Ruslan; Muterko, Alexandr; Shamekova, Malika; Zhambakin, Kabyl

2017-01-01

The polymerase chain reaction (PCR) is fundamental to molecular biology and is the most important practical molecular technique for the research laboratory. The principle of this technique has been further used and applied in plenty of other simple or complex nucleic acid amplification technologies (NAAT). In parallel to laboratory "wet bench" experiments for nucleic acid amplification technologies, in silico or virtual (bioinformatics) approaches have been developed, among which in silico PCR analysis. In silico NAAT analysis is a useful and efficient complementary method to ensure the specificity of primers or probes for an extensive range of PCR applications from homology gene discovery, molecular diagnosis, DNA fingerprinting, and repeat searching. Predicting sensitivity and specificity of primers and probes requires a search to determine whether they match a database with an optimal number of mismatches, similarity, and stability. In the development of in silico bioinformatics tools for nucleic acid amplification technologies, the prospects for the development of new NAAT or similar approaches should be taken into account, including forward-looking and comprehensive analysis that is not limited to only one PCR technique variant. The software FastPCR and the online Java web tool are integrated tools for in silico PCR of linear and circular DNA, multiple primer or probe searches in large or small databases and for advanced search. These tools are suitable for processing of batch files that are essential for automation when working with large amounts of data. The FastPCR software is available for download at http://primerdigital.com/fastpcr.html and the online Java version at http://primerdigital.com/tools/pcr.html .

Recombinase polymerase and enzyme-linked immunosorbent assay as a DNA amplification-detection strategy for food analysis.

PubMed

Santiago-Felipe, S; Tortajada-Genaro, L A; Puchades, R; Maquieira, A

2014-02-06

Polymerase chain reaction in conjunction with enzyme-linked immunosorbent assay (PCR-ELISA) is a well-established technique that provides a suitable rapid, sensitive, and selective method for a broad range of applications. However, the need for precise rapid temperature cycling of PCR is an important drawback that can be overcome by employing isothermal amplification reactions such as recombinase polymerase amplification (RPA). The RPA-ELISA combination is proposed for amplification at a low, constant temperature (40°C) in a short time (40 min), for the hybridisation of labelled products to specific 5'-biotinylated probes/streptavidin in coated microtiter plates at room temperature, and for detection by colorimetric immunoassay. RPA-ELISA was applied to screen common safety threats in foodstuffs, such as allergens (hazelnut, peanut, soybean, tomato, and maize), genetically modified organisms (P35S and TNOS), pathogenic bacteria (Salmonella sp. and Cronobacter sp.), and fungi (Fusarium sp.). Satisfactory sensitivity and reproducibility results were achieved for all the targets. The RPA-ELISA technique does away with thermocycling and provides a suitable sensitive, specific, and cost-effective method for routine applications, and proves particularly useful for resource-limited settings. Copyright © 2013 Elsevier B.V. All rights reserved.

Protective role of humic acids against picloram-induced genomic instability and DNA methylation in Phaseolus vulgaris.

PubMed

Taspinar, Mahmut Sinan; Aydin, Murat; Sigmaz, Burcu; Yildirim, Nalan; Agar, Guleray

2017-10-01

Picloram (4-amino-3,5,6-trichloropicolinic acid) is a liquid auxinic herbicide used to control broad-leaved weeds. Picloram is representing a possible hazard to ecosystems and human health. Therefore, in this study, DNA methylation changes and DNA damage levels in Phaseolus vulgaris exposed to picloram, as well as whether humic acid (HA) has preventive effects on these changes were investigated. Random amplified polymorphic DNA (RAPD) techniques were used for identification of DNA damage and coupled restriction enzyme digestion-random amplification (CRED-RA) techniques were used to detect the changed pattern of DNA methylation. According to the obtained results, picloram (5, 10, 20, and 40 mg/l) caused DNA damage profile changes (RAPDs) increasing, DNA hypomethylation and genomic template stability (GTS) decreasing. On the other hand, different concentrations of applied HA (2, 4, 6, 8, and 10%) reduced hazardous effects of picloram. The results of the experiment have explicitly indicated that HAs could be an alternative for reducing genetic damage in plants. In addition to the alleviate effects of humic acid on genetic damage, its epigenetic effect is hypomethylation.

Isothermal Recombinase Polymerase amplification (RPA) of Schistosoma haematobium DNA and oligochromatographic lateral flow detection.

PubMed

Rosser, A; Rollinson, D; Forrest, M; Webster, B L

2015-09-04

Accurate diagnosis of urogenital schistosomiasis is vital for surveillance/control programs. Amplification of schistosome DNA in urine by PCR is sensitive and specific but requires infrastructure, financial resources and skilled personnel, often not available in endemic areas. Recombinase Polymerase Amplification (RPA) is an isothermal DNA amplification/detection technology that is simple, rapid, portable and needs few resources. Here a Schistosoma haematobium RPA assay was developed and adapted so that DNA amplicons could be detected using oligochromatographic Lateral Flow (LF) strips. The assay successfully amplified S. haematobium DNA at 30-45 °C in 10 mins and was sensitive to a lower limit of 100 fg of DNA. The assay was also successful with the addition of crude urine, up to 5% of the total reaction volume. Cross amplification occurred with other schistosome species but not with other common urine microorganisms. The LF-RPA assay developed here can amplify and detect low levels of S. haematobium DNA. Reactions are rapid, require low temperatures and positive reactions are interpreted using lateral flow strips, reducing the need for infrastructure and resources. This together with an ability to withstand inhibitors within urine makes RPA a promising technology for further development as a molecular diagnostic tool for urogenital schistosomiasis.

An integrated direct loop-mediated isothermal amplification microdevice incorporated with an immunochromatographic strip for bacteria detection in human whole blood and milk without a sample preparation step.

PubMed

Lee, Dohwan; Kim, Yong Tae; Lee, Jee Won; Kim, Do Hyun; Seo, Tae Seok

2016-05-15

We have developed an integrated direct loop-mediated isothermal amplification (Direct LAMP) microdevice incorporated with an immunochromatographic strip (ICS) to identify bacteria contaminated in real samples. The Direct LAMP is a novel isothermal DNA amplification technique which does not require thermal cycling steps as well as any sample preparation steps such as cell lysis and DNA extraction for amplifying specific target genes. In addition, the resultant amplicons were colorimetrically detected on the ICS, thereby enabling the entire genetic analysis process to be simplified. The two functional units (Direct LAMP and ICS) were integrated on a single device without use of the tedious and complicated microvalve and tubing systems. The utilization of a slidable plate allows us to manipulate the fluidic control in the microchannels manually and the sequential operation of the Direct LAMP and ICS detection could be performed by switching the slidable plate to each functional unit. Thus, the combination of the direct isothermal amplification without any sample preparation and thermal cycling steps, the ICS based amplicon detection by naked eyes, and the slidable plate to eliminate the microvalves in the integrated microdevice would be an ideal platform for point-of-care DNA diaganotics. On the integrated Direct LAMP-ICS microdevice, we could analyze Staphylococcus aureus (S. aureus) and Escherichia coli O157:H7 (E. coli O157:H7) contaminated in human whole blood or milk at a single-cell level within 1h. Copyright © 2015 Elsevier B.V. All rights reserved.

Amplification of the entire kanamycin biosynthetic gene cluster during empirical strain improvement of Streptomyces kanamyceticus.

PubMed

Yanai, Koji; Murakami, Takeshi; Bibb, Mervyn

2006-06-20

Streptomyces kanamyceticus 12-6 is a derivative of the wild-type strain developed for industrial kanamycin (Km) production. Southern analysis and DNA sequencing revealed amplification of a large genomic segment including the entire Km biosynthetic gene cluster in the chromosome of strain 12-6. At 145 kb, the amplifiable unit of DNA (AUD) is the largest AUD reported in Streptomyces. Striking repetitive DNA sequences belonging to the clustered regularly interspaced short palindromic repeats family were found in the AUD and may play a role in its amplification. Strain 12-6 contains a mixture of different chromosomes with varying numbers of AUDs, sometimes exceeding 36 copies and producing an amplified region >5.7 Mb. The level of Km production depended on the copy number of the Km biosynthetic gene cluster, suggesting that DNA amplification occurred during strain improvement as a consequence of selection for increased Km resistance. Amplification of DNA segments including entire antibiotic biosynthetic gene clusters might be a common mechanism leading to increased antibiotic production in industrial strains.

Detection of mycobacterium tuberculosis in paraffin-embedded pleural biopsy specimens by commercial ribosomal RNA and DNA amplification kits.

PubMed

Ruiz-Manzano, J; Manterola, J M; Gamboa, F; Calatrava, A; Monsó, E; Martínez, C; Ausina, V

2000-09-01

To evaluate the utility of two gene amplification systems in historical paraffin-embedded pleural biopsy (PEB) tissues from patients with pleural tuberculosis, and to compare the results to those obtained with conventional histologic and microbiological methods. A retrospective study. Seventy-four formalin-fixed PEB tissues collected and stored over 12 years (1984 through 1995) were retrieved. Gene amplifications were performed in 57 tissues from patients with diagnoses of pleural tuberculosis and in 17 from patients with carcinoma as controls, using the first version of the Amplified Mycobacterium tuberculosis Direct Test (AMTDT; Gen-Probe; San Diego, CA) and the LCx Mycobacterium tuberculosis Assay (LCxMTB; Abbott Laboratories; Abbott Park, IL). The sensitivities of the AMTDT and LCxMTB were 52.6% and 63.2%, respectively (p = not statistically significant). The specificity of both tests was 100%. Twenty tissue samples (35.1%) were positive by both systems, and 10 tissues (17.5%) were positive only by the AMTDT, while 16 tissues (28.1%) were positive only by the LCxMTB. Both tests gave negative results for 11 specimens (19.3%). When both tests were used, a positive diagnosis was achieved in 80.7% of the samples. Diagnosis of 73.7% of patient conditions had previously been made by smear examination of pleural biopsy and sputum, pleural liquid, or biopsy culture. The overall diagnostic yield with both culture and amplification techniques was 96.5% (55 of 57 patients) for pleural tuberculosis, with amplification techniques adding 22.8% of the diagnoses. Amplification techniques are useful in archival PEB tissues, providing additional diagnoses beyond culturing, although the sensitivity should be improved, possibly by standardizing protocols.

Rapid screening for human-pathogenic Mucorales using rolling circle amplification.

PubMed

Dolatabadi, S; Najafzadeh, M J; de Hoog, G S

2014-12-01

Mucormycosis has emerged as a relatively common severe mycosis in patients with haematological and allogeneic stem cell transplantation. Source of transmission is from unidentified sources in the environment. Early diagnosis of infection and its source of contamination are paramount for rapid and appropriate therapy. In this study, rolling circle amplification (RCA) is introduced as a sensitive, specific and reproducible isothermal DNA amplification technique for rapid molecular identification of six of the most virulent species (Rhizopus microsporus, R. arrhizus var. arrhizus, R. arrhizus var. delemar, Mucor irregularis, Mucor circinelloides, Lichtheimia ramosa, Lichtheimia corymbifera). DNAs of target species were successfully amplified, with no cross reactivity between species. RCA can be considered as a rapid detection method with high specificity and sensitivity, suitable for large screening. © 2014 Blackwell Verlag GmbH.

Origins of DNA Replication and Amplification in the Breast Cancer Genome

DTIC Science & Technology

2011-09-01

AD_________________ Award Number: W81XWH-10-1-0463 TITLE: Origins of DNA Replication and...hypothesis we need to map origins of DNA replication in the genome and ask which of these coincide with sites of DNA amplification and with ER...Spring Harbor DNA Replication meetings this summer/earlyfall. Figures from the posters and also the abstracts are attached. The samples have been

Label-free genotyping of cytochrome P450 2D6*10 using ligation-mediated strand displacement amplification with DNAzyme-based chemiluminescence detection.

PubMed

Wang, Hong-Qi; Wu, Zhan; Zhang, Yan; Tang, Li-Juan; Yu, Ru-Qin; Jiang, Jian-Hui

2012-01-13

Genotyping of cytochrome P450 monooxygenase 2D6*10 (CYP2D6*10) plays an important role in pharmacogenomics, especially in clinical drug therapy of Asian populations. This work reported a novel label-free technique for genotyping of CYP2D6*10 based on ligation-mediated strand displacement amplification (SDA) with DNAzyme-based chemiluminescence detection. Discrimination of single-base mismatch is firstly accomplished using DNA ligase to generate a ligation product. The ligated product then initiates a SDA reaction to produce aptamer sequences against hemin, which can be probed by chemiluminescence detection. The proposed strategy is used for the assay of CYP2D6*10 target and the genomic DNA. The results reveal that the proposed technique displays chemiluminescence responses in linear correlation to the concentrations of DNA target within the range from 1 pM to 1 nM. A detection limit of 0.1 pM and a signal-to-background ratio of 57 are achieved. Besides such high sensitivity, the proposed CYP2D6*10 genotyping strategy also offers superb selectivity, great robustness, low cost and simplified operations due to its label-free, homogeneous, and chemiluminescence-based detection format. These advantages suggest this technique may hold considerable potential for clinical CYP2D6*10 genotyping and association studies. Copyright © 2011 Elsevier B.V. All rights reserved.

Tip-enhanced fluorescence with radially polarized illumination for monitoring loop-mediated isothermal amplification on Hepatitis C virus cDNA

NASA Astrophysics Data System (ADS)

Wei, Shih-Chung; Chuang, Tsung-Liang; Wang, Da-Shin; Lu, Hui-Hsin; Gu, Frank X.; Sung, Kung-Bin; Lin, Chii-Wann

2015-02-01

A tip nanobiosensor for monitoring DNA replication was presented. The effects of excitation power and polarization on tip-enhanced fluorescence (TEF) were assessed with the tip immersed in fluorescein isothiocyanate solution first. The photon count rose on average fivefold with radially polarized illumination at 50 mW. We then used polymerase-functionalized tips for monitoring loop-mediated isothermal amplification on Hepatitis C virus cDNA. The amplicon-SYBR Green I complex was detected and compared to real-time loop-mediated isothermal amplification. The signals of the reaction using 4 and 0.004 ng/μl templates were detected 10 and 30 min earlier, respectively. The results showed the potential of TEF in developing a nanobiosensor for real-time DNA amplification.

Vanadium accelerates polymerase chain reaction and expands the applicability of forensic DNA testing.

PubMed

Kaminiwa, Junko; Honda, Katsuya; Sugano, Yukiko; Yano, Shizue; Nishi, Takeki; Sekine, Yuko

2013-05-01

Polymerase chain reaction (PCR) has been rapidly established as one of the most widely used techniques in molecular biology. Because most DNA analysis is PCR-based, the analysis of unamplifiable DNA of poor quality or low quantity is nearly impossible. However, we observed that if an appropriate concentration of vanadium chloride is added to the standard reaction mixture, the enzymatic amplification of DNA could be enhanced. Using multiplex PCR with the addition of vanadium, DNA typing was possible from even trace amounts of DNA that we were unable to amplify using normal reaction conditions. This method might be an effective tool for not only criminal investigations and ancient DNA analysis, but also for nearly all fields using DNA technology. Copyright © 2012 Elsevier Ltd and Faculty of Forensic and Legal Medicine. All rights reserved.

Novel selection methods for DNA-encoded chemical libraries.

PubMed

Chan, Alix I; McGregor, Lynn M; Liu, David R

2015-06-01

Driven by the need for new compounds to serve as biological probes and leads for therapeutic development and the growing accessibility of DNA technologies including high-throughput sequencing, many academic and industrial groups have begun to use DNA-encoded chemical libraries as a source of bioactive small molecules. In this review, we describe the technologies that have enabled the selection of compounds with desired activities from these libraries. These methods exploit the sensitivity of in vitro selection coupled with DNA amplification to overcome some of the limitations and costs associated with conventional screening methods. In addition, we highlight newer techniques with the potential to be applied to the high-throughput evaluation of DNA-encoded chemical libraries. Copyright © 2015 Elsevier Ltd. All rights reserved.

Ultrasensitive Electrochemical Detection of mRNA Using Branched DNA Amplifiers

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mao, Xun; Liu, Guodong; Wang, Shengfu

2008-11-01

We describe here an ultrasensitive electrochemical detection of m RNA protocol without RNA purification and PCR amplification. The new m RNA electrical detection capability is coupled to the amplification feature of branched DNA (bDNA) technology and with the nagnetic beads based electrochemical bioassay.

Analysis of raw meats and fats of pigs using polymerase chain reaction for Halal authentication.

PubMed

Aida, A A; Che Man, Y B; Wong, C M V L; Raha, A R; Son, R

2005-01-01

A method for species identification from pork and lard samples using polymerase chain reaction (PCR) analysis of a conserved region in the mitochondrial (mt) cytochrome b (cyt b) gene has been developed. Genomic DNA of pork and lard were extracted using Qiagen DNeasy(®) Tissue Kits and subjected to PCR amplification targeting the mt cyt b gene. The genomic DNA from lard was found to be of good quality and produced clear PCR products on the amplification of the mt cyt b gene of approximately 360 base pairs. To distinguish between species, the amplified PCR products were cut with restriction enzyme BsaJI resulting in porcine-specific restriction fragment length polymorphisms (RFLP). The cyt b PCR-RFLP species identification assay yielded excellent results for identification of pig species. It is a potentially reliable technique for detection of pig meat and fat from other animals for Halal authentication.

Co-amplification at lower denaturation temperature-PCR: methodology and applications.

PubMed

Liang, Hui; Chen, Guo-Jie; Yu, Yan; Xiong, Li-Kuan

2018-03-20

Co-amplification at lower denaturation temperature-polymerase chain reaction (COLD-PCR) is a novel form of PCR that selectively denatures and amplifies low-abundance mutations from mixtures of wild-type and mutation-containing sequences, enriching the mutation 10 to 100 folds. Due to the slightly altered melting temperature (Tm) of the double-stranded DNA and the formation of the mutation/wild-type heteroduplex DNA, COLD-PCR methods are sensitive, specific, accurate, cost-effective and easy to maneuver, and can enrich mutations of any type and at any position, even unknown mutations within amplicons. COLD-PCR and its improved methods are now applied in cancer, microorganisms, prenatal screening, animals and plants. They are extremely useful for early diagnosis, monitoring the prognosis of disease and the efficiency of the treatment, drug selection, prediction of prognosis, plant breeding and etc. In this review, we introduce the principles, key techniques, derived methods and applications of COLD-PCR.

Controlled Microwave Heating Accelerates Rolling Circle Amplification.

PubMed

Yoshimura, Takeo; Suzuki, Takamasa; Mineki, Shigeru; Ohuchi, Shokichi

2015-01-01

Rolling circle amplification (RCA) generates single-stranded DNAs or RNA, and the diverse applications of this isothermal technique range from the sensitive detection of nucleic acids to analysis of single nucleotide polymorphisms. Microwave chemistry is widely applied to increase reaction rate as well as product yield and purity. The objectives of the present research were to apply microwave heating to RCA and indicate factors that contribute to the microwave selective heating effect. The microwave reaction temperature was strictly controlled using a microwave applicator optimized for enzymatic-scale reactions. Here, we showed that microwave-assisted RCA reactions catalyzed by either of the four thermostable DNA polymerases were accelerated over 4-folds compared with conventional RCA. Furthermore, the temperatures of the individual buffer components were specifically influenced by microwave heating. We concluded that microwave heating accelerated isothermal RCA of DNA because of the differential heating mechanisms of microwaves on the temperatures of reaction components, although the overall reaction temperatures were the same.

Single Molecule Bioelectronics and Their Application to Amplification-Free Measurement of DNA Lengths

PubMed Central

Gül, O. Tolga; Pugliese, Kaitlin M.; Choi, Yongki; Sims, Patrick C.; Pan, Deng; Rajapakse, Arith J.; Weiss, Gregory A.; Collins, Philip G.

2016-01-01

As biosensing devices shrink smaller and smaller, they approach a scale in which single molecule electronic sensing becomes possible. Here, we review the operation of single-enzyme transistors made using single-walled carbon nanotubes. These novel hybrid devices transduce the motions and catalytic activity of a single protein into an electronic signal for real-time monitoring of the protein’s activity. Analysis of these electronic signals reveals new insights into enzyme function and proves the electronic technique to be complementary to other single-molecule methods based on fluorescence. As one example of the nanocircuit technique, we have studied the Klenow Fragment (KF) of DNA polymerase I as it catalytically processes single-stranded DNA templates. The fidelity of DNA polymerases makes them a key component in many DNA sequencing techniques, and here we demonstrate that KF nanocircuits readily resolve DNA polymerization with single-base sensitivity. Consequently, template lengths can be directly counted from electronic recordings of KF’s base-by-base activity. After measuring as few as 20 copies, the template length can be determined with <1 base pair resolution, and different template lengths can be identified and enumerated in solutions containing template mixtures. PMID:27348011

Single Molecule Bioelectronics and Their Application to Amplification-Free Measurement of DNA Lengths.

PubMed

Gül, O Tolga; Pugliese, Kaitlin M; Choi, Yongki; Sims, Patrick C; Pan, Deng; Rajapakse, Arith J; Weiss, Gregory A; Collins, Philip G

2016-06-24

As biosensing devices shrink smaller and smaller, they approach a scale in which single molecule electronic sensing becomes possible. Here, we review the operation of single-enzyme transistors made using single-walled carbon nanotubes. These novel hybrid devices transduce the motions and catalytic activity of a single protein into an electronic signal for real-time monitoring of the protein's activity. Analysis of these electronic signals reveals new insights into enzyme function and proves the electronic technique to be complementary to other single-molecule methods based on fluorescence. As one example of the nanocircuit technique, we have studied the Klenow Fragment (KF) of DNA polymerase I as it catalytically processes single-stranded DNA templates. The fidelity of DNA polymerases makes them a key component in many DNA sequencing techniques, and here we demonstrate that KF nanocircuits readily resolve DNA polymerization with single-base sensitivity. Consequently, template lengths can be directly counted from electronic recordings of KF's base-by-base activity. After measuring as few as 20 copies, the template length can be determined with <1 base pair resolution, and different template lengths can be identified and enumerated in solutions containing template mixtures.

An evaluation of long-term preservation methods for brown bear (Ursus arctos) faecal DNA samples

USGS Publications Warehouse

Murphy, M.A.; Waits, L.P.; Kendall, K.C.; Wasser, S.K.; Higbee, J.A.; Bogden, R.

2002-01-01

Relatively few large-scale faecal DNA studies have been initiated due to difficulties in amplifying low quality and quantity DNA template. To improve brown bear faecal DNA PCR amplification success rates and to determine post collection sample longevity, five preservation methods were evaluated: 90% ethanol, DETs buffer, silica-dried, oven-dried stored at room temperature, and oven-dried stored at -20??C. Preservation effectiveness was evaluated for 50 faecal samples by PCR amplification of a mitochondrial DNA (mtDNA) locus (???146 bp) and a nuclear DNA (nDNA) locus (???200 bp) at time points of one week, one month, three months and six months. Preservation method and storage time significantly impacted mtDNA and nDNA amplification success rates. For mtDNA, all preservation methods had ??? 75% success at one week, but storage time had a significant impact on the effectiveness of the silica preservation method. Ethanol preserved samples had the highest success rates for both mtDNA (86.5%) and nDNA (84%). Nuclear DNA amplification success rates ranged from 26-88%, and storage time had a significant impact on all methods but ethanol. Preservation method and storage time should be important considerations for researchers planning projects utilizing faecal DNA. We recommend preservation of faecal samples in 90% ethanol when feasible, although when collecting in remote field conditions or for both DNA and hormone assays a dry collection method may be advantageous.

Invasive reaction assisted strand-displacement signal amplification for sensitive DNA detection.

PubMed

Zou, Bingjie; Song, Qinxin; Wang, Jianping; Liu, Yunlong; Zhou, Guohua

2014-11-18

A novel DNA detection assay was proposed by invasive reaction coupled with molecular beacon assisted strand-displacement signal amplification (IRASA). Target DNAs are firstly hybridized to two probes to initiate invasive reaction to produce amplified flaps. Then these flaps are further amplified by strand-displacement signal amplification. The detection limit was around 0.2 pM.

Application of advanced cytometric and molecular technologies to minimal residual disease monitoring

NASA Astrophysics Data System (ADS)

Leary, James F.; He, Feng; Reece, Lisa M.

2000-04-01

Minimal residual disease monitoring presents a number of theoretical and practical challenges. Recently it has been possible to meet some of these challenges by combining a number of new advanced biotechnologies. To monitor the number of residual tumor cells requires complex cocktails of molecular probes that collectively provide sensitivities of detection on the order of one residual tumor cell per million total cells. Ultra-high-speed, multi parameter flow cytometry is capable of analyzing cells at rates in excess of 100,000 cells/sec. Residual tumor selection marker cocktails can be optimized by use of receiver operating characteristic analysis. New data minimizing techniques when combined with multi variate statistical or neural network classifications of tumor cells can more accurately predict residual tumor cell frequencies. The combination of these techniques can, under at least some circumstances, detect frequencies of tumor cells as low as one cell in a million with an accuracy of over 98 percent correct classification. Detection of mutations in tumor suppressor genes requires insolation of these rare tumor cells and single-cell DNA sequencing. Rare residual tumor cells can be isolated at single cell level by high-resolution single-cell cell sorting. Molecular characterization of tumor suppressor gene mutations can be accomplished using a combination of single- cell polymerase chain reaction amplification of specific gene sequences followed by TA cloning techniques and DNA sequencing. Mutations as small as a single base pair in a tumor suppressor gene of a single sorted tumor cell have been detected using these methods. Using new amplification procedures and DNA micro arrays it should be possible to extend the capabilities shown in this paper to screening of multiple DNA mutations in tumor suppressor and other genes on small numbers of sorted metastatic tumor cells.

Target-triggering multiple-cycle signal amplification strategy for ultrasensitive detection of DNA based on QCM and SPR.

PubMed

Song, Weiling; Yin, Wenshuo; Sun, Wenbo; Guo, Xiaoyan; He, Peng; Yang, Xiaoyan; Zhang, Xiaoru

2018-04-24

Detection of ultralow concentrations of nucleic acid sequences is a central challenge in the early diagnosis of genetic diseases. Herein, we developed a target-triggering cascade multiple cycle amplification for ultrasensitive DNA detection using quartz crystal microbalance (QCM) and surface plasmon resonance (SPR). It was based on the exonuclease Ⅲ (Exo Ⅲ)-assisted signal amplification and the hybridization chain reaction (HCR). The streptavidin-coated Au-NPs (Au-NPs-SA) were assembled on the HCR products as recognition element. Upon sensing of target DNA, the duplex DNA probe triggered the Exo Ⅲ cleavage process, accompanied by generating a new secondary target DNA and releasing target DNA. The released target DNA and the secondary target DNA were recycled. Simultaneously, numerous single strands were liberated and acted as the trigger of HCR to generate further signal amplification, resulting in the immobilization of abundant Au-NPs-SA on the gold substrate. The QCM sensor results were found to be comparable to that achieved using a SPR sensor platform. This method exhibited a high sensitivity toward target DNA with a detection limit of 0.70 fM. The high sensitivity and specificity make this method a great potential for detecting DNA with trace amounts in bioanalysis and clinical biomedicine. Copyright © 2018 Elsevier Inc. All rights reserved.

[Quantitative PCR in the diagnosis of Leishmania].

PubMed

Mortarino, M; Franceschi, A; Mancianti, F; Bazzocchi, C; Genchi, C; Bandi, C

2004-06-01

Polymerase chain reaction (PCR) is a sensitive and rapid method for the diagnosis of canine Leishmania infection and can be performed on a variety of biological samples, including peripheral blood, lymph node, bone marrow and skin. Standard PCR requires electrophoretic analysis of the amplification products and is usually not suitable for quantification of the template DNA (unless competitor-based or other methods are developed), being of reduced usefulness when accurate monitoring of target DNA is required. Quantitative real-time PCR allows the continuous monitoring of the accumulation of PCR products during the amplification reaction. This allows the identification of the cycle of near-logarithmic PCR product generation (threshold cycle) and, by inference, the relative quantification of the template DNA present at the start of the reaction. Since the amplification product are monitored in "real-time" as they form cycle-by-cycle, no post-amplification handling is required. The absolute quantification is performed according either to an internal standard co-amplified with the sample DNA, or to an external standard curve obtained by parallel amplification of serial known concentrations of a reference DNA sequence. From the quantification of the template DNA, an estimation of the relative load of parasites in the different samples can be obtained. The advantages compared to standard and semi-quantitative PCR techniques are reduction of the assay's time and contamination risks, and improved sensitivity. As for standard PCR, the minimal components of the quantitative PCR reaction mixture are the DNA target of the amplification, an oligonucleotide primer pair flanking the target sequence, a suitable DNA polymerase, deoxynucleotides, buffer and salts. Different technologies have been set up for the monitoring of amplification products, generally based on the use of fluorescent probes. For instance, SYBR Green technology is a non-specific detection system based on a fluorescent dsDNA intercalator and it is applicable to all potential targets. TaqMan technology is more specific since performs the direct assessment of the amount of amplified DNA using a fluorescent probe specific for the target sequence flanked by the primer pair. This probe is an oligonucleotide labelled with a reporter dye (fluorescent) and a quencher (which absorbs the fluorescent signal generated by the reporter). The thermic protocol of amplification allows the binding of the fluorescent probe to the target sequence before the binding of the primers and the starting of the polymerization by Taq polymerase. During polymerization, 5'-3' exonuclease activity of Taq polymerase digests the probe and in this way the reporter dye is released from the probe and a fluorescent signal is detected. The intensity of the signal accumulates at the end of each cycle and is related to the amount of the amplification product. In recent years, quantitative PCR methods based either on SYBR Green or TaqMan technology have been set up for the quantification of Leishmania in mouse liver, mouse skin and human peripheral blood, targeting either single-copy chromosomal or multi-copy minicircle sequences with high sensitivity and reproducibility. In particular, real-time PCR seems to be a reliable, rapid and noninvasive method for the diagnosis and follow up of visceral leishmaniasis in humans. At present, the application of real-time PCR for research and clinical diagnosis of Leishmania infection in dogs is still foreseable. As for standard PCR, the high sensitivity of real-time PCR could allow the use of blood sampling that is less invasive and easily performed for monitoring the status of the dogs. The development of a real-time PCR assay for Leishmania infantum infection in dogs could support the standard and optimized serological and PCR methods currenly in use for the diagnosis and follow-up of canine leishmaniasis, and perhaps prediction of recurrences associated with tissue loads of residual pathogens after treatment. At this regard, a TaqMan Real Time PCR method developed for the quantification of Leishmania infantum minicircle DNA in peripheral blood of naturally infected dogs sampled before and at different time points after the beginning of a standard antileishmanial therapy will be illustrated.

The effect of main urine inhibitors on the activity of different DNA polymerases in loop-mediated isothermal amplification.

PubMed

Jevtuševskaja, Jekaterina; Krõlov, Katrin; Tulp, Indrek; Langel, Ülo

2017-04-01

The use of rapid amplification methods to detect pathogens in biological samples is mainly limited by the amount of pathogens present in the sample and the presence of inhibiting substances. Inhibitors can affect the amplification efficiency by either binding to the polymerase, interacting with the DNA, or interacting with the polymerase during primer extension. Amplification is performed using DNA polymerase enzymes and even small changes in their activity can influence the sensitivity and robustness of molecular assays Methods: The main purpose of this research was to examine which compounds present in urine inhibit polymerases with strand displacement activity. To quantify the inhibition, we employed quantitative loop-mediated isothermal amplification Results: The authors found that the presence of BSA, Mg 2+, and urea at physiologically relevant concentrations, as well as acidic or alkaline conditions did not affect the activity of any of the tested polymerases. However, addition of salt significantly affected the activity of the tested polymerases. These findings may aid in the development of more sensitive, robust, cost effective isothermal amplification based molecular assays suitable for both point-of-care testing and on-site screening of pathogens directly from unprocessed urine which avoid the need for long and tedious DNA purification steps prior to amplification.

Real-time electrochemical monitoring of isothermal helicase-dependent amplification of nucleic acids.

PubMed

Kivlehan, Francine; Mavré, François; Talini, Luc; Limoges, Benoît; Marchal, Damien

2011-09-21

We described an electrochemical method to monitor in real-time the isothermal helicase-dependent amplification of nucleic acids. The principle of detection is simple and well-adapted to the development of portable, easy-to-use and inexpensive nucleic acids detection technologies. It consists of monitoring a decrease in the electrochemical current response of a reporter DNA intercalating redox probe during the isothermal DNA amplification. The method offers the possibility to quantitatively analyze target nucleic acids in less than one hour at a single constant temperature, and to perform at the end of the isothermal amplification a DNA melt curve analysis for differentiating between specific and non-specific amplifications. To illustrate the potentialities of this approach for the development of a simple, robust and low-cost instrument with high throughput capability, the method was validated with an electrochemical system capable of monitoring up to 48 real-time isothermal HDA reactions simultaneously in a disposable microplate consisting of 48-electrochemical microwells. Results obtained with this approach are comparable to that obtained with a well-established but more sophisticated and expensive fluorescence-based method. This makes for a promising alternative detection method not only for real-time isothermal helicase-dependent amplification of nucleic acid, but also for other isothermal DNA amplification strategies.

Highly sensitive and selective microRNA detection based on DNA-bio-bar-code and enzyme-assisted strand cycle exponential signal amplification.

PubMed

Dong, Haifeng; Meng, Xiangdan; Dai, Wenhao; Cao, Yu; Lu, Huiting; Zhou, Shufeng; Zhang, Xueji

2015-04-21

Herein, a highly sensitive and selective microRNA (miRNA) detection strategy using DNA-bio-bar-code amplification (BCA) and Nb·BbvCI nicking enzyme-assisted strand cycle for exponential signal amplification was designed. The DNA-BCA system contains a locked nucleic acid (LNA) modified DNA probe for improving hybridization efficiency, while a signal reported molecular beacon (MB) with an endonuclease recognition site was designed for strand cycle amplification. In the presence of target miRNA, the oligonucleotides functionalized magnetic nanoprobe (MNP-DNA) and gold nanoprobe (AuNP-DNA) with numerous reported probes (RP) can hybridize with target miRNA, respectively, to form a sandwich structure. After sandwich structures were separated from the solution by the magnetic field, the RP were released under high temperature to recognize the MB and cleaved the hairpin DNA to induce the dissociation of RP. The dissociated RP then triggered the next strand cycle to produce exponential fluorescent signal amplification for miRNA detection. Under optimized conditions, the exponential signal amplification system shows a good linear range of 6 orders of magnitude (from 0.3 pM to 3 aM) with limit of detection (LOD) down to 52.5 zM, while the sandwich structure renders the system with high selectivity. Meanwhile, the feasibility of the proposed strategy for cell miRNA detection was confirmed by analyzing miRNA-21 in HeLa lysates. Given the high-performance for miRNA analysis, the strategy has a promising application in biological detection and in clinical diagnosis.

De novo amplification within a silent human cholinesterase gene in a family subjected to prolonged exposure to organophosphorus insecticides

DOE Office of Scientific and Technical Information (OSTI.GOV)

Prody, C.A.; Dreyfus, P.; Soreq, H.

1989-01-01

A 100-fold DNA amplification in the CHE gene, coding for serum butyrylcholinesterase (BtChoEase), was found in a farmer expressing silent CHE phenotype. Individuals homozygous for this gene display a defective serum BtChoEase and are particularly vulnerable to poisoning by agricultural organophosphorus insecticides, to which all members of this family had long been exposed. DNA blot hybridization with regional BtChoEase cDNA probes suggested that the amplification was most intense in regions encoding central sequences within BtChoEase cDNA, whereas distal sequences were amplified to a much lower extent. This is in agreement with the onion skin model, based on amplification of genesmore » in cultured cells and primary tumors. The amplification was absent in the grandparents but present at the same extent in one of their sons and in a grandson, with similar DNA blot hybridization patterns. In situ hybridization experiments localized the amplified sequences to the long arm of chromosome 3, close to the site where the authors previously mapped the CHE gene. Altogether, these observations suggest that the initial amplification event occurred early in embryogenesis, spermatogenesis, or oogenesis, where the CHE gene is intensely active and where cholinergic functioning was indicated to be physiologically necessary. These findings demonstrate a de novo amplification in apparently healthy individuals within an autosomal gene producing a target protein to an inhibitor.« less

An ARMS-based technique for sex determination of red panda (Ailurus fulgens).

PubMed

Li, Yuzhi; Xu, Xiao; Zhang, Liang; Zhang, Zhihe; Shen, Fujun; Zhang, Wenping; Yue, Bisong

2011-03-01

Molecular sexing is a key component in the investigation of wild populations. In this study, we developed a fast, accurate and reliable amplification refractory mutation system (ARMS) technique for sex determination of red panda based on the exon 4 of the ZFX/ZFY gene. The amplicons were distinguished simply by agarose gel electrophoresis, exhibiting one fragment in females (X: 300 bp) and two in males (X: 300 bp, Y: 166 bp). Robustness of this ARMS system was confirmed by testing both 43 captive red pandas using DNA samples with known-sex and 10 wild red pandas using faecal DNA samples with unknown sex. © 2010 Blackwell Publishing Ltd.

In vivo analysis of polyadenylation in prokaryotes.

PubMed

Mohanty, Bijoy K; Kushner, Sidney R

2014-01-01

Polyadenylation at the 3' ends of mRNAs, tRNAs, rRNAs, and sRNAs plays important roles in RNA metabolism in both prokaryotes and eukaryotes. However, the nature of poly(A) tails in prokaryotes is distinct compared to their eukaryotic counterparts. Specifically, depending on the organism, eukaryotic poly(A) tails average between 50 and >200 nt and can easily be isolated by several techniques involving oligo(dT)-dependent cDNA amplification. In contrast, the bulk of the poly(A) tails present on prokaryotic transcripts is relatively short (<10 nt) and is difficult to characterize using similar techniques. This chapter describes methods that can circumvent these problems. For example, we discuss how to isolate total RNA and characterize its overall polyadenylation status employing a poly(A) sizing assay. Furthermore, we describe a technique involving RNase H treatment of total RNA followed by northern analysis in order to distinguish length of poly(A) tails on various types of transcripts. Finally, we outline a useful procedure to clone the poly(A) tails of specific transcripts using 5'-3' end-ligated RNA, which is independent of oligo(dT)-dependent cDNA amplification. These approaches are particularly helpful in analyzing transcripts with either short or long poly(A) tails both in prokaryotes and eukaryotes.

Development of real-time recombinase polymerase amplification assay for rapid and sensitive detection of canine parvovirus 2.

PubMed

Geng, Yunyun; Wang, Jianchang; Liu, Libing; Lu, Yan; Tan, Ke; Chang, Yan-Zhong

2017-11-06

Canine parvovirus 2, a linear single-stranded DNA virus belonging to the genus Parvovirus within the family Parvoviridae, is a highly contagious pathogen of domestic dogs and several wild canidae species. Early detection of canine parvovirus (CPV-2) is crucial to initiating appropriate outbreak control strategies. Recombinase polymerase amplification (RPA), a novel isothermal gene amplification technique, has been developed for the molecular detection of diverse pathogens. In this study, a real-time RPA assay was developed for the detection of CPV-2 using primers and an exo probe targeting the CPV-2 nucleocapsid protein gene. The real-time RPA assay was performed successfully at 38 °C, and the results were obtained within 4-12 min for 10 5 -10 1 molecules of template DNA. The assay only detected CPV-2, and did not show cross-detection of other viral pathogens, demonstrating a high level of specificity. The analytical sensitivity of the real-time RPA was 10 1 copies/reaction of a standard DNA template, which was 10 times more sensitive than the common RPA method. The clinical sensitivity of the real-time RPA assay matched 100% (n = 91) to the real-time PCR results. The real-time RPA assay is a simple, rapid, reliable and affordable method that can potentially be applied for the detection of CPV-2 in the research laboratory and point-of-care diagnosis.

PCR performance of a thermostable heterodimeric archaeal DNA polymerase

PubMed Central

Killelea, Tom; Ralec, Céline; Bossé, Audrey; Henneke, Ghislaine

2014-01-01

DNA polymerases are versatile tools used in numerous important molecular biological core technologies like the ubiquitous polymerase chain reaction (PCR), cDNA cloning, genome sequencing, and nucleic acid based diagnostics. Taking into account the multiple DNA amplification techniques in use, different DNA polymerases must be optimized for each type of application. One of the current tendencies is to reengineer or to discover new DNA polymerases with increased performance and broadened substrate spectra. At present, there is a great demand for such enzymes in applications, e.g., forensics or paleogenomics. Current major limitations hinge on the inability of conventional PCR enzymes, such as Taq, to amplify degraded or low amounts of template DNA. Besides, a wide range of PCR inhibitors can also impede reactions of nucleic acid amplification. Here we looked at the PCR performances of the proof-reading D-type DNA polymerase from P. abyssi, Pab-polD. Fragments, 3 kilobases in length, were specifically PCR-amplified in its optimized reaction buffer. Pab-polD showed not only a greater resistance to high denaturation temperatures than Taq during cycling, but also a superior tolerance to the presence of potential inhibitors. Proficient proof-reading Pab-polD enzyme could also extend a primer containing up to two mismatches at the 3' primer termini. Overall, we found valuable biochemical properties in Pab-polD compared to the conventional Taq, which makes the enzyme ideally suited for cutting-edge PCR-applications. PMID:24847315

Highly efficient nuclear DNA typing of the World War II skeletal remains using three new autosomal short tandem repeat amplification kits with the extended European Standard Set of loci

PubMed Central

Zupanič Pajnič, Irena; Gornjak Pogorelc, Barbara; Balažic, Jože; Zupanc, Tomaž; Štefanič, Borut

2012-01-01

Aim To perform an efficiency study of three new amplification kits with the extended European Standard Set (ESS) of loci for autosomal short tandem repeat (STR) typing of skeletal remains excavated from the World War II mass graves in Slovenia. Methods In the beginning of the 2011, we analyzed 102 bones and teeth using the PowerPlex ESX 17 System (Promega), AmpFiSTR NGM PCR Amplification Kit (Applied Biosystems), and Investigator ESSplex Kit (Qiagen). We cleaned the bones and teeth, removed surface contamination, and ground them into a powder using liquid nitrogen. Prior to DNA isolation with Biorobot EZ1 (Qiagen), 0.5 g bone or tooth powder was decalcified. Nuclear DNA of the samples was quantified using real-time polymerase chain reaction. All three kits used the same extract with the amplification conditions recommended by the manufacturers. Results We extracted up to 131 ng DNA/g of powder from the bones and teeth. All three amplification kits showed very similar efficiency, since DNA typing was successful with all amplification kits in 101 out of 102 bones and teeth, which represents a 99% success rate. Conclusion The commercially available ESX 17, ESSplex, and NGM kits are highly reliable for STR typing of World War II skeletal remains with the DNA extraction method optimized in our laboratory. PMID:22351574

Amplification and chromosomal dispersion of human endogenous retroviral sequences

DOE Office of Scientific and Technical Information (OSTI.GOV)

Steele, P.E.; Martin, M.A.; Rabson, A.B.

1986-09-01

Endogenous retroviral sequences have undergone amplification events involving both viral and flanking cellular sequences. The authors cloned members of an amplified family of full-length endogenous retroviral sequences. Genomic blotting, employing a flanking cellular DNA probe derived from a member of this family, revealed a similar array of reactive bands in both humans and chimpanzees, indicating that an amplification event involving retroviral and associated cellular DNA sequences occurred before the evolutionary separation of these two primates. Southern analyses of restricted somatic cell hybrid DNA preparations suggested that endogenous retroviral segments are widely dispersed in the human genome and that amplification andmore » dispersion events may be linked.« less

Nanopore-CMOS Interfaces for DNA Sequencing

PubMed Central

Magierowski, Sebastian; Huang, Yiyun; Wang, Chengjie; Ghafar-Zadeh, Ebrahim

2016-01-01

DNA sequencers based on nanopore sensors present an opportunity for a significant break from the template-based incumbents of the last forty years. Key advantages ushered by nanopore technology include a simplified chemistry and the ability to interface to CMOS technology. The latter opportunity offers substantial promise for improvement in sequencing speed, size and cost. This paper reviews existing and emerging means of interfacing nanopores to CMOS technology with an emphasis on massively-arrayed structures. It presents this in the context of incumbent DNA sequencing techniques, reviews and quantifies nanopore characteristics and models and presents CMOS circuit methods for the amplification of low-current nanopore signals in such interfaces. PMID:27509529

Nanopore-CMOS Interfaces for DNA Sequencing.

PubMed

Magierowski, Sebastian; Huang, Yiyun; Wang, Chengjie; Ghafar-Zadeh, Ebrahim

2016-08-06

DNA sequencers based on nanopore sensors present an opportunity for a significant break from the template-based incumbents of the last forty years. Key advantages ushered by nanopore technology include a simplified chemistry and the ability to interface to CMOS technology. The latter opportunity offers substantial promise for improvement in sequencing speed, size and cost. This paper reviews existing and emerging means of interfacing nanopores to CMOS technology with an emphasis on massively-arrayed structures. It presents this in the context of incumbent DNA sequencing techniques, reviews and quantifies nanopore characteristics and models and presents CMOS circuit methods for the amplification of low-current nanopore signals in such interfaces.

Plant genotyping using fluorescently tagged inter-simple sequence repeats (ISSRs): basic principles and methodology.

PubMed

Prince, Linda M

2015-01-01

Inter-simple sequence repeat PCR (ISSR-PCR) is a fast, inexpensive genotyping technique based on length variation in the regions between microsatellites. The method requires no species-specific prior knowledge of microsatellite location or composition. Very small amounts of DNA are required, making this method ideal for organisms of conservation concern, or where the quantity of DNA is extremely limited due to organism size. ISSR-PCR can be highly reproducible but requires careful attention to detail. Optimization of DNA extraction, fragment amplification, and normalization of fragment peak heights during fluorescent detection are critical steps to minimizing the downstream time spent verifying and scoring the data.

Rapid screening method for male DNA by using the loop-mediated isothermal amplification assay.

PubMed

Kitamura, Masashi; Kubo, Seiji; Tanaka, Jin; Adachi, Tatsushi

2017-08-12

Screening for male-derived biological material from collected samples plays an important role in criminal investigations, especially those involving sexual assaults. We have developed a loop-mediated isothermal amplification (LAMP) assay targeting multi-repeat sequences of the Y chromosome for detecting male DNA. Successful amplification occurred with 0.5 ng of male DNA under isothermal conditions of 61 to 67 °C, but no amplification occurred with up to 10 ng of female DNA. Under the optimized conditions, the LAMP reaction initiated amplification within 10 min and amplified for 20 min. The LAMP reaction was sensitive at levels as low as 1-pg male DNA, and a quantitative LAMP assay could be developed because of the strong correlation between the reaction time and the amount of template DNA in the range of 10 pg to 10 ng. Furthermore, to apply the LAMP assay to on-site screening for male-derived samples, we evaluated a protocol using a simple DNA extraction method and a colorimetric intercalating dye that allows detection of the LAMP reaction by evaluating the change in color of the solution. Using this protocol, samples of male-derived blood and saliva stains were processed in approximately 30 min from DNA extraction to detection. Because our protocol does not require much hands-on time or special equipment, this LAMP assay promises to become a rapid and simple screening method for male-derived samples in forensic investigations.

Parvovirus B19 is a bystander in adult myocarditis.

PubMed

Koepsell, Scott A; Anderson, Daniel R; Radio, Stanley J

2012-01-01

The genomic DNA of parvovirus B19, a small single-stranded DNA virus of the genus Erythrovirus, has been shown to persist in solid tissues of constitutionally healthy, immunocompetent individuals. Despite these data, many case reports and series have linked the presence of parvovirus B19 genomic DNA, detected through nucleic acid amplification testing, with myocarditis and cardiomyopathy. Herein, we use multiple tools to better assess the relationship between parvovirus B19 and myocarditis and cardiomyopathy. Nucleic acid amplification testing, immunohistochemistry, in situ hybridization, and electron microscopy were used to assess the location and activity of parvovirus B19 in cases of myocarditis and in cases with no significant cardiac disease. Nucleic acid amplification testing for parvovirus B19 genomic DNA was positive in 73% of patients with myocarditis/cardiomyopathy and in 26% of patients with no significant disease. In situ hybridization and immunohistochemistry showed that, in cases with amplifiable parvovirus B19 DNA, parvovirus B19 genomic DNA and viral protein production were present in rare mononuclear cells. In a majority of cases of myocarditis and a significant number of otherwise normal hearts, nucleic acid amplification testing detected persistent parvovirus B19 genomic DNA that did not play a significant pathogenic role. The source of parvovirus B19 DNA appeared to be interstitial mononuclear inflammatory cells and not myocardial or endothelial cells. Therefore, nucleic acid amplification testing alone is not diagnostically helpful for determining the etiology of adult myocarditis. Copyright © 2012 Elsevier Inc. All rights reserved.

Detection and signal amplification in zebrafish RNA FISH.

PubMed

Hauptmann, Giselbert; Lauter, Gilbert; Söll, Iris

2016-04-01

In situ hybridization (ISH) has become an invaluable tool for the detection of RNA in cells, tissues and organisms. Due to improvements in target and signal amplification and in probe design remarkable progress has been made concerning sensitivity, specificity and resolution of chromogenic and fluorescent ISH (FISH). These advancements allow for exquisite cellular and sub-cellular resolution and for detecting multiple RNA species at a time by multiplexing. In zebrafish (F)ISH non-enzymatic and enzymatic amplification systems have been employed to obtain enhanced signal intensities and signal-to-noise ratios. These amplification strategies include branched DNA-based RNAscope and in situ hybridization chain reaction (HCR) techniques, as well as alkaline phosphatase (AP)- and horseradish peroxidase (PO)-based immunoassays. For practical application, we provide proven multiplex FISH protocols for AP- and PO-based visualization of mRNAs at high resolution. The protocols take advantage of optimized tyramide signal amplification (TSA) conditions of the PO assay and long-lasting high signal-to-noise ratio of the AP reaction, thereby enabling detection of less abundant transcripts. Copyright © 2016 Elsevier Inc. All rights reserved.

Internal validation of the GlobalFiler™ Express PCR Amplification Kit for the direct amplification of reference DNA samples on a high-throughput automated workflow.

PubMed

Flores, Shahida; Sun, Jie; King, Jonathan; Budowle, Bruce

2014-05-01

The GlobalFiler™ Express PCR Amplification Kit uses 6-dye fluorescent chemistry to enable multiplexing of 21 autosomal STRs, 1 Y-STR, 1 Y-indel and the sex-determining marker amelogenin. The kit is specifically designed for processing reference DNA samples in a high throughput manner. Validation studies were conducted to assess the performance and define the limitations of this direct amplification kit for typing blood and buccal reference DNA samples on various punchable collection media. Studies included thermal cycling sensitivity, reproducibility, precision, sensitivity of detection, minimum detection threshold, system contamination, stochastic threshold and concordance. Results showed that optimal amplification and injection parameters for a 1.2mm punch from blood and buccal samples were 27 and 28 cycles, respectively, combined with a 12s injection on an ABI 3500xL Genetic Analyzer. Minimum detection thresholds were set at 100 and 120RFUs for 27 and 28 cycles, respectively, and it was suggested that data from positive amplification controls provided a better threshold representation. Stochastic thresholds were set at 250 and 400RFUs for 27 and 28 cycles, respectively, as stochastic effects increased with cycle number. The minimum amount of input DNA resulting in a full profile was 0.5ng, however, the optimum range determined was 2.5-10ng. Profile quality from the GlobalFiler™ Express Kit and the previously validated AmpFlSTR(®) Identifiler(®) Direct Kit was comparable. The validation data support that reliable DNA typing results from reference DNA samples can be obtained using the GlobalFiler™ Express PCR Amplification Kit. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Multiplexed enrichment of rare DNA variants via sequence-selective and temperature-robust amplification

PubMed Central

Wu, Lucia R.; Chen, Sherry X.; Wu, Yalei; Patel, Abhijit A.; Zhang, David Yu

2018-01-01

Rare DNA-sequence variants hold important clinical and biological information, but existing detection techniques are expensive, complex, allele-specific, or don’t allow for significant multiplexing. Here, we report a temperature-robust polymerase-chain-reaction method, which we term blocker displacement amplification (BDA), that selectively amplifies all sequence variants, including single-nucleotide variants (SNVs), within a roughly 20-nucleotide window by 1,000-fold over wild-type sequences. This allows for easy detection and quantitation of hundreds of potential variants originally at ≤0.1% in allele frequency. BDA is compatible with inexpensive thermocycler instrumentation and employs a rationally designed competitive hybridization reaction to achieve comparable enrichment performance across annealing temperatures ranging from 56 °C to 64 °C. To show the sequence generality of BDA, we demonstrate enrichment of 156 SNVs and the reliable detection of single-digit copies. We also show that the BDA detection of rare driver mutations in cell-free DNA samples extracted from the blood plasma of lung-cancer patients is highly consistent with deep sequencing using molecular lineage tags, with a receiver operator characteristic accuracy of 95%. PMID:29805844

DNA recovery from microhymenoptera using six non-destructive methodologies with considerations for subsequent preparation of museum slides.

PubMed

Guzmán-Larralde, Adriana J; Suaste-Dzul, Alba P; Gallou, Adrien; Peña-Carrillo, Kenzy I

2017-01-01

Because of the tiny size of microhymenoptera, successful morphological identification typically requires specific mounting protocols that require time, skills, and experience. Molecular taxonomic identification is an alternative, but many DNA extraction protocols call for maceration of the whole specimen, which is not compatible with preserving museum vouchers. Thus, non-destructive DNA isolation methods are attractive alternatives for obtaining DNA without damaging sample individuals. However, their performance needs to be assessed in microhymenopterans. We evaluated six non-destructive methods: (A) DNeasy® Blood & Tissue Kit; (B) DNeasy® Blood & Tissue Kit, modified; (C) Protocol with CaCl 2 buffer; (D) Protocol with CaCl 2 buffer, modified; (E) HotSHOT; and (F) Direct PCR. The performance of each DNA extraction method was tested across several microhymenopteran species by attempting to amplify the mitochondrial gene COI from insect specimens of varying ages: 1 day, 4 months, 3 years, 12 years, and 23 years. Methods B and D allowed COI amplification in all insects, while methods A, C, and E were successful in DNA amplification from insects up to 12 years old. Method F, the fastest, was useful in insects up to 4 months old. Finally, we adapted permanent slide preparation in Canada balsam for every technique. The results reported allow for combining morphological and molecular methodologies for taxonomic studies.

Binding-induced DNA walker for signal amplification in highly selective electrochemical detection of protein.

PubMed

Ji, Yuhang; Zhang, Lei; Zhu, Longyi; Lei, Jianping; Wu, Jie; Ju, Huangxian

2017-10-15

A binding-induced DNA walker-assisted signal amplification was developed for highly selective electrochemical detection of protein. Firstly, the track of DNA walker was constructed by self-assembly of the high density ferrocene (Fc)-labeled anchor DNA and aptamer 1 on the gold electrode surface. Sequentially, a long swing-arm chain containing aptamer 2 and walking strand DNA was introduced onto gold electrode through aptamers-target specific recognition, and thus initiated walker strand sequences to hybridize with anchor DNA. Then, the DNA walker was activated by the stepwise cleavage of the hybridized anchor DNA by nicking endonuclease to release multiple Fc molecules for signal amplification. Taking thrombin as the model target, the Fc-generated electrochemical signal decreased linearly with logarithm value of thrombin concentration ranging from 10pM to 100nM with a detection limit of 2.5pM under the optimal conditions. By integrating the specific recognition of aptamers to target with the enzymatic cleavage of nicking endonuclease, the aptasensor showed the high selectivity. The binding-induced DNA walker provides a promising strategy for signal amplification in electrochemical biosensor, and has the extensive applications in sensitive and selective detection of the various targets. Copyright © 2017 Elsevier B.V. All rights reserved.

Calibrating genomic and allelic coverage bias in single-cell sequencing.

PubMed

Zhang, Cheng-Zhong; Adalsteinsson, Viktor A; Francis, Joshua; Cornils, Hauke; Jung, Joonil; Maire, Cecile; Ligon, Keith L; Meyerson, Matthew; Love, J Christopher

2015-04-16

Artifacts introduced in whole-genome amplification (WGA) make it difficult to derive accurate genomic information from single-cell genomes and require different analytical strategies from bulk genome analysis. Here, we describe statistical methods to quantitatively assess the amplification bias resulting from whole-genome amplification of single-cell genomic DNA. Analysis of single-cell DNA libraries generated by different technologies revealed universal features of the genome coverage bias predominantly generated at the amplicon level (1-10 kb). The magnitude of coverage bias can be accurately calibrated from low-pass sequencing (∼0.1 × ) to predict the depth-of-coverage yield of single-cell DNA libraries sequenced at arbitrary depths. We further provide a benchmark comparison of single-cell libraries generated by multi-strand displacement amplification (MDA) and multiple annealing and looping-based amplification cycles (MALBAC). Finally, we develop statistical models to calibrate allelic bias in single-cell whole-genome amplification and demonstrate a census-based strategy for efficient and accurate variant detection from low-input biopsy samples.

Calibrating genomic and allelic coverage bias in single-cell sequencing

PubMed Central

Francis, Joshua; Cornils, Hauke; Jung, Joonil; Maire, Cecile; Ligon, Keith L.; Meyerson, Matthew; Love, J. Christopher

2016-01-01

Artifacts introduced in whole-genome amplification (WGA) make it difficult to derive accurate genomic information from single-cell genomes and require different analytical strategies from bulk genome analysis. Here, we describe statistical methods to quantitatively assess the amplification bias resulting from whole-genome amplification of single-cell genomic DNA. Analysis of single-cell DNA libraries generated by different technologies revealed universal features of the genome coverage bias predominantly generated at the amplicon level (1–10 kb). The magnitude of coverage bias can be accurately calibrated from low-pass sequencing (~0.1 ×) to predict the depth-of-coverage yield of single-cell DNA libraries sequenced at arbitrary depths. We further provide a benchmark comparison of single-cell libraries generated by multi-strand displacement amplification (MDA) and multiple annealing and looping-based amplification cycles (MALBAC). Finally, we develop statistical models to calibrate allelic bias in single-cell whole-genome amplification and demonstrate a census-based strategy for efficient and accurate variant detection from low-input biopsy samples. PMID:25879913

A novel electrochemical biosensor for ultrasensitive and specific detection of DNA based on molecular beacon mediated circular strand displacement and rolling circle amplification.

PubMed

Cheng, Wei; Zhang, Wei; Yan, Yurong; Shen, Bo; Zhu, Dan; Lei, Pinhua; Ding, Shijia

2014-12-15

A novel electrochemical biosensing strategy was developed for ultrasensitive and specific detection of target DNA using a cascade signal amplification based on molecular beacon (MB) mediated circular strand displacement (CSD), rolling circle amplification (RCA), biotin-strepavidin system, and enzymatic amplification. The target DNA hybridized with the loop portion of MB probe immobilized on the gold electrode and triggered the CSD, leading to multiple biotin-tagged DNA duplex. Furthermore, via biotin-streptavidin interaction, the RCA was implemented, producing long massive tandem-repeat DNA sequences for binding numerous biotinylated detection probes. This enabled an ultrasensitive electrochemical readout by further employing the streptavidin-alkaline phosphatase. The proposed biosensor showed very high sensitivity and selectivity with a dynamic response range from 1 fM to 100 pM. The proposed strategy could have the potential for applying in clinical molecular diagnostics and environmental monitoring. Copyright © 2014 Elsevier B.V. All rights reserved.

'Mitominis': multiplex PCR analysis of reduced size amplicons for compound sequence analysis of the entire mtDNA control region in highly degraded samples.

PubMed

Eichmann, Cordula; Parson, Walther

2008-09-01

The traditional protocol for forensic mitochondrial DNA (mtDNA) analyses involves the amplification and sequencing of the two hypervariable segments HVS-I and HVS-II of the mtDNA control region. The primers usually span fragment sizes of 300-400 bp each region, which may result in weak or failed amplification in highly degraded samples. Here we introduce an improved and more stable approach using shortened amplicons in the fragment range between 144 and 237 bp. Ten such amplicons were required to produce overlapping fragments that cover the entire human mtDNA control region. These were co-amplified in two multiplex polymerase chain reactions and sequenced with the individual amplification primers. The primers were carefully selected to minimize binding on homoplasic and haplogroup-specific sites that would otherwise result in loss of amplification due to mis-priming. The multiplexes have successfully been applied to ancient and forensic samples such as bones and teeth that showed a high degree of degradation.

Clustered Regularly Interspaced Short Palindromic Repeats/Cas9 Triggered Isothermal Amplification for Site-Specific Nucleic Acid Detection.

PubMed

Huang, Mengqi; Zhou, Xiaoming; Wang, Huiying; Xing, Da

2018-02-06

A novel CRISPR/Cas9 triggered isothermal exponential amplification reaction (CAS-EXPAR) strategy based on CRISPR/Cas9 cleavage and nicking endonuclease (NEase) mediated nucleic acids amplification was developed for rapid and site-specific nucleic acid detection. CAS-EXPAR was primed by the target DNA fragment produced by cleavage of CRISPR/Cas9, and the amplification reaction performed cyclically to generate a large number of DNA replicates which were detected using a real-time fluorescence monitoring method. This strategy that combines the advantages of CRISPR/Cas9 and exponential amplification showed high specificity as well as rapid amplification kinetics. Unlike conventional nucleic acids amplification reactions, CAS-EXPAR does not require exogenous primers, which often cause target-independent amplification. Instead, primers are first generated by Cas9/sgRNA directed site-specific cleavage of target and accumulated during the reaction. It was demonstrated this strategy gave a detection limit of 0.82 amol and showed excellent specificity in discriminating single-base mismatch. Moreover, the applicability of this method to detect DNA methylation and L. monocytogenes total RNA was also verified. Therefore, CAS-EXPAR may provide a new paradigm for efficient nucleic acid amplification and hold the potential for molecular diagnostic applications.

Development of a Rapid Point-of-Use DNA Test for the Screening of Genuity® Roundup Ready 2 Yield® Soybean in Seed Samples

PubMed Central

Chandu, Dilip; Paul, Sudakshina; Parker, Mathew; Dudin, Yelena; King-Sitzes, Jennifer; Perez, Tim; Mittanck, Don W.; Shah, Manali; Glenn, Kevin C.; Piepenburg, Olaf

2016-01-01

Testing for the presence of genetically modified material in seed samples is of critical importance for all stakeholders in the agricultural industry, including growers, seed manufacturers, and regulatory bodies. While rapid antibody-based testing for the transgenic protein has fulfilled this need in the past, the introduction of new variants of a given transgene demands new diagnostic regimen that allows distinguishing different traits at the nucleic acid level. Although such molecular tests can be performed by PCR in the laboratory, their requirement for expensive equipment and sophisticated operation have prevented its uptake in point-of-use applications. A recently developed isothermal DNA amplification technique, recombinase polymerase amplification (RPA), combines simple sample preparation and amplification work-flow procedures with the use of minimal detection equipment in real time. Here, we report the development of a highly sensitive and specific RPA-based detection system for Genuity Roundup Ready 2 Yield (RR2Y) material in soybean (Glycine max) seed samples and present the results of studies applying the method in both laboratory and field-type settings. PMID:27314015

Detection of the free-living forms of sulfide-oxidizing gill endosymbionts in the lucinid habitat (Thalassia testudinum environment).

PubMed

Gros, Olivier; Liberge, Martine; Heddi, Abdelaziz; Khatchadourian, Chaqué; Felbeck, Horst

2003-10-01

Target DNA from the uncultivable Codakia orbicularis endosymbiont was PCR amplified from sea-grass sediment. To confirm that such amplifications originated from intact bacterial cells rather than free DNA, whole-cell hybridization (fluorescence in situ hybridization technique) with the specific probe Symco2 was performed along with experimental infection of aposymbiotic juveniles placed in contact with the same sediment. Taken together, the data demonstrate that the sulfide-oxidizing gill endosymbiont of Codakia orbicularis is present in the environment as a free-living uncultivable form.

Nuclease-resistant double-stranded DNA controls or standards for hepatitis B virus nucleic acid amplification assays

PubMed Central

2009-01-01

Background Identical blood samples tested using different kits can give markedly different hepatitis B virus (HBV) DNA levels, which can cause difficulty in the interpretation of viral load. A universal double-stranded DNA control or standard that can be used in all commercial HBV DNA nucleic acid amplification assay kits is urgently needed. By aligning all HBV genotypes (A-H), we found that the surface antigen gene and precore-core gene regions of HBV are the most conserved regions among the different HBV genotypes. We constructed a chimeric fragment by overlapping extension polymerase chain reaction and obtained a 1,349-bp HBVC+S fragment. We then packaged the fragment into lambda phages using a traditional lambda phage cloning procedure. Results The obtained armored DNA was resistant to DNase I digestion and was stable, noninfectious to humans, and could be easily extracted using commercial kits. More importantly, the armored DNA may be used with all HBV DNA nucleic acid amplification assay kits. Conclusions The lambda phage packaging system can be used as an excellent expression platform for armored DNA. The obtained armored DNA possessed all characteristics of an excellent positive control or standard. In addition, this armored DNA is likely to be appropriate for all commercial HBV DNA nucleic acid amplification detection kits. Thus, the constructed armored DNA can probably be used as a universal positive control or standard in HBV DNA assays. PMID:20025781

DNA polymerase preference determines PCR priming efficiency.

PubMed

Pan, Wenjing; Byrne-Steele, Miranda; Wang, Chunlin; Lu, Stanley; Clemmons, Scott; Zahorchak, Robert J; Han, Jian

2014-01-30

Polymerase chain reaction (PCR) is one of the most important developments in modern biotechnology. However, PCR is known to introduce biases, especially during multiplex reactions. Recent studies have implicated the DNA polymerase as the primary source of bias, particularly initiation of polymerization on the template strand. In our study, amplification from a synthetic library containing a 12 nucleotide random portion was used to provide an in-depth characterization of DNA polymerase priming bias. The synthetic library was amplified with three commercially available DNA polymerases using an anchored primer with a random 3' hexamer end. After normalization, the next generation sequencing (NGS) results of the amplified libraries were directly compared to the unamplified synthetic library. Here, high throughput sequencing was used to systematically demonstrate and characterize DNA polymerase priming bias. We demonstrate that certain sequence motifs are preferred over others as primers where the six nucleotide sequences at the 3' end of the primer, as well as the sequences four base pairs downstream of the priming site, may influence priming efficiencies. DNA polymerases in the same family from two different commercial vendors prefer similar motifs, while another commercially available enzyme from a different DNA polymerase family prefers different motifs. Furthermore, the preferred priming motifs are GC-rich. The DNA polymerase preference for certain sequence motifs was verified by amplification from single-primer templates. We incorporated the observed DNA polymerase preference into a primer-design program that guides the placement of the primer to an optimal location on the template. DNA polymerase priming bias was characterized using a synthetic library amplification system and NGS. The characterization of DNA polymerase priming bias was then utilized to guide the primer-design process and demonstrate varying amplification efficiencies among three commercially available DNA polymerases. The results suggest that the interaction of the DNA polymerase with the primer:template junction during the initiation of DNA polymerization is very important in terms of overall amplification bias and has broader implications for both the primer design process and multiplex PCR.

Application of heteroduplex analysis for detecting variation within the growth hormone 2 gene in Salmo trutta L. (brown trout).

PubMed

Gross, R; Nilsson, J

1995-03-01

A new method to detect variation at a single copy nuclear gene in brown trout, Salmo trutta L., is provided. The technique entails (i) selective gene amplification by the polymerase chain reaction (PCR), (ii) digestion of amplification products by restriction endonucleases to obtain fragments of suitable size, (iii) hybridization with heterologous DNA followed by denaturation and reannealing to obtain heteroduplex molecules, and (iv) screening for variation in polyacrylamide gels. Variation was studied within a growth hormone 2 gene 1489 bp segment and polymorphism was detected in two HinfI-digested fragments. Formation of different heteroduplex patterns in experimental mixtures of digested amplification products from brown trout and Atlantic salmon, Salmo salar L., allowed us to determine the genotype of the brown trout. Polymorphism was observed in four out of six studied populations.

Pre-amplification in the context of high-throughput qPCR gene expression experiment.

PubMed

Korenková, Vlasta; Scott, Justin; Novosadová, Vendula; Jindřichová, Marie; Langerová, Lucie; Švec, David; Šídová, Monika; Sjöback, Robert

2015-03-11

With the introduction of the first high-throughput qPCR instrument on the market it became possible to perform thousands of reactions in a single run compared to the previous hundreds. In the high-throughput reaction, only limited volumes of highly concentrated cDNA or DNA samples can be added. This necessity can be solved by pre-amplification, which became a part of the high-throughput experimental workflow. Here, we focused our attention on the limits of the specific target pre-amplification reaction and propose the optimal, general setup for gene expression experiment using BioMark instrument (Fluidigm). For evaluating different pre-amplification factors following conditions were combined: four human blood samples from healthy donors and five transcripts having high to low expression levels; each cDNA sample was pre-amplified at four cycles (15, 18, 21, and 24) and five concentrations (equivalent to 0.078 ng, 0.32 ng, 1.25 ng, 5 ng, and 20 ng of total RNA). Factors identified as critical for a success of cDNA pre-amplification were cycle of pre-amplification, total RNA concentration, and type of gene. The selected pre-amplification reactions were further tested for optimal Cq distribution in a BioMark Array. The following concentrations combined with pre-amplification cycles were optimal for good quality samples: 20 ng of total RNA with 15 cycles of pre-amplification, 20x and 40x diluted; and 5 ng and 20 ng of total RNA with 18 cycles of pre-amplification, both 20x and 40x diluted. We set up upper limits for the bulk gene expression experiment using gene expression Dynamic Array and provided an easy-to-obtain tool for measuring of pre-amplification success. We also showed that variability of the pre-amplification, introduced into the experimental workflow of reverse transcription-qPCR, is lower than variability caused by the reverse transcription step.

Ultrasensitive photoelectrochemical biosensor for the detection of HTLV-I DNA: A cascade signal amplification strategy integrating λ-exonuclease aided target recycling with hybridization chain reaction and enzyme catalysis.

PubMed

Shi, Xiao-Mei; Fan, Gao-Chao; Tang, Xueying; Shen, Qingming; Zhu, Jun-Jie

2018-06-30

Sensitive and specific detection of DNA is of great significance for clinical diagnosis. In this paper, an effective cascade signal amplification strategy was introduced into photoelectrochemical (PEC) biosensor for ultrasensitive detection of human T-cell lymphotropic virus type I (HTLV-I) DNA. This proposed signal amplification strategy integrates λ-exonuclease (λ-Exo) aided target recycling with hybridization chain reaction (HCR) and enzyme catalysis. In the presence of target DNA (tDNA) of HTLV-I, the designed hairpin DNA (h 1 DNA) hybridized with tDNA, subsequently recognized and cleaved by λ-Exo to set free tDNA. With the λ-Exo aided tDNA recycling, an increasing number of DNA fragments (output DNA, oDNA) were released from the digestion of h 1 DNA. Then, triggered by the hybridization of oDNA with capture DNA (cDNA), numerous biotin-labeled hairpin DNAs (h 2 DNA and h 3 DNA) could be loaded onto the photoelectrode via the HCR. Finally, avidin-labeled alkaline phosphatase (avidin-ALP) could be introduced onto the electrode by specific interaction between biotin and avidin. The ALP could catalyze dephosphorylation of phospho-L-ascorbic acid trisodium salt (AAP) to generate an efficient electron donor of ascorbic acid (AA), and thereby greatly increasing the photocurrent signal. By utilizing the proposed cascade signal amplification strategy, the fabricated PEC biosensor exhibited an ultrasensitive and specific detection of HTLV-I DNA down to 11.3 aM, and it also offered an effective strategy to detect other DNAs at ultralow levels. Copyright © 2018 Elsevier B.V. All rights reserved.

Methylation-Sensitive Amplification Length Polymorphism (MS-AFLP) Microarrays for Epigenetic Analysis of Human Genomes.

PubMed

Alonso, Sergio; Suzuki, Koichi; Yamamoto, Fumiichiro; Perucho, Manuel

2018-01-01

Somatic, and in a minor scale also germ line, epigenetic aberrations are fundamental to carcinogenesis, cancer progression, and tumor phenotype. DNA methylation is the most extensively studied and arguably the best understood epigenetic mechanisms that become altered in cancer. Both somatic loss of methylation (hypomethylation) and gain of methylation (hypermethylation) are found in the genome of malignant cells. In general, the cancer cell epigenome is globally hypomethylated, while some regions-typically gene-associated CpG islands-become hypermethylated. Given the profound impact that DNA methylation exerts on the transcriptional profile and genomic stability of cancer cells, its characterization is essential to fully understand the complexity of cancer biology, improve tumor classification, and ultimately advance cancer patient management and treatment. A plethora of methods have been devised to analyze and quantify DNA methylation alterations. Several of the early-developed methods relied on the use of methylation-sensitive restriction enzymes, whose activity depends on the methylation status of their recognition sequences. Among these techniques, methylation-sensitive amplification length polymorphism (MS-AFLP) was developed in the early 2000s, and successfully adapted from its original gel electrophoresis fingerprinting format to a microarray format that notably increased its throughput and allowed the quantification of the methylation changes. This array-based platform interrogates over 9500 independent loci putatively amplified by the MS-AFLP technique, corresponding to the NotI sites mapped throughout the human genome.

Whole Genome Amplification of Day 3 or Day 5 Human Embryos Biopsies Provides a Suitable DNA Template for PCR-Based Techniques for Genotyping, a Complement of Preimplantation Genetic Testing.

PubMed

Schaeffer, Elizabeth; López-Bayghen, Bruno; Neumann, Adina; Porchia, Leonardo M; Camacho, Rafael; Garrido, Efraín; Gómez, Rocío; Camargo, Felipe; López-Bayghen, Esther

2017-01-01

Our objective was to determine if whole genome amplification (WGA) provides suitable DNA for qPCR-based genotyping for human embryos. Single blastomeres (Day 3) or trophoblastic cells (Day 5) were isolated from 342 embryos for WGA. Comparative Genomic Hybridization determined embryo sex as well as Trisomy 18 or Trisomy 21. To determine the embryo's sex, qPCR melting curve analysis for SRY and DYS14 was used. Logistic regression indicated a 4.4%, 57.1%, or 98.8% probability of a male embryo when neither gene, SRY only, or both genes were detected, respectively (accuracy = 94.1%, kappa = 0.882, and p < 0.001). Fluorescent Capillary Electrophoresis for the amelogenin genes (AMEL) was also used to determine sex. AMELY peak's height was higher and this peak's presence was highly predictive of male embryos (AUC = 0.93, accuracy = 81.7%, kappa = 0.974, and p < 0.001). Trisomy 18 and Trisomy 21 were determined using the threshold cycle difference for RPL17 and TTC3, respectively, which were significantly lower in the corresponding embryos. The Ct difference for TTC3 specifically determined Trisomy 21 (AUC = 0.89) and RPL17 for Trisomy 18 (AUC = 0.94). Here, WGA provides adequate DNA for PCR-based techniques for preimplantation genotyping.

The Development and Use of Internal Amplification Controls (IACs) with DNA Profiling Kits for Forensic DNA Analysis.

PubMed

Zahra, Nathalie; Goodwin, William

2016-01-01

Biological samples recovered for forensic investigations are often degraded and/or have low amounts of DNA; in addition, in some instances the samples may be contaminated with chemicals that can act as PCR inhibitors. As a consequence this can make interpretation of the results challenging with the possibility of having partial profiles and false negative results. Because of the impact of DNA analysis on forensic investigations, it is important to monitor the process of DNA profiling, in particular the amplification reaction. In this chapter we describe a method for the in-house generation and use of internal amplification controls (IACs) with DNA profiling kits to monitor the success of the PCR proces. In the example we show the use of the SGM Plus® kit. These controls can also be used to aid the interpretation of the DNA profile.

A paper and plastic device for the combined isothermal amplification and lateral flow detection of Plasmodium DNA.

PubMed

Cordray, Michael S; Richards-Kortum, Rebecca R

2015-11-26

Isothermal amplification techniques are emerging as a promising method for malaria diagnosis since they are capable of detecting extremely low concentrations of parasite target while mitigating the need for infrastructure and training required by other nucleic acid based tests. Recombinase polymerase amplification (RPA) is promising for further development since it operates in a short time frame (<30 min) and produces a product that can be visually detected on a lateral flow dipstick. A self-sealing paper and plastic system that performs both the amplification and detection of a malaria DNA sequence is presented. Primers were designed using the NCBI nBLAST tools and screened using gel electrophoresis. Paper and plastic devices were prototyped using commercial design software and parts were cut using a laser cutter and assembled by hand. Synthetic copies of the Plasmodium 18S gene were spiked into solution and used as targets for the RPA reaction. To test the performance of the device the same samples spiked with synthetic target were run in parallel both in the paper and plastic devices and using conventional bench top methods. Novel RPA primers were developed that bind to sequences present in the four species of Plasmodium which infect humans. The paper and plastic devices were found to be capable of detecting as few as 5 copies/µL of synthetic Plasmodium DNA (50 copies total), comparable to the same reaction run on the bench top. The devices produce visual results in an hour, cost approximately $1, and are self-contained once the device is sealed. The device was capable of carrying out the RPA reaction and detecting meaningful amounts of synthetic Plasmodium DNA in a self-sealing and self-contained device. This device may be a step towards making nucleic acid tests more accessible for malaria detection.

Ancient pathogens in museal dry bone specimens: analysis of paleocytology and aDNA.

PubMed

Gaul, Johanna Sophia; Winter, Eduard; Grossschmidt, Karl

2015-04-01

Bone samples investigated in this study derive from the pathologic-anatomical collection of the Natural History Museum of Vienna. In order to explore the survival of treponemes and treponemal ancient DNA in museal dry bone specimens, we analyzed three individuals known to have been infected with Treponema pallidum pallidum. No reproducible evidence of surviving pathogen's ancient DNA (aDNA) was obtained, despite the highly sensitive extraction and amplification techniques (TPP15 and arp). Additionally, decalcification fluid of bone sections was smear stained with May-Gruenwald-Giemsa. The slides were examined using direct light microscope and dark field illumination. Remnants of spirochetal structures were detectable in every smear. Our results demonstrate that aDNA is unlikely to survive, but spirochetal remains are stainable and thus detectable.

Novel approach to quantitative polymerase chain reaction using real-time detection: application to the detection of gene amplification in breast cancer.

PubMed

Bièche, I; Olivi, M; Champème, M H; Vidaud, D; Lidereau, R; Vidaud, M

1998-11-23

Gene amplification is a common event in the progression of human cancers, and amplified oncogenes have been shown to have diagnostic, prognostic and therapeutic relevance. A kinetic quantitative polymerase-chain-reaction (PCR) method, based on fluorescent TaqMan methodology and a new instrument (ABI Prism 7700 Sequence Detection System) capable of measuring fluorescence in real-time, was used to quantify gene amplification in tumor DNA. Reactions are characterized by the point during cycling when PCR amplification is still in the exponential phase, rather than the amount of PCR product accumulated after a fixed number of cycles. None of the reaction components is limited during the exponential phase, meaning that values are highly reproducible in reactions starting with the same copy number. This greatly improves the precision of DNA quantification. Moreover, real-time PCR does not require post-PCR sample handling, thereby preventing potential PCR-product carry-over contamination; it possesses a wide dynamic range of quantification and results in much faster and higher sample throughput. The real-time PCR method, was used to develop and validate a simple and rapid assay for the detection and quantification of the 3 most frequently amplified genes (myc, ccndl and erbB2) in breast tumors. Extra copies of myc, ccndl and erbB2 were observed in 10, 23 and 15%, respectively, of 108 breast-tumor DNA; the largest observed numbers of gene copies were 4.6, 18.6 and 15.1, respectively. These results correlated well with those of Southern blotting. The use of this new semi-automated technique will make molecular analysis of human cancers simpler and more reliable, and should find broad applications in clinical and research settings.

Dispersion quality of amine functionalized multiwall carbon nanotubes plays critical roles in polymerase chain reaction enhancement

NASA Astrophysics Data System (ADS)

Yuce, Meral; Budak, Hikmet

2014-12-01

Impact of dispersion quality of NH2-MWCNTs (13-18 nm in diameter with a length between 1 and 12 µm, >99 % purity) in the amplification efficiency of a random DNA oligonucleotide library (96 bp) was investigated. Amplification yield in the presence of non-filtered NH2-MWCNT dispersion, filtered NH2-MWCNT dispersion and surface-attached NH2-MWCNTs was explored, and physical interactions between NH2-MWCNTs and major PCR reagents including DNA template, wild type Taq DNA polymerase enzyme and primers were determined using high resolution polyacrylamide gel electrophoresis, dynamic light scattering, UV-Vis-NIR spectroscopy and scanning electron microscopy techniques. The results revealed that presence of NH2-MWCNT dispersion which was sonicated, centrifuged and filtered, enhanced the total PCR efficiency up to 70 % while the presence of NH2-MWCNT only centrifuged after sonication, inhibited the reaction significantly at similar concentrations. Furthermore, the NH2-MWCNTs coupled covalently onto magnetic microspheres, contributed for the specificity enhancement whilst decreasing the amplification efficiency by 30 % at the maximum concentration, which suggests a removable enhancement system for sensitive applications. On the other hand, the relative hydrodynamic size distribution measurements displayed a clear difference between the filtered NH2 and non-filtered NH2-MWCNT water dispersions, which justifies the inhibition of the amplification by the non-filtered NH2-MWCNTs containing big agglomerates and bundles. Finally, we demonstrated that major PCR components adsorb onto the NH2-MWCNTs with diverse affinities, and maintain their functions after adsorption, which provides a good framework to further develop tunable NH2-MWCNT-carriers to be utilized in various nanobiotechnology and material science applications.

A DNA logic gate based on strand displacement reaction and rolling circle amplification, responding to multiple low-abundance DNA fragment input signals, and its application in detecting miRNAs.

PubMed

Chen, Yuqi; Song, Yanyan; Wu, Fan; Liu, Wenting; Fu, Boshi; Feng, Bingkun; Zhou, Xiang

2015-04-25

A conveniently amplified DNA AND logic gate platform was designed for the highly sensitive detection of low-abundance DNA fragment inputs based on strand displacement reaction and rolling circle amplification strategy. Compared with others, this system can detect miRNAs in biological samples. The success of this strategy demonstrates the potential of DNA logic gates in disease diagnosis.

[PCR-based evaluation of sequence specificity of DNA fragmentation by ultrasound].

PubMed

Garafutdinov, R R; Galimova, A A; Sakhabutdinova, A R; Chemeris, A V

2016-01-01

Ultrasonic fragmentation, which is a simple and convenient method for the mechanical degradation of DNA, is widely used in modern genome studies as one of the sample preparation steps. It has been recently found that the DNA breaks occur more often in the regions containing 5'-CG-3' dinucleotides. We studied the influence of the 5'-CG-3' dinucleotides on the efficiency of the 28S rRNA gene amplification during PCR with sonicated DNA of Mantis religiosa. It was shown that the amplification rate depends on the template length and the number of 5'-CG-3' dinucleotides. Amplification of the DNA regions with a higher 5'-CG-3' density is less efficient because of their higher sensitivity to ultrasound. The amount of the amplified DNA templates is inversely proportional to the 5'-CG-3'number.

Identification of Prostate Cancer-Specific microDNAs

DTIC Science & Technology

2016-02-01

circular DNA by rolling circle amplification (RCA) and then amplified DNA fragments were subject to deep sequencing. Deep sequencing of the...demonstrate the existence of microDNAs in prostate cancer. We adopted multiple displacement amplification (MDA) with random 2 primers for enriched...prostate cancer cells through multiple displacement amplification and next generation sequencing. R e la ti v e c e ll g ro w th ( % ) 0 20

Development and Application of a Loop-Mediated Isothermal Amplification (LAMP) Approach for the Rapid Detection of Dirofilaria repens from Biological Samples

PubMed Central

Raele, Donato Antonio; Pugliese, Nicola; Galante, Domenico; Latorre, Laura Maria; Cafiero, Maria Assunta

2016-01-01

Dirofilariasis by Dirofilaria repens is an important mosquito vector borne parasitosis, and the dog represents the natural host and reservoir of the parasite. This filarial nematode can also induce disease in humans, and in the last decades an increasing number of cases have been being reported. The present study describes the first loop mediated isothermal amplification (LAMP) assay to detect D. repens DNA in blood and mosquitoes. Two versions of the technique have been developed and described: in the first, the amplification is followed point by point through a real time PCR instrument (ReT-LAMP); in the second, the amplification is visualized by checking UV fluorescence of the reaction mixture after addition of propidium iodide (PI-LAMP). The two variants use the same set of 4 primers targeting the D. repens cytochrome oxidase subunit I (COI) gene. To assess the specificity of the method, reactions were carried out by using DNA from the major zoonotic parasites of the family of Onchocercidae, and no amplification was observed. The lower limit of detection of the ReT-LAMP assay was 0.15 fg/μl (corresponding to about 50 copy of COI gene per μl). Results suggest that the described assay is specific, and its sensitivity is higher than the conventional PCR based on the same gene. It is also provide a rapid and cost-effective molecular detection of D. repens, mainly when PI-LAMP is applied, and it should be performed in areas where this emerging parasitosis is endemic. PMID:27341205

Sex and PRNP genotype determination in preimplantation caprine embryos.

PubMed

Guignot, F; Perreau, C; Cavarroc, C; Touzé, J-L; Pougnard, J-L; Dupont, F; Beckers, J-F; Rémy, B; Babilliot, J-M; Bed'Hom, B; Lamorinière, J M; Mermillod, P; Baril, G

2011-08-01

The objective of this study was to test the accuracy of genotype diagnosis after whole amplification of DNA extracted from biopsies obtained by trimming goat embryos and to evaluate the viability of biopsied embryos after vitrification/warming and transfer. Whole genome amplification (WGA) was performed using Multiple Displacement Amplification (MDA). Sex and prion protein (PRNP) genotypes were determined. Sex diagnosis was carried out by PCR amplification of ZFX/ZFY and Y chromosome-specific sequences. Prion protein genotype determination was performed on codons 142, 154, 211, 222 and 240. Embryos were collected at day 7 after oestrus and biopsied either immediately after collection (blastocysts and expanded blastocysts) or after 24 h of in vitro culture (compacted morulae). Biopsied embryos were frozen by vitrification. Vitrified whole embryos were kept as control. DNA of biopsies was extracted and amplified using MDA. Sex diagnosis was efficient for 97.4% of biopsies and PRNP genotyping was determined in 78.7% of biopsies. After embryo transfer, no significant difference was observed in kidding rate between biopsied and vitrified control embryos, whereas embryo survival rate was different between biopsied and whole vitrified embryos (p = 0.032). At birth, 100% of diagnosed sex and 98.2% of predetermined codons were correct. Offspring PRNP profiles were in agreement with parental genotype. Whole genome amplification with MDA kit coupled with sex diagnosis and PRNP genotype predetermination are very accurate techniques to genotype goat embryos before transfer. These novel results allow us to plan selection of scrapie-resistant genotypes and kid sex before transfer of cryopreserved embryo. © 2010 Blackwell Verlag GmbH.

Isothermal circular-strand-displacement polymerization of DNA and microRNA in digital microfluidic devices.

PubMed

Giuffrida, Maria Chiara; Zanoli, Laura Maria; D'Agata, Roberta; Finotti, Alessia; Gambari, Roberto; Spoto, Giuseppe

2015-02-01

Nucleic-acid amplification is a crucial step in nucleic-acid-sequence-detection assays. The use of digital microfluidic devices to miniaturize amplification techniques reduces the required sample volume and the analysis time and offers new possibilities for process automation and integration in a single device. The recently introduced droplet polymerase-chain-reaction (PCR) amplification methods require repeated cycles of two or three temperature-dependent steps during the amplification of the nucleic-acid target sequence. In contrast, low-temperature isothermal-amplification methods have no need for thermal cycling, thus requiring simplified microfluidic-device features. Here, the combined use of digital microfluidics and molecular-beacon (MB)-assisted isothermal circular-strand-displacement polymerization (ICSDP) to detect microRNA-210 sequences is described. MicroRNA-210 has been described as the most consistently and predominantly upregulated hypoxia-inducible factor. The nmol L(-1)-pmol L(-1) detection capabilities of the method were first tested by targeting single-stranded DNA sequences from the genetically modified Roundup Ready soybean. The ability of the droplet-ICSDP method to discriminate between full-matched, single-mismatched, and unrelated sequences was also investigated. The detection of a range of nmol L(-1)-pmol L(-1) microRNA-210 solutions compartmentalized in nanoliter-sized droplets was performed, establishing the ability of the method to detect as little as 10(-18) mol of microRNA target sequences compartmentalized in 20 nL droplets. The suitability of the method for biological samples was tested by detecting microRNA-210 from transfected K562 cells.

[Do Multiplex PCR techniques displace classical cultures in microbiology?].

PubMed

Auckenthaler, Raymond; Risch, Martin

2015-02-01

Multiplex PCR technologies progressively find their way in clinical microbiology. This technique allows the simultaneous amplification of multiple DNA targets in a single test run for the identification of pathogens up to the species level. Various pathogens of infectious diseases can be detected by a symptom-oriented approach clearly and quickly with high reliability. Essentially multiplex PCR panels are available for clarification of gastrointestinal, respiratory, sexually transmitted infections and meningitis. Today's offer from industry, university hospitals and large private laboratories of Switzerland is tabulated and commented.

Optimization of ultrahigh-speed multiplex PCR for forensic analysis.

PubMed

Gibson-Daw, Georgiana; Crenshaw, Karin; McCord, Bruce

2018-01-01

In this paper, we demonstrate the design and optimization of an ultrafast PCR amplification technique, used with a seven-locus multiplex that is compatible with conventional capillary electrophoresis systems as well as newer microfluidic chip devices. The procedure involves the use of a high-speed polymerase and a rapid cycling protocol to permit multiplex PCR amplification of forensic short tandem repeat loci in 6.5 min. We describe the selection and optimization of master mix reagents such as enzyme, buffer, MgCl 2 , and dNTPs, as well as primer ratios, total volume, and cycle conditions, in order to get the best profile in the shortest time possible. Sensitivity and reproducibility studies are also described. The amplification process utilizes a small high-speed thermocycler and compact laptop, making it portable and potentially useful for rapid, inexpensive on-site genotyping. The seven loci of the multiplex were taken from conventional STR genotyping kits and selected for their size and lack of overlap. Analysis was performed using conventional capillary electrophoresis and microfluidics with fluorescent detection. Overall, this technique provides a more rapid method for rapid sample screening of suspects and victims. Graphical abstract Rapid amplification of forensic DNA using high speed thermal cycling followed by capillary or microfluidic electrophoresis.

GMO detection in food and feed through screening by visual loop-mediated isothermal amplification assays.

PubMed

Wang, Cong; Li, Rong; Quan, Sheng; Shen, Ping; Zhang, Dabing; Shi, Jianxin; Yang, Litao

2015-06-01

Isothermal DNA/RNA amplification techniques are the primary methodology for developing on-spot rapid nucleic acid amplification assays, and the loop-mediated isothermal amplification (LAMP) technique has been developed and applied in the detection of foodborne pathogens, plant/animal viruses, and genetically modified (GM) food/feed contents. In this study, one set of LAMP assays targeting on eight frequently used universal elements, marker genes, and exogenous target genes, such as CaMV35S promoter, FMV35S promoter, NOS, bar, cry1Ac, CP4 epsps, pat, and NptII, were developed for visual screening of GM contents in plant-derived food samples with high efficiency and accuracy. For these eight LAMP assays, their specificity was evaluated by testing commercial GM plant events and their limits of detection were also determined, which are 10 haploid genome equivalents (HGE) for FMV35S promoter, cry1Ac, and pat assays, as well as five HGE for CaMV35S promoter, bar, NOS terminator, CP4 epsps, and NptII assays. The screening applicability of these LAMP assays was further validated successfully using practical canola, soybean, and maize samples. The results suggested that the established visual LAMP assays are applicable and cost-effective for GM screening in plant-derived food samples.

Single-tube analysis of DNA methylation with silica superparamagnetic beads.

PubMed

Bailey, Vasudev J; Zhang, Yi; Keeley, Brian P; Yin, Chao; Pelosky, Kristen L; Brock, Malcolm; Baylin, Stephen B; Herman, James G; Wang, Tza-Huei

2010-06-01

DNA promoter methylation is a signature for the silencing of tumor suppressor genes. Most widely used methods to detect DNA methylation involve 3 separate, independent processes: DNA extraction, bisulfite conversion, and methylation detection via a PCR method, such as methylation-specific PCR (MSP). This method includes many disconnected steps with associated losses of material, potentially reducing the analytical sensitivity required for analysis of challenging clinical samples. Methylation on beads (MOB) is a new technique that integrates DNA extraction, bisulfite conversion, and PCR in a single tube via the use of silica superparamagnetic beads (SSBs) as a common DNA carrier for facilitating cell debris removal and buffer exchange throughout the entire process. In addition, PCR buffer is used to directly elute bisulfite-treated DNA from SSBs for subsequent target amplifications. The diagnostic sensitivity of MOB was evaluated by methylation analysis of the CDKN2A [cyclin-dependent kinase inhibitor 2A (melanoma, p16, inhibits CDK4); also known as p16(INK4a)] promoter in serum DNA of lung cancer patients and compared with that of conventional methods. Methylation analysis consisting of DNA extraction followed by bisulfite conversion and MSP was successfully carried out within 9 h in a single tube. The median pre-PCR DNA yield was 6.61-fold higher with the MOB technique than with conventional techniques. Furthermore, MOB increased the diagnostic sensitivity in our analysis of the CDKN2A promoter in patient serum by successfully detecting methylation in 74% of cancer patients, vs the 45% detection rate obtained with conventional techniques. The MOB technique successfully combined 3 processes into a single tube, thereby allowing ease in handling and an increased detection throughput. The increased pre-PCR yield in MOB allowed efficient, diagnostically sensitive methylation detection.

Microbial Degradation of Forensic Samples of Biological Origin: Potential Threat to Human DNA Typing.

PubMed

Dash, Hirak Ranjan; Das, Surajit

2018-02-01

Forensic biology is a sub-discipline of biological science with an amalgam of other branches of science used in the criminal justice system. Any nucleated cell/tissue harbouring DNA, either live or dead, can be used as forensic exhibits, a source of investigation through DNA typing. These biological materials of human origin are rich source of proteins, carbohydrates, lipids, trace elements as well as water and, thus, provide a virtuous milieu for the growth of microbes. The obstinate microbial growth augments the degradation process and is amplified with the passage of time and improper storage of the biological materials. Degradation of these biological materials carriages a huge challenge in the downstream processes of forensic DNA typing technique, such as short tandem repeats (STR) DNA typing. Microbial degradation yields improper or no PCR amplification, heterozygous peak imbalance, DNA contamination from non-human sources, degradation of DNA by microbial by-products, etc. Consequently, the most precise STR DNA typing technique is nullified and definite opinion can be hardly given with degraded forensic exhibits. Thus, suitable precautionary measures should be taken for proper storage and processing of the biological exhibits to minimize their decaying process by micro-organisms.

Ancient DNA in historical parchments - identifying a procedure for extraction and amplification of genetic material.

PubMed

Lech, T

2016-05-06

Historical parchments in the form of documents, manuscripts, books, or letters, make up a large portion of cultural heritage collections. Their priceless historical value is associated with not only their content, but also the information hidden in the DNA deposited on them. Analyses of ancient DNA (aDNA) retrieved from parchments can be used in various investigations, including, but not limited to, studying their authentication, tracing the development of the culture, diplomacy, and technology, as well as obtaining information on the usage and domestication of animals. This article proposes and verifies a procedure for aDNA recovery from historical parchments and its appropriate preparation for further analyses. This study involved experimental selection of an aDNA extraction method with the highest efficiency and quality of extracted genetic material, from among the multi-stage phenol-chloroform extraction methods, and the modern, column-based techniques that use selective DNA-binding membranes. Moreover, current techniques to amplify entire genetic material were questioned, and the possibility of using mitochondrial DNA for species identification was analyzed. The usefulness of the proposed procedure was successfully confirmed in identification tests of historical parchments dating back to the 13-16th century AD.

Sensitive and inexpensive digital DNA analysis by microfluidic enrichment of rolling circle amplified single-molecules.

PubMed

Kühnemund, Malte; Hernández-Neuta, Iván; Sharif, Mohd Istiaq; Cornaglia, Matteo; Gijs, Martin A M; Nilsson, Mats

2017-05-05

Single molecule quantification assays provide the ultimate sensitivity and precision for molecular analysis. However, most digital analysis techniques, i.e. droplet PCR, require sophisticated and expensive instrumentation for molecule compartmentalization, amplification and analysis. Rolling circle amplification (RCA) provides a simpler means for digital analysis. Nevertheless, the sensitivity of RCA assays has until now been limited by inefficient detection methods. We have developed a simple microfluidic strategy for enrichment of RCA products into a single field of view of a low magnification fluorescent sensor, enabling ultra-sensitive digital quantification of nucleic acids over a dynamic range from 1.2 aM to 190 fM. We prove the broad applicability of our analysis platform by demonstrating 5-plex detection of as little as ∼1 pg (∼300 genome copies) of pathogenic DNA with simultaneous antibiotic resistance marker detection, and the analysis of rare oncogene mutations. Our method is simpler, more cost-effective and faster than other digital analysis techniques and provides the means to implement digital analysis in any laboratory equipped with a standard fluorescent microscope. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Recombinase Polymerase Amplification (RPA) of CaMV-35S Promoter and nos Terminator for Rapid Detection of Genetically Modified Crops

PubMed Central

Xu, Chao; Li, Liang; Jin, Wujun; Wan, Yusong

2014-01-01

Recombinase polymerase amplification (RPA) is a novel isothermal DNA amplification and detection technology that enables the amplification of DNA within 30 min at a constant temperature of 37–42 °C by simulating in vivo DNA recombination. In this study, based on the regulatory sequence of the cauliflower mosaic virus 35S (CaMV-35S) promoter and the Agrobacterium tumefaciens nopaline synthase gene (nos) terminator, which are widely incorporated in genetically modified (GM) crops, we designed two sets of RPA primers and established a real-time RPA detection method for GM crop screening and detection. This method could reliably detect as few as 100 copies of the target molecule in a sample within 15–25 min. Furthermore, the real-time RPA detection method was successfully used to amplify and detect DNA from samples of four major GM crops (maize, rice, cotton, and soybean). With this novel amplification method, the test time was significantly shortened and the reaction process was simplified; thus, this method represents an effective approach to the rapid detection of GM crops. PMID:25310647

Recombinase polymerase amplification (RPA) of CaMV-35S promoter and nos terminator for rapid detection of genetically modified crops.

PubMed

Xu, Chao; Li, Liang; Jin, Wujun; Wan, Yusong

2014-10-10

Recombinase polymerase amplification (RPA) is a novel isothermal DNA amplification and detection technology that enables the amplification of DNA within 30 min at a constant temperature of 37-42 °C by simulating in vivo DNA recombination. In this study, based on the regulatory sequence of the cauliflower mosaic virus 35S (CaMV-35S) promoter and the Agrobacterium tumefaciens nopaline synthase gene (nos) terminator, which are widely incorporated in genetically modified (GM) crops, we designed two sets of RPA primers and established a real-time RPA detection method for GM crop screening and detection. This method could reliably detect as few as 100 copies of the target molecule in a sample within 15-25 min. Furthermore, the real-time RPA detection method was successfully used to amplify and detect DNA from samples of four major GM crops (maize, rice, cotton, and soybean). With this novel amplification method, the test time was significantly shortened and the reaction process was simplified; thus, this method represents an effective approach to the rapid detection of GM crops.

DNA Amplification Techniques for the Detection of Toxoplasma gondii Tissue Cysts in Meat Producing Animals: A Narrative Review Article

PubMed Central

RIAZ, Farooq; RASHID, Imran; AKBAR, Haroon; SHEHZAD, Wasim; ISLAM, Saher; BAJWA, Amna Arshad; SAEED, Khalid; ASHRAF, Kamran

2016-01-01

Background: Toxoplasma gondii is an intracellular parasite, which infects one-third population of world. Humans and animals acquire infection by ingesting oocytes from feces of cats or by meat of other animals having cysts that may lead to congenital, ocular or cephalic toxoplasmosis. Either it is important to detect T. gondii from meat of food animals from retail shops or directly at slaughterhouses, which is meant for export. Methods: The current research was done without time limitation using such terms as follows: “Toxoplasma gondii”, “Meat”, “Tissue cyst”, “PCR”, “LAMP”, “Screening” and “Immunological assay” alone or in combination, in English language. The used electronic databases for searching included as follows: Pub-Med, Scopus, Google Scholar, Web of Science and Science Direct. The searches were limited to the published papers to English language. Results: Sensitivity of different molecular techniques for diagnosis of Toxoplasma is real-time PCR > LAMP > conventional PCR. In addition to these DNA analysis tools, bioassay in mice and cats is considered as “gold standard” to detect T. gondii. Conclusion: This review article will help the readers for grasping advantages and limitations of different diagnostic tools for screening meat samples for T. gondii. This review also makes bibliography about the type of meat sample to be processed for diagnosis and different primers or sequences to be targeted for T. gondii by number of researches for its detection from meat or tissue sample using DNA amplification techniques. PMID:28127354

Systematic evaluation of bias in microbial community profiles induced by whole genome amplification.

PubMed

Direito, Susana O L; Zaura, Egija; Little, Miranda; Ehrenfreund, Pascale; Röling, Wilfred F M

2014-03-01

Whole genome amplification methods facilitate the detection and characterization of microbial communities in low biomass environments. We examined the extent to which the actual community structure is reliably revealed and factors contributing to bias. One widely used [multiple displacement amplification (MDA)] and one new primer-free method [primase-based whole genome amplification (pWGA)] were compared using a polymerase chain reaction (PCR)-based method as control. Pyrosequencing of an environmental sample and principal component analysis revealed that MDA impacted community profiles more strongly than pWGA and indicated that this related to species GC content, although an influence of DNA integrity could not be excluded. Subsequently, biases by species GC content, DNA integrity and fragment size were separately analysed using defined mixtures of DNA from various species. We found significantly less amplification of species with the highest GC content for MDA-based templates and, to a lesser extent, for pWGA. DNA fragmentation also interfered severely: species with more fragmented DNA were less amplified with MDA and pWGA. pWGA was unable to amplify low molecular weight DNA (< 1.5 kb), whereas MDA was inefficient. We conclude that pWGA is the most promising method for characterization of microbial communities in low-biomass environments and for currently planned astrobiological missions to Mars. © 2013 Society for Applied Microbiology and John Wiley & Sons Ltd.

Developmental validation of a Cannabis sativa STR multiplex system for forensic analysis.

PubMed

Howard, Christopher; Gilmore, Simon; Robertson, James; Peakall, Rod

2008-09-01

A developmental validation study based on recommendations of the Scientific Working Group on DNA Analysis Methods (SWGDAM) was conducted on a multiplex system of 10 Cannabis sativa short tandem repeat loci. Amplification of the loci in four multiplex reactions was tested across DNA from dried root, stem, and leaf sources, and DNA from fresh, frozen, and dried leaf tissue with a template DNA range of 10.0-0.01 ng. The loci were amplified and scored consistently for all DNA sources when DNA template was in the range of 10.0-1.0 ng. Some allelic dropout and PCR failure occurred in reactions with lower template DNA amounts. Overall, amplification was best using 10.0 ng of template DNA from dried leaf tissue indicating that this is the optimal source material. Cross species amplification was observed in Humulus lupulus for three loci but there was no allelic overlap. This is the first study following SWGDAM validation guidelines to validate short tandem repeat markers for forensic use in plants.

Label-free and sensitive detection of T4 polynucleotide kinase activity via coupling DNA strand displacement reaction with enzymatic-aided amplification.

PubMed

Cheng, Rui; Tao, Mangjuan; Shi, Zhilu; Zhang, Xiafei; Jin, Yan; Li, Baoxin

2015-11-15

Several fluorescence signal amplification strategies have been developed for sensitive detection of T4 polynucleotide kinase (T4 PNK) activity, but they need fluorescence dye labeled DNA probe. We have addressed the limitation and report here a label-free strategy for sensitive detection of PNK activity by coupling DNA strand displacement reaction with enzymatic-aided amplification. A hairpin oligonucleotide (hpDNA) with blunt ends was used as the substrate for T4 PNK phosphorylation. In the presence of T4 PNK, the stem of hpDNA was phosphorylated and further degraded by lambda exonuclease (λ exo) from 5' to 3' direction to release a single-stranded DNA as a trigger of DNA strand displacement reaction (SDR). The trigger DNA can continuously displace DNA P2 from P1/P2 hybrid with the help of specific cleavage of nicking endonuclease (Nt.BbvCI). Then, DNA P2 can form G-quadruplex in the presence of potassium ions and quadruplex-selective fluorphore, N-methyl mesoporphyrin IX (NMM), resulting in a significant increase in fluorescence intensity of NMM. Thus, the accumulative release of DNA P2 led to fluorescence signal amplification for determining T4 PNK activity with a detection limit of 6.6×10(-4) U/mL, which is superior or comparative with established approaches. By ingeniously utilizing T4 PNK-triggered DNA SDR, T4 PNK activity can be specifically and facilely studied in homogeneous solution containing complex matrix without any external fluorescence labeling. Moreover, the influence of different inhibitors on the T4 PNK activity revealed that it also can be explored to screen T4 PNK inhibitors. Therefore, this label-free amplification strategy presents a facile and cost-effective approach for nucleic acid phosphorylation related research. Copyright © 2015 Elsevier B.V. All rights reserved.

Yoctomole electrochemical genosensing of Ebola virus cDNA by rolling circle and circle to circle amplification.

PubMed

Carinelli, S; Kühnemund, M; Nilsson, M; Pividori, M I

2017-07-15

This work addresses the design of an Ebola diagnostic test involving a simple, rapid, specific and highly sensitive procedure based on isothermal amplification on magnetic particles with electrochemical readout. Ebola padlock probes were designed to detect a specific L-gene sequence present in the five most common Ebola species. Ebola cDNA was amplified by rolling circle amplification (RCA) on magnetic particles. Further re-amplification was performed by circle-to-circle amplification (C2CA) and the products were detected in a double-tagging approach using a biotinylated capture probe for immobilization on magnetic particles and a readout probe for electrochemical detection by square-wave voltammetry on commercial screen-printed electrodes. The electrochemical genosensor was able to detect as low as 200 ymol, corresponding to 120 cDNA molecules of L-gene Ebola virus with a limit of detection of 33 cDNA molecules. The isothermal double-amplification procedure by C2CA combined with the electrochemical readout and the magnetic actuation enables the high sensitivity, resulting in a rapid, inexpensive, robust and user-friendly sensing strategy that offers a promising approach for the primary care in low resource settings, especially in less developed countries. Copyright © 2016 Elsevier B.V. All rights reserved.

A novel electrochemiluminescence strategy for ultrasensitive DNA assay using luminol functionalized gold nanoparticles multi-labeling and amplification of gold nanoparticles and biotin-streptavidin system.

PubMed

Chai, Ying; Tian, Dayong; Wang, Wei; Cui, Hua

2010-10-28

Luminol functionalized gold nanoparticles were used as labels for electrochemiluminescence signal amplification and an ultrasensitive, highly selective, convenient, low cost DNA detection strategy was developed.

Origins of DNA Replication and Amplification in the Breast Cancer Genome

DTIC Science & Technology

2012-09-01

W81XWH-10-1-0463 TITLE: Origins of DNA Replication and Amplification in the...2. REPORT TYPE Final 3. DATES COVERED 1 Sep 2010 – 31 Aug 2012 4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER Origins of DNA Replication and...described in the DOD funded parent grant, to test our hypothesis we need to map origins of DNA replication in the genome and ask which of these

Product differentiation by analysis of DNA melting curves during the polymerase chain reaction.

PubMed

Ririe, K M; Rasmussen, R P; Wittwer, C T

1997-02-15

A microvolume fluorometer integrated with a thermal cycler was used to acquire DNA melting curves during polymerase chain reaction by fluorescence monitoring of the double-stranded DNA specific dye SYBR Green I. Plotting fluorescence as a function of temperature as the thermal cycler heats through the dissociation temperature of the product gives a DNA melting curve. The shape and position of this DNA melting curve are functions of the GC/AT ratio, length, and sequence and can be used to differentiate amplification products separated by less than 2 degrees C in melting temperature. Desired products can be distinguished from undesirable products, in many cases eliminating the need for gel electrophoresis. Analysis of melting curves can extend the dynamic range of initial template quantification when amplification is monitored with double-stranded DNA specific dyes. Complete amplification and analysis of products can be performed in less than 15 min.

Multiplex, Rapid, and Sensitive Isothermal Detection of Nucleic-Acid Sequence by Endonuclease Restriction-Mediated Real-Time Multiple Cross Displacement Amplification.

PubMed

Wang, Yi; Wang, Yan; Zhang, Lu; Liu, Dongxin; Luo, Lijuan; Li, Hua; Cao, Xiaolong; Liu, Kai; Xu, Jianguo; Ye, Changyun

2016-01-01

We have devised a novel isothermal amplification technology, termed endonuclease restriction-mediated real-time multiple cross displacement amplification (ET-MCDA), which facilitated multiplex, rapid, specific and sensitive detection of nucleic-acid sequences at a constant temperature. The ET-MCDA integrated multiple cross displacement amplification strategy, restriction endonuclease cleavage and real-time fluorescence detection technique. In the ET-MCDA system, the functional cross primer E-CP1 or E-CP2 was constructed by adding a short sequence at the 5' end of CP1 or CP2, respectively, and the new E-CP1 or E-CP2 primer was labeled at the 5' end with a fluorophore and in the middle with a dark quencher. The restriction endonuclease Nb.BsrDI specifically recognized the short sequence and digested the newly synthesized double-stranded terminal sequences (5' end short sequences and their complementary sequences), which released the quenching, resulting on a gain of fluorescence signal. Thus, the ET-MCDA allowed real-time detection of single or multiple targets in only a single reaction, and the positive results were observed in as short as 12 min, detecting down to 3.125 fg of genomic DNA per tube. Moreover, the analytical specificity and the practical application of the ET-MCDA were also successfully evaluated in this study. Here, we provided the details on the novel ET-MCDA technique and expounded the basic ET-MCDA amplification mechanism.

Visualization of DNA in highly processed botanical materials.

PubMed

Lu, Zhengfei; Rubinsky, Maria; Babajanian, Silva; Zhang, Yanjun; Chang, Peter; Swanson, Gary

2018-04-15

DNA-based methods have been gaining recognition as a tool for botanical authentication in herbal medicine; however, their application in processed botanical materials is challenging due to the low quality and quantity of DNA left after extensive manufacturing processes. The low amount of DNA recovered from processed materials, especially extracts, is "invisible" by current technology, which has casted doubt on the presence of amplifiable botanical DNA. A method using adapter-ligation and PCR amplification was successfully applied to visualize the "invisible" DNA in botanical extracts. The size of the "invisible" DNA fragments in botanical extracts was around 20-220 bp compared to fragments of around 600 bp for the more easily visualized DNA in botanical powders. This technique is the first to allow characterization and visualization of small fragments of DNA in processed botanical materials and will provide key information to guide the development of appropriate DNA-based botanical authentication methods in the future. Copyright © 2017 Elsevier Ltd. All rights reserved.

DNA fingerprinting of Brassica juncea cultivars using microsatellite probes.

PubMed

Bhatia, S; Das, S; Jain, A; Lakshmikumaran, M

1995-09-01

The genetic variability in the Brassica juncea cultivars was detected by employing in-gel hybridization of restricted DNA to simple repetitive sequences such as (GATA)4, (GACA)4 and (CAC)5. The most informative probe/enzyme combination was (GATA)4/EcoRI, yielding highly polymorphic fingerprint patterns for the B. juncea cultivars. This technique was found to be dependable for establishing the variety specific patterns for most of the cultivars studied, a prerequisite for germplasm preservation. The results of the present study were compared with those reported in our earlier study in which random amplification of polymorphic DNA (RAPD) was used for assessing the genetic variability in the B. juncea cultivars.

Advanced DNA-Based Point-of-Care Diagnostic Methods for Plant Diseases Detection.

PubMed

Lau, Han Yih; Botella, Jose R

2017-01-01

Diagnostic technologies for the detection of plant pathogens with point-of-care capability and high multiplexing ability are an essential tool in the fight to reduce the large agricultural production losses caused by plant diseases. The main desirable characteristics for such diagnostic assays are high specificity, sensitivity, reproducibility, quickness, cost efficiency and high-throughput multiplex detection capability. This article describes and discusses various DNA-based point-of care diagnostic methods for applications in plant disease detection. Polymerase chain reaction (PCR) is the most common DNA amplification technology used for detecting various plant and animal pathogens. However, subsequent to PCR based assays, several types of nucleic acid amplification technologies have been developed to achieve higher sensitivity, rapid detection as well as suitable for field applications such as loop-mediated isothermal amplification, helicase-dependent amplification, rolling circle amplification, recombinase polymerase amplification, and molecular inversion probe. The principle behind these technologies has been thoroughly discussed in several review papers; herein we emphasize the application of these technologies to detect plant pathogens by outlining the advantages and disadvantages of each technology in detail.

Advanced DNA-Based Point-of-Care Diagnostic Methods for Plant Diseases Detection

PubMed Central

Lau, Han Yih; Botella, Jose R.

2017-01-01

Diagnostic technologies for the detection of plant pathogens with point-of-care capability and high multiplexing ability are an essential tool in the fight to reduce the large agricultural production losses caused by plant diseases. The main desirable characteristics for such diagnostic assays are high specificity, sensitivity, reproducibility, quickness, cost efficiency and high-throughput multiplex detection capability. This article describes and discusses various DNA-based point-of care diagnostic methods for applications in plant disease detection. Polymerase chain reaction (PCR) is the most common DNA amplification technology used for detecting various plant and animal pathogens. However, subsequent to PCR based assays, several types of nucleic acid amplification technologies have been developed to achieve higher sensitivity, rapid detection as well as suitable for field applications such as loop-mediated isothermal amplification, helicase-dependent amplification, rolling circle amplification, recombinase polymerase amplification, and molecular inversion probe. The principle behind these technologies has been thoroughly discussed in several review papers; herein we emphasize the application of these technologies to detect plant pathogens by outlining the advantages and disadvantages of each technology in detail. PMID:29375588

A DNA Barcode-Based RPA Assay (BAR-RPA) for Rapid Identification of the Dry Root of Ficus hirta (Wuzhimaotao).

PubMed

Tian, Enwei; Liu, Qianqian; Ye, Haoting; Li, Fang; Chao, Zhi

2017-12-18

Background: Wuzhimaotao (the dry root of Ficus hirta ) is used as both medicine and food ingredient by the locals in areas around Nanling Mountains of China. Due to its very similar external morphologies with Duanchangcao (the root of Gelsemium elegans , which contains gelsemine that is extremely neurotoxic) and the associated growth of these two plants, incidents of food poisoning and even death frequently occur, resulting from the misuse of Duanchangcao as Wuzhimaotao. The aim of this study is to develop a fast, even, on-spot approach to identification of Wuzhimaotao. Methods: We used DNA barcode-based recombinase polymerase amplification (BAR-RPA) with species-specific primers targeting the internal transcribed spacer (ITS) region of the rDNA of F. hirta. BAR-RPA reaction time and temperature were optimized and the specificity and sensitivity of BAR-RPA species-specific primers were assessed. Results: This technique showed a high specificity and sensitivity to amplify the genomic DNA of F. hirta and allowed for rapid amplification (within 15 min) of the ITS region under a constant and mild temperature range of 37-42 °C without using thermocyclers. Conclusions: The BAR-RPA assay with a fast DNA extraction protocol provides a simple, energy-saving, and rapid method for identification of Wuzhimaotao in both laboratory and field settings.

Logical Framework of Forensic Identification: Ability to Resist Fabricated DNA.

PubMed

Wang, Zheng; Zhou, Di; Zhang, Suhua; Bian, Yingnan; Hu, Zhen; Zhu, Ruxin; Lu, Daru; Li, Chengtao

2015-12-01

Over the past 30 years, DNA analysis has revolutionized forensic science and has become the most useful single tool in the multifaceted fight against crime. Today, DNA profiling with sets of highly polymorphic autosomal short tandem repeat markers is widely employed and accepted in the courts due to its high discriminating power and reliability. However, an artificial bloodstain purposefully created using molecular biology techniques succeeded in tricking a leading forensic DNA laboratory. The disturbing possibility that a forensic DNA profile can be faked shocked the general public and the mass media, and generated serious discussion about the credibility of DNA evidence. Herein, we present two exemplary assays based on tissue-specific methylation patterns and cell-specific mRNA expression, respectively. These two assays can be integrated into the DNA analysis pipelines without consumption of additional samples. We show that the two assays can not only distinguish between artificial and genuine samples, but also provide information on tissue origin. The two assays were tested on natural and artificial bloodstains (generated by polymerase chain reaction and whole genome amplification technique) and the results illustrated that the logical framework of forensic identification is still useful for forensic identification with the high credibility.

Identification of five highly priced tuna species by quantitative real-time polymerase chain reaction.

PubMed

Liu, Shasha; Xu, Kunhua; Wu, Zhigang; Xie, Xiao; Feng, Junli

2016-09-01

Tunas are economically important fishery worldwide, and are often used for commercial processed production. For effective fishery management and protection of consumers' rights, it is important to develop a molecular method to identify species in canned tuna products rapidly and reliably. Here, we have developed a duplex quantitative real-time PCR (qPCR) for identification of five highly priced tuna species (Thunnus maccoyii, Thunnus obesus, Thunnus albacares, Thunnus alalunga and Katsuwonus pelamis) from processed as well as fresh fish. After amplification and sequencing of seven genetic markers commonly used for species identification, 16S rDNA and control region (CR) of mitochondrial DNA were selected as the reference gene markers for genus Thunnus and tuna species identification, respectively. Subsequently, a 73 bp fragment of 16S rDNA and 85-99 bp fragment of CR were simultaneously amplified from each target species by qPCR. The qPCR efficiency of each reaction was calculated according to the standard curves, and the method was validated by amplification DNA extracted from single or mixed tuna specimen. The developed duplex qPCR system was applied to authenticate species of 14 commercial tuna products successfully, which demonstrated it was really a useful and academic technique to identify highly priced tuna species.

Colorimetric Nucleic Acid Detection on Paper Microchip Using Loop Mediated Isothermal Amplification and Crystal Violet Dye.

PubMed

Roy, Sharmili; Mohd-Naim, Noor Faizah; Safavieh, Mohammadali; Ahmed, Minhaz Uddin

2017-11-22

Nucleic acid detection is of paramount importance in monitoring of microbial pathogens in food safety and infectious disease diagnostic applications. To address these challenges, a rapid, cost-effective label-free technique for nucleic acid detection with minimal instrumentations is highly desired. Here, we present paper microchip to detect and quantify nucleic acid using colorimetric sensing modality. The extracted DNA from food samples of meat as well as microbial pathogens was amplified utilizing loop-mediated isothermal amplification (LAMP). LAMP amplicon was then detected and quantified on a paper microchip fabricated in a cellulose paper and a small wax chamber utilizing crystal violet dye. The affinity of crystal violet dye toward dsDNA and positive signal were identified by changing the color from colorless to purple. Using this method, detection of Sus scrofa (porcine) and Bacillus subtilis (bacteria) DNA was possible at concentrations as low as 1 pg/μL (3.43 × 10 -1 copies/μL) and 10 pg/μL (2.2 × 10 3 copies/μL), respectively. This strategy can be adapted for detection of other DNA samples, with potential for development of a new breed of simple and inexpensive paper microchip at the point-of-need.

Twin target self-amplification-based DNA machine for highly sensitive detection of cancer-related gene.

PubMed

Xu, Huo; Jiang, Yifan; Liu, Dengyou; Liu, Kai; Zhang, Yafeng; Yu, Suhong; Shen, Zhifa; Wu, Zai-Sheng

2018-06-29

The sensitive detection of cancer-related genes is of great significance for early diagnosis and treatment of human cancers, and previous isothermal amplification sensing systems were often based on the reuse of target DNA, the amplification of enzymatic products and the accumulation of reporting probes. However, no reporting probes are able to be transformed into target species and in turn initiate the signal of other probes. Herein we reported a simple, isothermal and highly sensitive homogeneous assay system for tumor suppressor p53 gene detection based on a new autonomous DNA machine, where the signaling probe, molecular beacon (MB), was able to execute the function similar to target DNA besides providing the common signal. In the presence of target p53 gene, the operation of DNA machine can be initiated, and cyclical nucleic acid strand-displacement polymerization (CNDP) and nicking/polymerization cyclical amplification (NPCA) occur, during which the MB was opened by target species and cleaved by restriction endonuclease. In turn, the cleaved fragments could activate the next signaling process as target DNA did. According to the functional similarity, the cleaved fragment was called twin target, and the corresponding fashion to amplify the signal was named twin target self-amplification. Utilizing this newly-proposed DNA machine, the target DNA could be detected down to 0.1 pM with a wide dynamic range (6 orders of magnitude) and single-base mismatched targets were discriminated, indicating a very high assay sensitivity and good specificity. In addition, the DNA machine was not only used to screen the p53 gene in complex biological matrix but also was capable of practically detecting genomic DNA p53 extracted from A549 cell line. This indicates that the proposed DNA machine holds the potential application in biomedical research and early clinical diagnosis. Copyright © 2018 Elsevier B.V. All rights reserved.

Development of a real-world direct interface for integrated DNA extraction and amplification in a microfluidic device.

PubMed

Shaw, Kirsty J; Joyce, Domino A; Docker, Peter T; Dyer, Charlotte E; Greenway, Gillian M; Greenman, John; Haswell, Stephen J

2011-02-07

Integrated DNA extraction and amplification have been carried out in a microfluidic device using electro-osmotic pumping (EOP) for fluidic control. All the necessary reagents for performing both DNA extraction and polymerase chain reaction (PCR) amplification were pre-loaded into the microfluidic device following encapsulation in agarose gel. Buccal cells were collected using OmniSwabs [Whatman™, UK] and manually added to a chaotropic binding/lysis solution pre-loaded into the microfluidic device. The released DNA was then adsorbed onto a silica monolith contained within the DNA extraction chamber and the microfluidic device sealed using polymer electrodes. The washing and elution steps for DNA extraction were carried out using EOP, resulting in transfer of the eluted DNA into the PCR chamber. Thermal cycling, achieved using a Peltier element, resulted in amplification of the Amelogenin locus as confirmed using conventional capillary gel electrophoresis. It was demonstrated that the PCR reagents could be stored in the microfluidic device for at least 8 weeks at 4 °C with no significant loss of activity. Such methodology lends itself to the production of 'ready-to-use' microfluidic devices containing all the necessary reagents for sample processing, with many obvious applications in forensics and clinical medicine.

A Simple Method for the Extraction, PCR-amplification, Cloning, and Sequencing of Pasteuria 16S rDNA from Small Numbers of Endospores.

PubMed

Atibalentja, N; Noel, G R; Ciancio, A

2004-03-01

For many years the taxonomy of the genus Pasteuria has been marred with confusion because the bacterium could not be cultured in vitro and, therefore, descriptions were based solely on morphological, developmental, and pathological characteristics. The current study sought to devise a simple method for PCR-amplification, cloning, and sequencing of Pasteuria 16S rDNA from small numbers of endospores, with no need for prior DNA purification. Results show that DNA extracts from plain glass bead-beating of crude suspensions containing 10,000 endospores at 0.2 x 10 endospores ml(-1) were sufficient for PCR-amplification of Pasteuria 16S rDNA, when used in conjunction with specific primers. These results imply that for P. penetrans and P. nishizawae only one parasitized female of Meloidogyne spp. and Heterodera glycines, respectively, should be sufficient, and as few as eight cadavers of Belonolaimus longicaudatus with an average number of 1,250 endospores of "Candidatus Pasteuria usgae" are needed for PCR-amplification of Pasteuria 16S rDNA. The method described in this paper should facilitate the sequencing of the 16S rDNA of the many Pasteuria isolates that have been reported on nematodes and, consequently, expedite the classification of those isolates through comparative sequence analysis.

SPERM RNA AMPLIFICATION FOR GENE EXPRESSION PROFILING BY DNA MICROARRAY TECHNOLOGY

EPA Science Inventory

Sperm RNA Amplification for Gene Expression Profiling by DNA Microarray Technology
Hongzu Ren, Kary E. Thompson, Judith E. Schmid and David J. Dix, Reproductive Toxicology Division, NHEERL, Office of Research and Development, US Environmental Protection Agency, Research Triang...

Integrated electrochemical microsystems for genetic detection of pathogens at the point of care.

PubMed

Hsieh, Kuangwen; Ferguson, B Scott; Eisenstein, Michael; Plaxco, Kevin W; Soh, H Tom

2015-04-21

The capacity to achieve rapid, sensitive, specific, quantitative, and multiplexed genetic detection of pathogens via a robust, portable, point-of-care platform could transform many diagnostic applications. And while contemporary technologies have yet to effectively achieve this goal, the advent of microfluidics provides a potentially viable approach to this end by enabling the integration of sophisticated multistep biochemical assays (e.g., sample preparation, genetic amplification, and quantitative detection) in a monolithic, portable device from relatively small biological samples. Integrated electrochemical sensors offer a particularly promising solution to genetic detection because they do not require optical instrumentation and are readily compatible with both integrated circuit and microfluidic technologies. Nevertheless, the development of generalizable microfluidic electrochemical platforms that integrate sample preparation and amplification as well as quantitative and multiplexed detection remains a challenging and unsolved technical problem. Recognizing this unmet need, we have developed a series of microfluidic electrochemical DNA sensors that have progressively evolved to encompass each of these critical functionalities. For DNA detection, our platforms employ label-free, single-step, and sequence-specific electrochemical DNA (E-DNA) sensors, in which an electrode-bound, redox-reporter-modified DNA "probe" generates a current change after undergoing a hybridization-induced conformational change. After successfully integrating E-DNA sensors into a microfluidic chip format, we subsequently incorporated on-chip genetic amplification techniques including polymerase chain reaction (PCR) and loop-mediated isothermal amplification (LAMP) to enable genetic detection at clinically relevant target concentrations. To maximize the potential point-of-care utility of our platforms, we have further integrated sample preparation via immunomagnetic separation, which allowed the detection of influenza virus directly from throat swabs and developed strategies for the multiplexed detection of related bacterial strains from the blood of septic mice. Finally, we developed an alternative electrochemical detection platform based on real-time LAMP, which not is only capable of detecting across a broad dynamic range of target concentrations, but also greatly simplifies quantitative measurement of nucleic acids. These efforts represent considerable progress toward the development of a true sample-in-answer-out platform for genetic detection of pathogens at the point of care. Given the many advantages of these systems, and the growing interest and innovative contributions from researchers in this field, we are optimistic that iterations of these systems will arrive in clinical settings in the foreseeable future.

Nucleic Acid Amplification Based Diagnostic of Lyme (Neuro-)borreliosis – Lost in the Jungle of Methods, Targets, and Assays?

PubMed Central

Nolte, Oliver

2012-01-01

Laboratory based diagnosis of infectious diseases usually relies on culture of the disease causing micro-organism, followed by identification and susceptibility testing. Since Borrelia burgdorferi sensu lato, the etiologic agent of Lyme disease or Lyme borreliosis, requires very specific culture conditions (e.g. specific liquid media, long term cul-ture) traditional bacteriology is often not done on a routine basis. Instead, confirmation of the clinical diagnosis needs ei-ther indirect techniques (like serology or measurement of cellular activity in the presence of antigens) or direct but culture independent techniques, like microscopy or nucleic acid amplification techniques (NAT), with polymerase chain reaction (PCR) being the most frequently applied NAT method in routine laboratories. NAT uses nucleic acids of the disease causing micro-organism as template for amplification, isolated from various sources of clinical specimens. Although the underlying principle, adoption of the enzymatic process running during DNA duplication prior to prokaryotic cell division, is comparatively easy, a couple of ‘pitfalls’ is associated with the technique itself as well as with interpretation of the results. At present, no commercial, CE-marked and sufficiently validated PCR assay is available. A number of homebrew assays have been published, which are different in terms of target (i.e. the gene targeted by the amplification primers), method (nested PCR, PCR followed by hybridization, real-time PCR) and validation criteria. Inhibitory compounds may lead to false negative results, if no appropriate internal control is included. Carry-over of amplicons, insufficient handling and workflow and/or insufficiently validated targets/primers may result in false positive results. Different targets may yield different analytical sensitivity, depending, among other factors, of the redundancy of a target gene in the genome. Per-formance characteristics (e.g. analytical sensitivity and specificity, clinical sensitivity and specificity, reproducibility, etc.) are, if available, only applicable to a specific assay, running in a specific laboratory. Finally, not only the NAT/PCR method itself, but also the process of DNA isolation from the specimen, is highly diverse and may have fundamental im-pact on the (expected) PCR result. Of concern are distribution effects of DNA, in particular, if only low numbers of bacte-ria/genomes are present in a sample, as it is the case for instance in cerebrospinal fluids. For the ordering physician and for the patient requesting PCR analysis, these ‘pitfalls’ are usually invisible. As a conse-quence, the reported result (i.e. PCR negative or positive for B. burgdorferi) is hard to interpret, especially, if the reported PCR result is contradictory to the clinical diagnosis or other laboratory findings. Moreover, due to the high number of dif-ferent assays in use, two laboratories, testing the same specimen, might come to different PCR results. The current paper wants to summarize the available PCR/NAT assays for the detection of B. burgdorferi DNA in clinical specimens, with special attention to neurologic disorders, and to discuss the difficulties in PCR analysis and result inter-pretation, associated thereof. In view of growing numbers of patients who are diagnosed of having Lyme disease, and ac-knowledging a substantial growth in knowledge regarding other tick- or vector-borne pathogens, which might be able to induce symptoms comparable to Lyme (neuro-)borreliosis, efforts are urgently needed to standardize and harmonize methods for B. burgdorferi nucleic acid amplification. PMID:23230454

Rolling Circle Amplification of Complete Nematode Mitochondrial Genomes

PubMed Central

Tang, Sha; Hyman, Bradley C.

2005-01-01

To enable investigation of nematode mitochondrial DNA evolution, methodology has been developed to amplify intact nematode mitochondrial genomes in preparative yields using a rolling circle replication strategy. Successful reactions were generated from whole cell template DNA prepared by alkaline lysis of the rhabditid nematode Caenorhabditis elegans and a mermithid nematode, Thaumamermis cosgrovei. These taxa, representing the two major nematode classes Chromodorea and Enoplea, maintain mitochondrial genomes of 13.8 kb and 20.0 kb, respectively. Efficient amplifications were conducted on template DNA isolated from individual or pooled nematodes that were alive or stored at -80°C. Unexpectedly, these experiments revealed that multiple T. cosgrovei mitochondrial DNA haplotypes are maintained in our local population. Rolling circle amplification products can be used as templates for standard PCR reactions with specific primers that target mitochondrial genes or for direct DNA sequencing. PMID:19262866

Molecular barcodes detect redundancy and contamination in hairpin-bisulfite PCR

PubMed Central

Miner, Brooks E.; Stöger, Reinhard J.; Burden, Alice F.; Laird, Charles D.; Hansen, R. Scott

2004-01-01

PCR amplification of limited amounts of DNA template carries an increased risk of product redundancy and contamination. We use molecular barcoding to label each genomic DNA template with an individual sequence tag prior to PCR amplification. In addition, we include molecular ‘batch-stamps’ that effectively label each genomic template with a sample ID and analysis date. This highly sensitive method identifies redundant and contaminant sequences and serves as a reliable method for positive identification of desired sequences; we can therefore capture accurately the genomic template diversity in the sample analyzed. Although our application described here involves the use of hairpin-bisulfite PCR for amplification of double-stranded DNA, the method can readily be adapted to single-strand PCR. Useful applications will include analyses of limited template DNA for biomedical, ancient DNA and forensic purposes. PMID:15459281

Nanoparticles Affect PCR Primarily via Surface Interactions with PCR Components: Using Amino-Modified Silica-Coated Magnetic Nanoparticles as a Main Model.

PubMed

Bai, Yalong; Cui, Yan; Paoli, George C; Shi, Chunlei; Wang, Dapeng; Shi, Xianming

2015-06-24

Nanomaterials have been widely reported to affect the polymerase chain reaction (PCR). However, many studies in which these effects were observed were not comprehensive, and many of the proposed mechanisms have been primarily speculative. In this work, we used amino-modified silica-coated magnetic nanoparticles (ASMNPs, which can be collected very easily using an external magnetic field) as a model and compared them with gold nanoparticles (AuNPs, which have been studied extensively) to reveal the mechanisms by which nanoparticles affect PCR. We found that nanoparticles affect PCR primarily by binding to PCR components: (1) inhibition, (2) specifity, and (3) efficiency and yield of PCR are impacted. (1) Excess nanomaterials inhibit PCR by adsorbing to DNA polymerase, Mg(2+), oligonucleotide primers, or DNA templates. Nanoparticle surface-active groups are particularly important to this effect. (2, a) Nanomaterials do not inhibit nonspecific amplification products caused by false priming as previously surmised. It was shown that relatively low concentrations of nanoparticles inhibited the amplification of long amplicons, and increasing the amount of nanoparticles inhibited the amplification of short amplicons. This concentration phenomenon appears to be the result of the formation of "joints" upon the adsorption of ASMNPs to DNA templates. (b) Nanomaterials are able to inhibit nonspecific amplification products due to incomplete amplification by preferably adsorbing single-stranded incomplete amplification products. (3) Some types of nanomaterials, such as AuNPs, enhance the efficiency and yield of PCR because these types of nanoparticles can adsorb to single-stranded DNA more strongly than to double-stranded DNA. This behavior assists in the rapid and thorough denaturation of double-stranded DNA templates. Therefore, the interaction between the surface of nanoparticles and PCR components is sufficient to explain most of the effects of nanoparticles on PCR.

Amplified DNAs in laboratory stocks of Leishmania tarentolae: extrachromosomal circles structurally and functionally similar to the inverted-H-region amplification of methotrexate-resistant Leishmania major

DOE Office of Scientific and Technical Information (OSTI.GOV)

Petrillo-Peixoto, M.L.; Beverley, S.M.

1988-12-01

We describe the structure of amplified DNA that was discovered in two laboratory stocks of the protozoan parasite Leishmania tarentolae. Restriction mapping and molecular cloning revealed that a region of 42 kilobases was amplified 8- to 30-fold in these lines. Southern blot analyses of digested DNAs or chromosomes separated by pulsed-field electrophoresis showed that the amplified DNA corresponded to the H region, a locus defined originally by its amplification in methotrexate-resistant Leishmania major. Similarities between the amplified DNA of the two species included (i) extensive cross-hybridization; (ii) approximate conservation of sequence order; (iii) extrachromosomal localization; (iv) an overall inverted, head-to-headmore » configuration as a circular 140-kilobase tetrameric molecule; (v) two regions of DNA sequence rearrangement, each of which was closely associated with the two centers of the inverted repeats; (vi) association with methotrexate resistance; and (vii) phenotypically conservative amplification, in which the wild-type chromosomal arrangement was retained without apparent modification. Our data showed that amplified DNA mediating drug resistance arose in unselected L. tarentolae, although the pressures leading to apparently spontaneous amplification and maintenance of the H region are not known. The simple structure and limited extent of DNA amplified in these and other Leishmania lines suggests that the study of gene amplification in Leishmania spp. offers an attractive model system for the study of amplification in cultured mammalian cells and tumors. We also introduced a method for measuring the size of large circular DNAs, using gamma-irradiation to introduce limited double-strand breaks followed by sizing of the linear DNAs by pulsed-field electrophoresis.« less

Quantitative Single-Cell mRNA Analysis in Hydrogel Beads.

PubMed

Rakszewska, Agata; Stolper, Rosa J; Kolasa, Anna B; Piruska, Aigars; Huck, Wilhelm T S

2016-06-01

In recent years, technologies capable of analyzing single cells have emerged that are transforming many fields of biological research. Herein we report how DNA-functionalized hydrogel beads can serve as a matrix to capture mRNA from lysed single cells. mRNA quantification free of pre-amplification bias is ensured by using padlock probes and rolling circle amplification followed by hybridization with fluorescent probes. The number of transcripts in individual cells is assessed by simply counting fluorescent dots inside gel beads. The method extends the potential of existing techniques and provides a general platform for capturing molecules of interest from single cells. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Nanoroughened plasmonic films for enhanced biosensing detection

NASA Astrophysics Data System (ADS)

LeMoal, Eric; Lévêque-Fort, Sandrine; Potier, Marie-Claude; Fort, Emmanuel

2009-06-01

Although fluorescence is the prevailing labeling technique in biosensing applications, sensitivity improvement is still a striving challenge. We show that coating standard microscope slides with nanoroughened silver films provides a high fluorescence signal enhancement due to plasmonic interactions. As a proof of concept, we applied these films with tailored plasmonic properties to DNA microarrays. Using common optical scanning devices, we achieved signal amplifications of more than 40-fold.

Comparing efficiency of American Fisheries Society standard snorkeling techniques to environmental DNA sampling techniques

USGS Publications Warehouse

Ulibarri, Roy M.; Bonar, Scott A.; Rees, Christopher B.; Amberg, Jon J.; Ladell, Bridget; Jackson, Craig

2017-01-01

Analysis of environmental DNA (eDNA) is an emerging technique used to detect aquatic species through water sampling and the extraction of biological material for amplification. Our study compared the efficacy of eDNA methodology to American Fisheries Society (AFS) standard snorkeling surveys with regard to detecting the presence of rare fish species. Knowing which method is more efficient at detecting target species will help managers to determine the best way to sample when both traditional sampling methods and eDNA sampling are available. Our study site included three Navajo Nation streams that contained Navajo Nation Genetic Subunit Bluehead Suckers Catostomus discobolus and Zuni Bluehead Suckers C. discobolus yarrowi. We first divided the entire wetted area of streams into consecutive 100-m reaches and then systematically selected 10 reaches/stream for snorkel and eDNA surveys. Surface water samples were taken in 10-m sections within each 100-m reach, while fish presence was noted via snorkeling in each 10-m section. Quantitative PCR was run on each individual water sample in quadruplicate to test for the presence or absence of the target species. With eDNA sampling techniques, we were able to positively detect both species in two out of the three streams. Snorkeling resulted in positive detection of both species in all three streams. In streams where the target species were detected with eDNA sampling, snorkeling detected fish at 11–29 sites/stream, whereas eDNA detected fish at 3–12 sites/stream. Our results suggest that AFS standard snorkeling is more effective than eDNA for detecting target fish species. To improve our eDNA procedures, the amount of water collected and tested should be increased. Additionally, filtering water on-site may improve eDNA techniques for detecting fish. Future research should focus on standardization of eDNA sampling to provide a widely operational sampling tool.

In Situ Detection of Anaplasma spp. by DNA Target-Primed Rolling-Circle Amplification of a Padlock Probe and Intracellular Colocalization with Immunofluorescently Labeled Host Cell von Willebrand Factor ▿

PubMed Central

Wamsley, Heather L.; Barbet, Anthony F.

2008-01-01

Endothelial cell culture and preliminary immunofluorescent staining of Anaplasma-infected tissues suggest that endothelial cells may be an in vivo nidus of mammalian infection. To investigate endothelial cells and other potentially cryptic sites of Anaplasma sp. infection in mammalian tissues, a sensitive and specific isothermal in situ technique to detect localized Anaplasma gene sequences by using rolling-circle amplification of circularizable, linear, oligonucleotide probes (padlock probes) was developed. Cytospin preparations of uninfected or Anaplasma-infected cell cultures were examined using this technique. Via fluorescence microscopy, the technique described here, and a combination of differential interference contrast microscopy and von Willebrand factor immunofluorescence, Anaplasma phagocytophilum and Anaplasma marginale were successfully localized in situ within intact cultured mammalian cells. This work represents the first application of this in situ method for the detection of a microorganism and forms the foundation for future applications of this technique to detect, localize, and analyze Anaplasma nucleotide sequences in the tissues of infected mammalian and arthropod hosts and in cell cultures. PMID:18495855

Effective PCR-based detection of Naegleria fowleri from cultured sample and PAM-developed mouse.

PubMed

Kang, Heekyoung; Seong, Gi-Sang; Sohn, Hae-Jin; Kim, Jong-Hyun; Lee, Sang-Eun; Park, Mi Yeoun; Lee, Won-Ja; Shin, Ho-Joon

2015-10-01

Increasing numbers of Primary Amoebic Meningoencephalitis (PAM) cases due to Naegleria fowleri are becoming a serious issue in subtropical and tropical countries as a Neglected Tropical Disease (NTD). To establish a rapid and effective diagnostic tool, a PCR-based detection technique was developed based on previous PCR methods. Four kinds of primer pairs, Nfa1, Nae3, Nf-ITS, and Naegl, were employed in the cultured amoebic trophozoites and a mouse with PAM experimentally developed by N. fowleri inoculation (PAM-mouse). For the extraction of genomic DNA from N. fowleri trophozoites (1×10(6)), simple boiling with 10μl of PBS (pH 7.4) at 100°C for 30min was found to be the most rapid and efficient procedure, allowing amplification of 2.5×10(2) trophozoites using the Nfa-1 primer. The primers Nfa1 and Nae3 amplified only N. fowleri DNA, whereas the ITS primer detected N. fowleri and N. gruberi DNA. Using the PAM-mouse brain tissue, the Nfa1 primer was able to amplify the N. fowleri DNA 4 days post infection with 1ng/μl of genomic DNA being detectable. Using the PAM-mouse CSF, amplification of the N. fowleri DNA with the Nae3 primer was possible 5 days post infection showing a better performance than the Nfa1 primer at day 6. Copyright © 2015 Elsevier GmbH. All rights reserved.

A general method to eliminate laboratory induced recombinants during massive, parallel sequencing of cDNA library.

PubMed

Waugh, Caryll; Cromer, Deborah; Grimm, Andrew; Chopra, Abha; Mallal, Simon; Davenport, Miles; Mak, Johnson

2015-04-09

Massive, parallel sequencing is a potent tool for dissecting the regulation of biological processes by revealing the dynamics of the cellular RNA profile under different conditions. Similarly, massive, parallel sequencing can be used to reveal the complexity of viral quasispecies that are often found in the RNA virus infected host. However, the production of cDNA libraries for next-generation sequencing (NGS) necessitates the reverse transcription of RNA into cDNA and the amplification of the cDNA template using PCR, which may introduce artefact in the form of phantom nucleic acids species that can bias the composition and interpretation of original RNA profiles. Using HIV as a model we have characterised the major sources of error during the conversion of viral RNA to cDNA, namely excess RNA template and the RNaseH activity of the polymerase enzyme, reverse transcriptase. In addition we have analysed the effect of PCR cycle on detection of recombinants and assessed the contribution of transfection of highly similar plasmid DNA to the formation of recombinant species during the production of our control viruses. We have identified RNA template concentrations, RNaseH activity of reverse transcriptase, and PCR conditions as key parameters that must be carefully optimised to minimise chimeric artefacts. Using our optimised RT-PCR conditions, in combination with our modified PCR amplification procedure, we have developed a reliable technique for accurate determination of RNA species using NGS technology.

Low Cost Extraction and Isothermal Amplification of DNA for Infectious Diarrhea Diagnosis

PubMed Central

Huang, Shichu; Do, Jaephil; Mahalanabis, Madhumita; Fan, Andy; Zhao, Lei; Jepeal, Lisa; Singh, Satish K.; Klapperich, Catherine M.

2013-01-01

In order to counter the common perception that molecular diagnostics are too complicated to work in low resource settings, we have performed a difficult sample preparation and DNA amplification protocol using instrumentation designed to be operated without wall or battery power. In this work we have combined a nearly electricity-free nucleic acid extraction process with an electricity-free isothermal amplification assay to detect the presence of Clostridium difficile (C. difficile) DNA in the stool of infected patients. We used helicase-dependent isothermal amplification (HDA) to amplify the DNA in a low-cost, thermoplastic reaction chip heated with a pair of commercially available toe warmers, while using a simple Styrofoam insulator. DNA was extracted from known positive and negative stool samples. The DNA extraction protocol utilized an air pressure driven solid phase extraction device run using a standard bicycle pump. The simple heater setup required no electricity or battery and was capable of maintaining the temperature at 65°C±2°C for 55 min, suitable for repeatable HDA amplification. Experiments were performed to explore the adaptability of the system for use in a range of ambient conditions. When compared to a traditional centrifuge extraction protocol and a laboratory thermocycler, this disposable, no power platform achieved approximately the same lower limit of detection (1.25×10−2 pg of C. difficile DNA) while requiring much less raw material and a fraction of the lab infrastructure and cost. This proof of concept study could greatly impact the accessibility of molecular assays for applications in global health. PMID:23555883

Label-Free Sensitive Detection of DNA Methyltransferase by Target-Induced Hyperbranched Amplification with Zero Background Signal.

PubMed

Zhang, Yan; Wang, Xin-Yan; Zhang, Qianyi; Zhang, Chun-Yang

2017-11-21

DNA methyltransferases (MTases) may specifically recognize the short palindromic sequences and transfer a methyl group from S-adenosyl-l-methionine to target cytosine/adenine. The aberrant DNA methylation is linked to the abnormal DNA MTase activity, and some DNA MTases have become promising targets of anticancer/antimicrobial drugs. However, the reported DNA MTase assays often involve laborious operation, expensive instruments, and radio-labeled substrates. Here, we develop a simple and label-free fluorescent method to sensitively detect DNA adenine methyltransferase (Dam) on the basis of terminal deoxynucleotidyl transferase (TdT)-activated Endonuclease IV (Endo IV)-assisted hyperbranched amplification. We design a hairpin probe with a palindromic sequence in the stem as the substrate and a NH 2 -modified 3' end for the prevention of nonspecific amplification. The substrate may be methylated by Dam and subsequently cleaved by DpnI, producing three single-stranded DNAs, two of which with 3'-OH termini may be amplified by hyperbranched amplification to generate a distinct fluorescence signal. Because high exactitude of TdT enables the amplification only in the presence of free 3'-OH termini and Endo IV only hydrolyzes the intact apurinic/apyrimidinic sites in double-stranded DNAs, zero background signal can be achieved. This method exhibits excellent selectivity and high sensitivity with a limit of detection of 0.003 U/mL for pure Dam and 9.61 × 10 -6 mg/mL for Dam in E. coli cells. Moreover, it can be used to screen the Dam inhibitors, holding great potentials in disease diagnosis and drug development.

Simulated rRNA/DNA Ratios Show Potential To Misclassify Active Populations as Dormant

DOE Office of Scientific and Technical Information (OSTI.GOV)

Steven, Blaire; Hesse, Cedar; Soghigian, John

The use of rRNA/DNA ratios derived from surveys of rRNA sequences in RNA and DNA extracts is an appealing but poorly validated approach to infer the activity status of environmental microbes. To improve the interpretation of rRNA/DNA ratios, we performed simulations to investigate the effects of community structure, rRNA amplification, and sampling depth on the accuracy of rRNA/DNA ratios in classifying bacterial populations as “active” or “dormant.” Community structure was an insignificant factor. In contrast, the extent of rRNA amplification that occurs as cells transition from dormant to growing had a significant effect (P < 0.0001) on classification accuracy, withmore » misclassification errors ranging from 16 to 28%, depending on the rRNA amplification model. The error rate increased to 47% when communities included a mixture of rRNA amplification models, but most of the inflated error was false negatives (i.e., active populations misclassified as dormant). Sampling depth also affected error rates (P < 0.001). Inadequate sampling depth produced various artifacts that are characteristic of rRNA/DNA ratios generated from real communities. These data show important constraints on the use of rRNA/DNA ratios to infer activity status. Whereas classification of populations as active based on rRNA/DNA ratios appears generally valid, classification of populations as dormant is potentially far less accurate.« less

Ultrasensitive electrochemical detection of DNA based on Zn²⁺ assistant DNA recycling followed with hybridization chain reaction dual amplification.

PubMed

Qian, Yong; Wang, Chunyan; Gao, Fenglei

2015-01-15

A new strategy to combine Zn(2+) assistant DNA recycling followed with hybridization chain reaction dual amplification was designed for highly sensitive electrochemical detection of target DNA. A gold electrode was used to immobilize molecular beacon (MB) as the recognition probe and perform the amplification procedure. In the presence of the target DNA, the hairpin probe 1 was opened, and the DNAzyme was liberated from the caged structure. The activated DNAzyme hybridized with the MB and catalyzed its cleavage in the presence of Zn(2+) cofactor and resulting in a free DNAzyme strand. Finally, each target-induced activated DNAzyme underwent many cycles triggering the cleavage of MB, thus forming numerous MB fragments. The MB fragments triggered the HCR and formed a long double-helix DNA structure. Because both H1 and H2 were labeled by biotin, a lot of SA-ALP was captured on the electrode surface, thus catalyzing a silver deposition process for electrochemical stripping analysis. This novel cascade signal amplification strategy can detect target DNA down to the attomolar level with a dynamic range spanning 6 orders of magnitude. This highly sensitive and specific assay has a great potential to become a promising DNA quantification method in biomedical research and clinical diagnosis. Copyright © 2014 Elsevier B.V. All rights reserved.

Simulated rRNA/DNA Ratios Show Potential To Misclassify Active Populations as Dormant

DOE PAGES

Steven, Blaire; Hesse, Cedar; Soghigian, John; ...

2017-03-31

The use of rRNA/DNA ratios derived from surveys of rRNA sequences in RNA and DNA extracts is an appealing but poorly validated approach to infer the activity status of environmental microbes. To improve the interpretation of rRNA/DNA ratios, we performed simulations to investigate the effects of community structure, rRNA amplification, and sampling depth on the accuracy of rRNA/DNA ratios in classifying bacterial populations as “active” or “dormant.” Community structure was an insignificant factor. In contrast, the extent of rRNA amplification that occurs as cells transition from dormant to growing had a significant effect (P < 0.0001) on classification accuracy, withmore » misclassification errors ranging from 16 to 28%, depending on the rRNA amplification model. The error rate increased to 47% when communities included a mixture of rRNA amplification models, but most of the inflated error was false negatives (i.e., active populations misclassified as dormant). Sampling depth also affected error rates (P < 0.001). Inadequate sampling depth produced various artifacts that are characteristic of rRNA/DNA ratios generated from real communities. These data show important constraints on the use of rRNA/DNA ratios to infer activity status. Whereas classification of populations as active based on rRNA/DNA ratios appears generally valid, classification of populations as dormant is potentially far less accurate.« less

Enzymatic repair of selected cross-linked homoduplex molecules enhances nuclear gene rescue from Pompeii and Herculaneum remains.

PubMed

Di Bernardo, Giovanni; Del Gaudio, Stefania; Cammarota, Marcella; Galderisi, Umberto; Cascino, Antonino; Cipollaro, Marilena

2002-02-15

Ancient DNA (aDNA) samples extracted from the bone remains of six equids buried by the Vesuvius eruption in 79 AD were investigated to test pre-amplification and enzymatic repair procedures designed to enhance the rescue of nuclear genes. The extracts, which proved all positive for Equidae mtDNA amplification, proved positive only four times out of 18 when tested for single-copy Equidae nuclear genes (epsilon globin, p53 and gamma interferon). Pre-amplification did not change the number of retrieved aDNA sequences but 10 times out of 14 enzymatic repair restored the amplifiability of the genes analysed, proving that repair increases the rate of successful rescue from 22 to alpha(lambda)mu(omicron)sigma(tau) 80%. These findings support the hypothesis that some of these cross-linked aDNA molecules, which are not completely separated when DNA is extracted under denaturing conditions, become homoduplex substrates for Pol I and/or T4 ligase action upon renaturation. aDNA authenticity is proved by the homology of the nucleotide sequences of loci tested to the corresponding modern Equidae sequences. Data also indicate that cross-linked homoduplex molecules selected by denaturation of the extract are repaired without any chimera formation. The general features of aDNA amplification with and without denaturation and enzymatic repair are discussed.

Enzymatic repair of selected cross-linked homoduplex molecules enhances nuclear gene rescue from Pompeii and Herculaneum remains

PubMed Central

Di Bernardo, Giovanni; Del Gaudio, Stefania; Cammarota, Marcella; Galderisi, Umberto; Cascino, Antonino; Cipollaro, Marilena

2002-01-01

Ancient DNA (aDNA) samples extracted from the bone remains of six equids buried by the Vesuvius eruption in 79 AD were investigated to test pre-amplification and enzymatic repair procedures designed to enhance the rescue of nuclear genes. The extracts, which proved all positive for Equidae mtDNA amplification, proved positive only four times out of 18 when tested for single-copy Equidae nuclear genes (ɛ globin, p53 and γ interferon). Pre-amplification did not change the number of retrieved aDNA sequences but 10 times out of 14 enzymatic repair restored the amplifiability of the genes analysed, proving that repair increases the rate of successful rescue from 22 to αλµοστ 80%. These findings support the hypothesis that some of these cross-linked aDNA molecules, which are not completely separated when DNA is extracted under denaturing conditions, become homoduplex substrates for Pol I and/or T4 ligase action upon renaturation. aDNA authenticity is proved by the homology of the nucleotide sequences of loci tested to the corresponding modern Equidae sequences. Data also indicate that cross-linked homoduplex molecules selected by denaturation of the extract are repaired without any chimera formation. The general features of aDNA amplification with and without denaturation and enzymatic repair are discussed. PMID:11842122

A method for release and multiple strand amplification of small quantities of DNA from endospores of the fastidious bacterium Pasteuria penetrans.

PubMed

Mauchline, T H; Mohan, S; Davies, K G; Schaff, J E; Opperman, C H; Kerry, B R; Hirsch, P R

2010-05-01

To establish a reliable protocol to extract DNA from Pasteuria penetrans endospores for use as template in multiple strand amplification, thus providing sufficient material for genetic analyses. To develop a highly sensitive PCR-based diagnostic tool for P. penetrans. An optimized method to decontaminate endospores, release and purify DNA enabled multiple strand amplification. DNA purity was assessed by cloning and sequencing gyrB and 16S rRNA gene fragments obtained from PCR using generic primers. Samples indicated to be 100%P. penetrans by the gyrB assay were estimated at 46% using the 16S rRNA gene. No bias was detected on cloning and sequencing 12 housekeeping and sporulation gene fragments from amplified DNA. The detection limit by PCR with Pasteuria-specific 16S rRNA gene primers following multiple strand amplification of DNA extracted using the method was a single endospore. Generation of large quantities DNA will facilitate genomic sequencing of P. penetrans. Apparent differences in sample purity are explained by variations in 16S rRNA gene copy number in Eubacteria leading to exaggerated estimations of sample contamination. Detection of single endospores will facilitate investigations of P. penetrans molecular ecology. These methods will advance studies on P. penetrans and facilitate research on other obligate and fastidious micro-organisms where it is currently impractical to obtain DNA in sufficient quantity and quality.

Automated real-time detection of drug-resistant Mycobacterium tuberculosis on a lab-on-a-disc by Recombinase Polymerase Amplification.

PubMed

Law, I L G; Loo, J F C; Kwok, H C; Yeung, H Y; Leung, C C H; Hui, M; Wu, S Y; Chan, H S; Kwan, Y W; Ho, H P; Kong, S K

2018-03-01

With the emergence of multi- and extensive-drug (MDR/XDR) resistant Mycobacterium tuberculosis (M. tb), tuberculosis (TB) persists as one of the world's leading causes of death. Recently, isothermal DNA amplification methods received much attention due to their ease of translation onto portable point-of-care (POC) devices for TB diagnosis. In this study, we aimed to devise a simple yet robust detection method for M. tb. Amongst the numerous up-and-coming isothermal techniques, Recombinase Polymerase Amplification (RPA) was chosen for a real-time detection of TB with or without MDR. In our platform, real-time RPA (RT-RPA) was integrated on a lab-on-a-disc (LOAD) with on-board power to maintain temperature for DNA amplification. Sputa collected from healthy volunteers were spiked with respective target M. tb samples for testing. A limit of detection of 10 2  colony-forming unit per millilitre in 15 min was achieved, making early detection and differentiation of M. tb strains highly feasible in extreme POC settings. Our RT-RPA LOAD platform has also been successfully applied in the differentiation of MDR-TB from H37Ra, an attenuated TB strain. In summary, a quantitative RT-RPA on LOAD assay with a high level of sensitivity was developed as a foundation for further developments in medical bedside and POC diagnostics. Copyright © 2018 Elsevier Inc. All rights reserved.

Applying DNA Barcodes to Identify Closely Related Species of Ferns: A Case Study of the Chinese Adiantum (Pteridaceae)

PubMed Central

Wen, Jun; Ebihara, Atsushi; Li, De-Zhu

2016-01-01

DNA barcoding is a fast-developing technique to identify species by using short and standard DNA sequences. Universal selection of DNA barcodes in ferns remains unresolved. In this study, five plastid regions (rbcL, matK, trnH-psbA, trnL-F and rps4-trnS) and eight nuclear regions (ITS, pgiC, gapC, LEAFY, ITS2, IBR3_2, DET1, and SQD1_1) were screened and evaluated in the fern genus Adiantum from China and neighboring areas. Due to low primer universality (matK) and/or the existence of multiple copies (ITS), the commonly used barcodes matK and ITS were not appropriate for Adiantum. The PCR amplification rate was extremely low in all nuclear genes except for IBR3_2. rbcL had the highest PCR amplification rate (94.33%) and sequencing success rate (90.78%), while trnH-psbA had the highest species identification rate (75%). With the consideration of discriminatory power, cost-efficiency and effort, the two-barcode combination of rbcL+ trnH-psbA seems to be the best choice for barcoding Adiantum, and perhaps basal polypod ferns in general. The nuclear IBR3_2 showed 100% PCR amplification success rate in Adiantum, however, it seemed that only diploid species could acquire clean sequences without cloning. With cloning, IBR3_2 can successfully distinguish cryptic species and hybrid species from their related species. Because hybridization and allopolyploidy are common in ferns, we argue for including a selected group of nuclear loci as barcodes, especially via the next-generation sequencing, as it is much more efficient to obtain single-copy nuclear loci without the cloning procedure. PMID:27603700

Controlled Microwave Heating Accelerates Rolling Circle Amplification

PubMed Central

Yoshimura, Takeo; Suzuki, Takamasa; Mineki, Shigeru; Ohuchi, Shokichi

2015-01-01

Rolling circle amplification (RCA) generates single-stranded DNAs or RNA, and the diverse applications of this isothermal technique range from the sensitive detection of nucleic acids to analysis of single nucleotide polymorphisms. Microwave chemistry is widely applied to increase reaction rate as well as product yield and purity. The objectives of the present research were to apply microwave heating to RCA and indicate factors that contribute to the microwave selective heating effect. The microwave reaction temperature was strictly controlled using a microwave applicator optimized for enzymatic-scale reactions. Here, we showed that microwave-assisted RCA reactions catalyzed by either of the four thermostable DNA polymerases were accelerated over 4-folds compared with conventional RCA. Furthermore, the temperatures of the individual buffer components were specifically influenced by microwave heating. We concluded that microwave heating accelerated isothermal RCA of DNA because of the differential heating mechanisms of microwaves on the temperatures of reaction components, although the overall reaction temperatures were the same. PMID:26348227

Detection of the Free-Living Forms of Sulfide-Oxidizing Gill Endosymbionts in the Lucinid Habitat (Thalassia testudinum Environment)

PubMed Central

Gros, Olivier; Liberge, Martine; Heddi, Abdelaziz; Khatchadourian, Chaqué; Felbeck, Horst

2003-01-01

Target DNA from the uncultivable Codakia orbicularis endosymbiont was PCR amplified from sea-grass sediment. To confirm that such amplifications originated from intact bacterial cells rather than free DNA, whole-cell hybridization (fluorescence in situ hybridization technique) with the specific probe Symco2 was performed along with experimental infection of aposymbiotic juveniles placed in contact with the same sediment. Taken together, the data demonstrate that the sulfide-oxidizing gill endosymbiont of Codakia orbicularis is present in the environment as a free-living uncultivable form. PMID:14532089

Nanoliter reactors improve multiple displacement amplification of genomes from single cells.

PubMed

Marcy, Yann; Ishoey, Thomas; Lasken, Roger S; Stockwell, Timothy B; Walenz, Brian P; Halpern, Aaron L; Beeson, Karen Y; Goldberg, Susanne M D; Quake, Stephen R

2007-09-01

Since only a small fraction of environmental bacteria are amenable to laboratory culture, there is great interest in genomic sequencing directly from single cells. Sufficient DNA for sequencing can be obtained from one cell by the Multiple Displacement Amplification (MDA) method, thereby eliminating the need to develop culture methods. Here we used a microfluidic device to isolate individual Escherichia coli and amplify genomic DNA by MDA in 60-nl reactions. Our results confirm a report that reduced MDA reaction volume lowers nonspecific synthesis that can result from contaminant DNA templates and unfavourable interaction between primers. The quality of the genome amplification was assessed by qPCR and compared favourably to single-cell amplifications performed in standard 50-microl volumes. Amplification bias was greatly reduced in nanoliter volumes, thereby providing a more even representation of all sequences. Single-cell amplicons from both microliter and nanoliter volumes provided high-quality sequence data by high-throughput pyrosequencing, thereby demonstrating a straightforward route to sequencing genomes from single cells.

Droplet digital polymerase chain reaction (PCR) outperforms real-time PCR in the detection of environmental DNA from an invasive fish species.

PubMed

Doi, Hideyuki; Takahara, Teruhiko; Minamoto, Toshifumi; Matsuhashi, Saeko; Uchii, Kimiko; Yamanaka, Hiroki

2015-05-05

Environmental DNA (eDNA) has been used to investigate species distributions in aquatic ecosystems. Most of these studies use real-time polymerase chain reaction (PCR) to detect eDNA in water; however, PCR amplification is often inhibited by the presence of organic and inorganic matter. In droplet digital PCR (ddPCR), the sample is partitioned into thousands of nanoliter droplets, and PCR inhibition may be reduced by the detection of the end-point of PCR amplification in each droplet, independent of the amplification efficiency. In addition, real-time PCR reagents can affect PCR amplification and consequently alter detection rates. We compared the effectiveness of ddPCR and real-time PCR using two different PCR reagents for the detection of the eDNA from invasive bluegill sunfish, Lepomis macrochirus, in ponds. We found that ddPCR had higher detection rates of bluegill eDNA in pond water than real-time PCR with either of the PCR reagents, especially at low DNA concentrations. Limits of DNA detection, which were tested by spiking the bluegill DNA to DNA extracts from the ponds containing natural inhibitors, found that ddPCR had higher detection rate than real-time PCR. Our results suggest that ddPCR is more resistant to the presence of PCR inhibitors in field samples than real-time PCR. Thus, ddPCR outperforms real-time PCR methods for detecting eDNA to document species distributions in natural habitats, especially in habitats with high concentrations of PCR inhibitors.

DNA Quantity and Quality in Remnants of Traffic-Killed Specimens of an Endangered Longhorn Beetle: A Comparison of Different Methods.

PubMed

Rusterholz, Hans-Peter; Ursenbacher, Sylvain; Coray, Armin; Weibel, Urs; Baur, Bruno

2015-01-01

The sampling of living insects should be avoided in highly endangered species when the sampling would further increase the risk of population extinction. Nonlethal sampling (wing clips or leg removals) can be an alternative to obtain DNA of individuals for population genetic studies. However, nonlethal sampling may not be possible for all insect species. We examined whether remnants of traffic-killed specimens of the endangered and protected flightless longhorn beetle Iberodorcadion fuliginator (L., 1758) can be used as a resource for population genetic analyses. Using insect fragments of traffic-killed specimens collected over 15 yr, we determined the most efficient DNA extraction method in relation to the state of the specimens (crushed, fragment, or intact), preservation (dried, airtight, or in ethanol), storage duration, and weight of the sample by assessing the quantity and quality of genomic DNA. A modified cetyltrimethyl ammonium bromide method provided the highest recovery rate of genomic DNA and the largest yield and highest quality of DNA. We further used traffic-killed specimens to evaluate two DNA amplification techniques (quantitative polymerase chain reaction [qPCR] and microsatellites). Both qPCR and microsatellites revealed successful DNA amplification in all degraded specimens or beetle fragments examined. However, relative qPCR concentration and peak height of microsatellites were affected by the state of specimen and storage duration but not by specimen weight. Our investigation demonstrates that degraded remnants of traffic-killed beetle specimens can serve as a source of high-quality genomic DNA, which allows to address conservation genetic issues. © The Author 2015. Published by Oxford University Press on behalf of the Entomological Society of America.

Periodic Assembly of Nanospecies on Repetitive DNA Sequences Generated on Gold Nanoparticles by Rolling Circle Amplification

NASA Astrophysics Data System (ADS)

Zhao, Weian; Brook, Michael A.; Li, Yingfu

Periodical assembly of nanospecies is desirable for the construction of nanodevices. We provide a protocol for the preparation of a gold nanoparticle (AuNP)/DNA scaffold on which nanospecies can be assembled in a periodical manner. AuNP/DNA scaffold is prepared by growing long single-stranded DNA (ssDNA) molecules (typically hundreds of nanometers to a few microns in length) on AuNPs via rolling circle amplification (RCA). Since these long ssDNA molecules contain many repetitive sequence units, complementary DNA-attached nanospecies can be assembled through specific hybridization in a controllable and periodical manner.

Cytochrome c oxidase I primers for corbiculate bees: DNA barcode and mini-barcode.

PubMed

Françoso, E; Arias, M C

2013-09-01

Bees (Apidae), of which there are more than 19 900 species, are extremely important for ecosystem services and economic purposes, so taxon identity is a major concern. The goal of this study was to optimize the DNA barcode technique based on the Cytochrome c oxidase (COI) mitochondrial gene region. This approach has previously been shown to be useful in resolving taxonomic inconsistencies and for species identification when morphological data are poor. Specifically, we designed and tested new primers and standardized PCR conditions to amplify the barcode region for bees, focusing on the corbiculate Apids. In addition, primers were designed to amplify small COI amplicons and tested with pinned specimens. Short barcode sequences were easily obtained for some Bombus century-old museum specimens and shown to be useful as mini-barcodes. The new primers and PCR conditions established in this study proved to be successful for the amplification of the barcode region for all species tested, regardless of the conditions of tissue preservation. We saw no evidence of Wolbachia or numts amplification by these primers, and so we suggest that these new primers are of broad value for corbiculate bee identification through DNA barcode. © 2013 John Wiley & Sons Ltd.

Random whole metagenomic sequencing for forensic discrimination of soils.

PubMed

Khodakova, Anastasia S; Smith, Renee J; Burgoyne, Leigh; Abarno, Damien; Linacre, Adrian

2014-01-01

Here we assess the ability of random whole metagenomic sequencing approaches to discriminate between similar soils from two geographically distinct urban sites for application in forensic science. Repeat samples from two parklands in residential areas separated by approximately 3 km were collected and the DNA was extracted. Shotgun, whole genome amplification (WGA) and single arbitrarily primed DNA amplification (AP-PCR) based sequencing techniques were then used to generate soil metagenomic profiles. Full and subsampled metagenomic datasets were then annotated against M5NR/M5RNA (taxonomic classification) and SEED Subsystems (metabolic classification) databases. Further comparative analyses were performed using a number of statistical tools including: hierarchical agglomerative clustering (CLUSTER); similarity profile analysis (SIMPROF); non-metric multidimensional scaling (NMDS); and canonical analysis of principal coordinates (CAP) at all major levels of taxonomic and metabolic classification. Our data showed that shotgun and WGA-based approaches generated highly similar metagenomic profiles for the soil samples such that the soil samples could not be distinguished accurately. An AP-PCR based approach was shown to be successful at obtaining reproducible site-specific metagenomic DNA profiles, which in turn were employed for successful discrimination of visually similar soil samples collected from two different locations.

NASBA: A detection and amplification system uniquely suited for RNA

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sooknanan, R.; Malek, L.T.

1995-06-01

The invention of PCR (polymerase chain reaction) has revolutionized our ability to amplify and manipulate a nucleic acid sequence in vitro. The commercial rewards of this revolution have driven the development of other nuclei acid amplification and detection methodologies. This has created an alphabet soup of technologies that use different amplification methods, including NASBA (nucleic acid sequence-based amplification), LCR (ligase chain reaction), SDA (strand displacement amplification), QBR (Q-beta replicase), CPR (cycling probe reaction), and bDNA (branched DNA). Despite the differences in their processes, these amplification systems can be separated into two broad categories based on how they achieve their goal:more » sequence-based amplification systems, such as PCR, NASBA, and SDA, amplify a target nucleic acid sequence. Signal-based amplification systems, such as LCR, QBR, CPR and bDNA, amplify or alter a signal from a detection reaction that is target-dependent. While the various methods have relative strengths and weaknesses, only NASBA offers the unique ability to homogeneously amplify an RNA analyte in the presence of homologous genomic DNA under isothermal conditions. Since the detection of RNA sequences almost invariably measures biological activity, it is an excellent prognostic indicator of activities as diverse as virus production, gene expression, and cell viability. The isothermal nature of the reaction makes NASBA especially suitable for large-scale manual screening. These features extend NASBA`s application range from research to commercial diagnostic applications. Field test kits are presently under development for human diagnostics as well as the burgeoning fields of food and environmental diagnostic testing. These developments suggest future integration of NASBA into robotic workstations for high-throughput screening as well. 17 refs., 1 tab.« less

Computational analysis of stochastic heterogeneity in PCR amplification efficiency revealed by single molecule barcoding

PubMed Central

Best, Katharine; Oakes, Theres; Heather, James M.; Shawe-Taylor, John; Chain, Benny

2015-01-01

The polymerase chain reaction (PCR) is one of the most widely used techniques in molecular biology. In combination with High Throughput Sequencing (HTS), PCR is widely used to quantify transcript abundance for RNA-seq, and in the context of analysis of T and B cell receptor repertoires. In this study, we combine DNA barcoding with HTS to quantify PCR output from individual target molecules. We develop computational tools that simulate both the PCR branching process itself, and the subsequent subsampling which typically occurs during HTS sequencing. We explore the influence of different types of heterogeneity on sequencing output, and compare them to experimental results where the efficiency of amplification is measured by barcodes uniquely identifying each molecule of starting template. Our results demonstrate that the PCR process introduces substantial amplification heterogeneity, independent of primer sequence and bulk experimental conditions. This heterogeneity can be attributed both to inherited differences between different template DNA molecules, and the inherent stochasticity of the PCR process. The results demonstrate that PCR heterogeneity arises even when reaction and substrate conditions are kept as constant as possible, and therefore single molecule barcoding is essential in order to derive reproducible quantitative results from any protocol combining PCR with HTS. PMID:26459131

Development of a loop-mediated isothermal amplification method for rapid mass-screening of sand flies for Leishmania infection.

PubMed

Nzelu, Chukwunonso O; Gomez, Eduardo A; Cáceres, Abraham G; Sakurai, Tatsuya; Martini-Robles, Luiggi; Uezato, Hiroshi; Mimori, Tatsuyuki; Katakura, Ken; Hashiguchi, Yoshihisa; Kato, Hirotomo

2014-04-01

Entomological monitoring of Leishmania infection in leishmaniasis endemic areas offers epidemiologic advantages for predicting the risk and expansion of the disease, as well as evaluation of the effectiveness of control programs. In this study, we developed a highly sensitive loop-mediated isothermal amplification (LAMP) method for the mass screening of sand flies for Leishmania infection based on the 18S rRNA gene. The LAMP technique could detect 0.01 parasites, which was more sensitive than classical PCR. The method was robust and could amplify the target DNA within 1h from a crude sand fly template without DNA purification. Amplicon detection could be accomplished by the newly developed colorimetric malachite green (MG)--mediated naked eye visualization. Pre-addition of MG to the LAMP reaction solution did not inhibit amplification efficiency. The field applicability of the colorimetric MG-based LAMP assay was demonstrated with 397 field-caught samples from the endemic areas of Ecuador and eight positive sand flies were detected. The robustness, superior sensitivity, and ability to produce better visual discriminatory reaction products than existing LAMP fluorescence and turbidity assays indicated the field potential usefulness of this new method for surveillance and epidemiological studies of leishmaniasis in developing countries. Copyright © 2013 Elsevier B.V. All rights reserved.

Nucleic acid purification from plants, animals and microbes in under 30 seconds

PubMed Central

Zou, Yiping; Wang, Yuling; Wee, Eugene; Turni, Conny; Blackall, Patrick J.; Trau, Matt; Botella, Jose Ramon

2017-01-01

Nucleic acid amplification is a powerful molecular biology tool, although its use outside the modern laboratory environment is limited due to the relatively cumbersome methods required to extract nucleic acids from biological samples. To address this issue, we investigated a variety of materials for their suitability for nucleic acid capture and purification. We report here that untreated cellulose-based paper can rapidly capture nucleic acids within seconds and retain them during a single washing step, while contaminants present in complex biological samples are quickly removed. Building on this knowledge, we have successfully created an equipment-free nucleic acid extraction dipstick methodology that can obtain amplification-ready DNA and RNA from plants, animals, and microbes from difficult biological samples such as blood and leaves from adult trees in less than 30 seconds. The simplicity and speed of this method as well as the low cost and availability of suitable materials (e.g., common paper towelling), means that nucleic acid extraction is now more accessible and affordable for researchers and the broader community. Furthermore, when combined with recent advancements in isothermal amplification and naked eye DNA visualization techniques, the dipstick extraction technology makes performing molecular diagnostic assays achievable in limited resource settings including university and high school classrooms, field-based environments, and developing countries. PMID:29161268

Linear nicking endonuclease-mediated strand-displacement DNA amplification.

PubMed

Joneja, Aric; Huang, Xiaohua

2011-07-01

We describe a method for linear isothermal DNA amplification using nicking endonuclease-mediated strand displacement by a DNA polymerase. The nicking of one strand of a DNA target by the endonuclease produces a primer for the polymerase to initiate synthesis. As the polymerization proceeds, the downstream strand is displaced into a single-stranded form while the nicking site is also regenerated. The combined continuous repetitive action of nicking by the endonuclease and strand-displacement synthesis by the polymerase results in linear amplification of one strand of the DNA molecule. We demonstrate that DNA templates up to 5000 nucleotides can be linearly amplified using a nicking endonuclease with 7-bp recognition sequence and Sequenase version 2.0 in the presence of single-stranded DNA binding proteins. We also show that a mixture of three templates of 500, 1000, and 5000 nucleotides in length is linearly amplified with the original molar ratios of the templates preserved. Moreover, we demonstrate that a complex library of hydrodynamically sheared genomic DNA from bacteriophage lambda can be amplified linearly. Copyright © 2011 Elsevier Inc. All rights reserved.

Linear nicking endonuclease-mediated strand displacement DNA amplification

PubMed Central

Joneja, Aric; Huang, Xiaohua

2011-01-01

We describe a method for linear isothermal DNA amplification using nicking endonuclease-mediated strand displacement by a DNA polymerase. The nicking of one strand of a DNA target by the endonuclease produces a primer for the polymerase to initiate synthesis. As the polymerization proceeds, the downstream strand is displaced into a single-stranded form while the nicking site is also regenerated. The combined continuous repetitive action of nicking by the endonuclease and strand displacement synthesis by the polymerase results in linear amplification of one strand of the DNA molecule. We demonstrate that DNA templates up to five thousand nucleotides can be linearly amplified using a nicking endonuclease with seven base-pair recognition sequence and Sequenase version 2.0 in the presence of single-stranded DNA binding proteins. We also show that a mixture of three templates of 500, 1000, and 5000 nucleotides in length are linearly amplified with the original molar ratios of the templates preserved. Moreover, we demonstrate that a complex library of hydrodynamically sheared genomic DNA from bacteriophage lambda can be amplified linearly. PMID:21342654

The impact of time and field conditions on brown bear (Ursus arctos) faecal DNA amplification

USGS Publications Warehouse

Murphy, M.A.; Kendall, K.C.; Robinson, A.; Waits, L.P.

2007-01-01

To establish longevity of faecal DNA samples under varying summer field conditions, we collected 53 faeces from captive brown bears (Ursus arctos) on a restricted vegetation diet. Each faeces was divided, and one half was placed on a warm, dry field site while the other half was placed on a cool, wet field site on Moscow Mountain, Idaho, USA. Temperature, relative humidity, and dew point data were collected on each site, and faeces were sampled for DNA extraction at <1, 3, 6, 14, 30, 45, and 60 days. Faecal DNA sample viability was assessed by attempting PCR amplification of a mitochondrial DNA (mtDNA) locus (???150 bp) and a nuclear DNA (nDNA) microsatellite locus (180-200 bp). Time in the field, temperature, and dew point impacted mtDNA and nDNA amplification success with the greatest drop in success rates occurring between 1 and 3 days. In addition, genotyping errors significantly increased over time at both field sites. Based on these results, we recommend collecting samples at frequent transect intervals and focusing sampling efforts during drier portions of the year when possible. ?? 2007 Springer Science+Business Media, Inc.

Quantification of HIV-1 DNA using real-time recombinase polymerase amplification.

PubMed

Crannell, Zachary Austin; Rohrman, Brittany; Richards-Kortum, Rebecca

2014-06-17

Although recombinase polymerase amplification (RPA) has many advantages for the detection of pathogenic nucleic acids in point-of-care applications, RPA has not yet been implemented to quantify sample concentration using a standard curve. Here, we describe a real-time RPA assay with an internal positive control and an algorithm that analyzes real-time fluorescence data to quantify HIV-1 DNA. We show that DNA concentration and the onset of detectable amplification are correlated by an exponential standard curve. In a set of experiments in which the standard curve and algorithm were used to analyze and quantify additional DNA samples, the algorithm predicted an average concentration within 1 order of magnitude of the correct concentration for all HIV-1 DNA concentrations tested. These results suggest that quantitative RPA (qRPA) may serve as a powerful tool for quantifying nucleic acids and may be adapted for use in single-sample point-of-care diagnostic systems.

DNA Extraction from Museum Specimens of Parasitic Hymenoptera

PubMed Central

Andersen, Jeremy C.; Mills, Nicholas J.

2012-01-01

At the same time that molecular researchers are improving techniques to extract DNA from museum specimens, this increased demand for access to museum specimens has created tension between the need to preserve specimens for maintaining collections and morphological research and the desire to conduct molecular analyses. To address these concerns, we examined the suitability of non-invasive DNA extraction techniques on three species of parasitic Hymenoptera (Braconidae), and test the effects of body size (parasitoid species), age (time since collection), and DNA concentration from each extract on the probability of amplifying meaningful fragments of two commonly used genetic loci. We found that age was a significant factor for determining the probability of success for sequencing both 28S and COI fragments. While the size of the braconid parasitoids significantly affected the total amount of extracted DNA, neither size nor DNA concentration were significant factors for the amplification of either gene region. We also tested several primer combinations of various lengths, but were unable to amplify fragments longer than ∼150 base pairs. These short fragments of 28S and COI were however sufficient for species identification, and for the discovery of within species genetic variation. PMID:23077493

Ultrasensitive and selective signal-on electrochemical DNA detection via exonuclease III catalysis and hybridization chain reaction amplification.

PubMed

Ren, Wang; Gao, Zhong Feng; Li, Nian Bing; Luo, Hong Qun

2015-01-15

This work reported a novel, ultrasensitive, and selective platform for electrochemical detection of DNA, employing an integration of exonuclease III (Exo-III) assisted target recycling and hybridization chain reaction (HCR) for the dual signal amplification strategy. The hairpin capture probe DNA (C-DNA) with an Exo-III 3' overhang end was self-assembled on a gold electrode. In the presence of target DNA (T-DNA), C-DNA hybridized with the T-DNA to form a duplex region, exposing its 5' complementary sequence (initiator). Exo-III was applied to selectively digest duplex region from its 3-hydroxyl termini until the duplex was fully consumed, leaving the remnant initiator. The intact T-DNA spontaneously dissociated from the structure and then initiated the next hybridization process as a result of catalysis of the Exo-III. HCR event was triggered by the initiator and two hairpin helper signal probes labeled with methylene blue, facilitating the polymerization of oligonucleotides into a long nicked dsDNA molecule. The numerous exposed remnant initiators can trigger more HCR events. Because of integration of dual signal amplification and the specific HCR process reaction, the resultant sensor showed a high sensitivity for the detection of the target DNA in a linear range from 1.0 fM to 1.0 nM, and a detection limit as low as 0.2 fM. The proposed dual signal amplification strategy provides a powerful tool for detecting different sequences of target DNA by changing the sequence of capture probe and signal probes, holding a great potential for early diagnosis in gene-related diseases. Copyright © 2014 Elsevier B.V. All rights reserved.

Absolute quantification of olive oil DNA by droplet digital-PCR (ddPCR): Comparison of isolation and amplification methodologies.

PubMed

Scollo, Francesco; Egea, Leticia A; Gentile, Alessandra; La Malfa, Stefano; Dorado, Gabriel; Hernandez, Pilar

2016-12-15

Olive oil is considered a premium product for its nutritional value and health benefits, and the ability to define its origin and varietal composition is a key step towards ensuring the traceability of the product. However, isolating the DNA from such a matrix is a difficult task. In this study, the quality and quantity of olive oil DNA, isolated using four different DNA isolation protocols, was evaluated using the qRT-PCR and ddPCR techniques. The results indicate that CTAB-based extraction methods were the best for unfiltered oil, while Nucleo Spin-based extraction protocols showed greater overall reproducibility. The use of both qRT-PCR and ddPCR led to the absolute quantification of the DNA copy number. The results clearly demonstrate the importance of the choice of DNA-isolation protocol, which should take into consideration the qualitative aspects of DNA and the evaluation of the amplified DNA copy number. Copyright © 2016 Elsevier Ltd. All rights reserved.

Ancient DNA in human bone remains from Pompeii archaeological site.

PubMed

Cipollaro, M; Di Bernardo, G; Galano, G; Galderisi, U; Guarino, F; Angelini, F; Cascino, A

1998-06-29

aDNA extraction and amplification procedures have been optimized for Pompeian human bone remains whose diagenesis has been determined by histological analysis. Single copy genes amplification (X and Y amelogenin loci and Y specific alphoid repeat sequences) have been performed and compared with anthropometric data on sexing.

The Effect of Storage and Extraction Methods on Amplification of Plasmodium falciparum DNA from Dried Blood Spots.

PubMed

Schwartz, Alanna; Baidjoe, Amrish; Rosenthal, Philip J; Dorsey, Grant; Bousema, Teun; Greenhouse, Bryan

2015-05-01

Extraction and amplification of DNA from dried blood spots (DBS) collected in field studies is commonly used for detection of Plasmodium falciparum. However, there have been few systematic efforts to determine the effects of storage and extraction methods on the sensitivity of DNA amplification. We investigated the effects of storage conditions, length of storage, and DNA extraction methods on amplification via three PCR-based assays using field samples and laboratory controls. Samples stored as DBS for 2 or more years at ambient temperature showed a significant loss of sensitivity that increased with time; after 10 years only 10% samples with parasite densities > 1,000 parasites/μL were detectable by nested polymerase chain reaction (PCR). Conversely, DBS and extracted DNA stored at -20°C showed no loss of sensitivity with time. Samples with low parasite densities amplified more successfully with saponin/Chelex compared with spin-column-based extraction, though the latter method performed better on samples with higher parasite densities stored for 2 years at ambient temperature. DNA extracted via both methods was stable after 20 freeze-thaw cycles. Our results suggest that DBS should be stored at -20°C or extracted immediately, especially if anticipating 2 or more years of storage. © The American Society of Tropical Medicine and Hygiene.

The Effect of Storage and Extraction Methods on Amplification of Plasmodium falciparum DNA from Dried Blood Spots

PubMed Central

Schwartz, Alanna; Baidjoe, Amrish; Rosenthal, Philip J.; Dorsey, Grant; Bousema, Teun; Greenhouse, Bryan

2015-01-01

Extraction and amplification of DNA from dried blood spots (DBS) collected in field studies is commonly used for detection of Plasmodium falciparum. However, there have been few systematic efforts to determine the effects of storage and extraction methods on the sensitivity of DNA amplification. We investigated the effects of storage conditions, length of storage, and DNA extraction methods on amplification via three PCR-based assays using field samples and laboratory controls. Samples stored as DBS for 2 or more years at ambient temperature showed a significant loss of sensitivity that increased with time; after 10 years only 10% samples with parasite densities > 1,000 parasites/μL were detectable by nested polymerase chain reaction (PCR). Conversely, DBS and extracted DNA stored at −20°C showed no loss of sensitivity with time. Samples with low parasite densities amplified more successfully with saponin/Chelex compared with spin-column-based extraction, though the latter method performed better on samples with higher parasite densities stored for 2 years at ambient temperature. DNA extracted via both methods was stable after 20 freeze-thaw cycles. Our results suggest that DBS should be stored at −20°C or extracted immediately, especially if anticipating 2 or more years of storage. PMID:25758652

Assessment of primer/template mismatch effects on real-time PCR amplification of target taxa for GMO quantification.

PubMed

Ghedira, Rim; Papazova, Nina; Vuylsteke, Marnik; Ruttink, Tom; Taverniers, Isabel; De Loose, Marc

2009-10-28

GMO quantification, based on real-time PCR, relies on the amplification of an event-specific transgene assay and a species-specific reference assay. The uniformity of the nucleotide sequences targeted by both assays across various transgenic varieties is an important prerequisite for correct quantification. Single nucleotide polymorphisms (SNPs) frequently occur in the maize genome and might lead to nucleotide variation in regions used to design primers and probes for reference assays. Further, they may affect the annealing of the primer to the template and reduce the efficiency of DNA amplification. We assessed the effect of a minor DNA template modification, such as a single base pair mismatch in the primer attachment site, on real-time PCR quantification. A model system was used based on the introduction of artificial mismatches between the forward primer and the DNA template in the reference assay targeting the maize starch synthase (SSIIb) gene. The results show that the presence of a mismatch between the primer and the DNA template causes partial to complete failure of the amplification of the initial DNA template depending on the type and location of the nucleotide mismatch. With this study, we show that the presence of a primer/template mismatch affects the estimated total DNA quantity to a varying degree.

Detection of the CLOCK/BMAL1 heterodimer using a nucleic acid probe with cycling probe technology.

PubMed

Nakagawa, Kazuhiro; Yamamoto, Takuro; Yasuda, Akio

2010-09-15

An isothermal signal amplification technique for specific DNA sequences, known as cycling probe technology (CPT), has enabled rapid acquisition of genomic information. Here we report an analogous technique for the detection of an activated transcription factor, a transcription element-binding assay with fluorescent amplification by apurinic/apyrimidinic (AP) site lysis cycle (TEFAL). This simple amplification assay can detect activated transcription factors by using a unique nucleic acid probe containing a consensus binding sequence and an AP site, which enables the CPT reaction with AP endonuclease. In this article, we demonstrate that this method detects the functional CLOCK/BMAL1 heterodimer via the TEFAL probe containing the E-box consensus sequence to which the CLOCK/BMAL1 heterodimer binds. Using TEFAL combined with immunoassays, we measured oscillations in the amount of CLOCK/BMAL1 heterodimer in serum-stimulated HeLa cells. Furthermore, we succeeded in measuring the circadian accumulation of the functional CLOCK/BMAL1 heterodimer in human buccal mucosa cells. TEFAL contributes greatly to the study of transcription factor activation in mammalian tissues and cell extracts and is a powerful tool for less invasive investigation of human circadian rhythms. 2010 Elsevier Inc. All rights reserved.

Simple and Sensitive Quantification of MicroRNAs via PS@Au Microspheres-Based DNA Probes and DSN-Assisted Signal Amplification Platform.

PubMed

Zhao, Qian; Piao, Jiafang; Peng, Weipan; Wang, Yang; Zhang, Bo; Gong, Xiaoqun; Chang, Jin

2018-01-31

Identifying the microRNA (miRNA) expression level can provide critical information for early diagnosis of cancers or monitoring the cancer therapeutic efficacy. This paper focused on a kind of gold-nanoparticle-coated polystyrene microbeads (PS@Au microspheres)-based DNA probe as miRNA capture and duplex-specific nuclease (DSN) signal amplification platform based on an RGB value readout for detection of miRNAs. In virtue of the outstanding selectivity and simple experimental operation, 5'-fluorochrome-labeled molecular beacons (MBs) were immobilized on PS@Au microspheres via their 3'-thiol, in the wake of the fluorescence quenching by nanoparticle surface energy transfer (NSET). Target miRNAs were captured by the PS@Au microspheres-based DNA probe through DNA/RNA hybridization. DSN enzyme subsequently selectively cleaved the DNA to recycle the target miRNA and release of fluorophores, thereby triggering the signal amplification with more free fluorophores. The RGB value measurement enabled a detection limit of 50 fM, almost 4 orders of magnitude lower than PS@Au microspheres-based DNA probe detection without DSN. Meanwhile, by different encoding of dyes, miRNA-21 and miRNA-10b were simultaneously detected in the same sample. Considering the ability for quantitation, high sensitivity, and convenient merits, the PS@Au microspheres-based DNA probe and DSN signal amplification platform supplied valuable information for early diagnosis of cancers.

Comparison of three methods of DNA extraction from peripheral blood mononuclear cells and lung fragments of equines.

PubMed

Santos, E M; Paula, J F R; Motta, P M C; Heinemann, M B; Leite, R C; Haddad, J P A; Del Puerto, H L; Reis, J K P

2010-08-17

We compared three different protocols for DNA extraction from horse peripheral blood mononuclear cells (PBMC) and lung fragments, determining average final DNA concentration, purity, percentage of PCR amplification using beta-actin, and cost. Thirty-four samples from PBMC, and 33 samples from lung fragments were submitted to DNA extraction by three different protocols. Protocol A consisted of a phenol-chloroform and isoamylic alcohol extraction, Protocol B used alkaline extraction with NaOH, and Protocol C used the DNAzol((R)) reagent kit. Protocol A was the best option for DNA extraction from lung fragments, producing high DNA concentrations, with high sensitivity in PCR amplification (100%), followed by Protocols C and B. On the other hand, for PBMC samples, Protocol B gave the highest sensitivity in PCR amplification (100%), followed by Protocols C and A. We conclude that Protocol A should be used for PCR diagnosis from lung fragment samples, while Protocol B should be used for PBMC.

Enrichment of methylated molecules using enhanced-ice-co-amplification at lower denaturation temperature-PCR (E-ice-COLD-PCR) for the sensitive detection of disease-related hypermethylation.

PubMed

Mauger, Florence; Kernaleguen, Magali; Lallemand, Céline; Kristensen, Vessela N; Deleuze, Jean-François; Tost, Jörg

2018-05-01

The detection of specific DNA methylation patterns bears great promise as biomarker for personalized management of cancer patients. Co-amplification at lower denaturation temperature-PCR (COLD-PCR) assays are sensitive methods, but have previously only been able to analyze loss of DNA methylation. Enhanced (E)-ice-COLD-PCR reactions starting from 2 ng of bisulfite-converted DNA were developed to analyze methylation patterns in two promoters with locked nucleic acid (LNA) probes blocking amplification of unmethylated CpGs. The enrichment of methylated molecules was compared to quantitative (q)PCR and quantified using serial dilutions. E-ice-COLD-PCR allowed the multiplexed enrichment and quantification of methylated DNA. Assays were validated in primary breast cancer specimens and circulating cell-free DNA from cancer patients. E-ice-COLD-PCR could prove a useful tool in the context of DNA methylation analysis for personalized medicine.

Single-Cell-Based Platform for Copy Number Variation Profiling through Digital Counting of Amplified Genomic DNA Fragments.

PubMed

Li, Chunmei; Yu, Zhilong; Fu, Yusi; Pang, Yuhong; Huang, Yanyi

2017-04-26

We develop a novel single-cell-based platform through digital counting of amplified genomic DNA fragments, named multifraction amplification (mfA), to detect the copy number variations (CNVs) in a single cell. Amplification is required to acquire genomic information from a single cell, while introducing unavoidable bias. Unlike prevalent methods that directly infer CNV profiles from the pattern of sequencing depth, our mfA platform denatures and separates the DNA molecules from a single cell into multiple fractions of a reaction mix before amplification. By examining the sequencing result of each fraction for a specific fragment and applying a segment-merge maximum likelihood algorithm to the calculation of copy number, we digitize the sequencing-depth-based CNV identification and thus provide a method that is less sensitive to the amplification bias. In this paper, we demonstrate a mfA platform through multiple displacement amplification (MDA) chemistry. When performing the mfA platform, the noise of MDA is reduced; therefore, the resolution of single-cell CNV identification can be improved to 100 kb. We can also determine the genomic region free of allelic drop-out with mfA platform, which is impossible for conventional single-cell amplification methods.

NAIMA: target amplification strategy allowing quantitative on-chip detection of GMOs.

PubMed

Morisset, Dany; Dobnik, David; Hamels, Sandrine; Zel, Jana; Gruden, Kristina

2008-10-01

We have developed a novel multiplex quantitative DNA-based target amplification method suitable for sensitive, specific and quantitative detection on microarray. This new method named NASBA Implemented Microarray Analysis (NAIMA) was applied to GMO detection in food and feed, but its application can be extended to all fields of biology requiring simultaneous detection of low copy number DNA targets. In a first step, the use of tailed primers allows the multiplex synthesis of template DNAs in a primer extension reaction. A second step of the procedure consists of transcription-based amplification using universal primers. The cRNA product is further on directly ligated to fluorescent dyes labelled 3DNA dendrimers allowing signal amplification and hybridized without further purification on an oligonucleotide probe-based microarray for multiplex detection. Two triplex systems have been applied to test maize samples containing several transgenic lines, and NAIMA has shown to be sensitive down to two target copies and to provide quantitative data on the transgenic contents in a range of 0.1-25%. Performances of NAIMA are comparable to singleplex quantitative real-time PCR. In addition, NAIMA amplification is faster since 20 min are sufficient to achieve full amplification.

NAIMA: target amplification strategy allowing quantitative on-chip detection of GMOs

PubMed Central

Morisset, Dany; Dobnik, David; Hamels, Sandrine; Žel, Jana; Gruden, Kristina

2008-01-01

We have developed a novel multiplex quantitative DNA-based target amplification method suitable for sensitive, specific and quantitative detection on microarray. This new method named NASBA Implemented Microarray Analysis (NAIMA) was applied to GMO detection in food and feed, but its application can be extended to all fields of biology requiring simultaneous detection of low copy number DNA targets. In a first step, the use of tailed primers allows the multiplex synthesis of template DNAs in a primer extension reaction. A second step of the procedure consists of transcription-based amplification using universal primers. The cRNA product is further on directly ligated to fluorescent dyes labelled 3DNA dendrimers allowing signal amplification and hybridized without further purification on an oligonucleotide probe-based microarray for multiplex detection. Two triplex systems have been applied to test maize samples containing several transgenic lines, and NAIMA has shown to be sensitive down to two target copies and to provide quantitative data on the transgenic contents in a range of 0.1–25%. Performances of NAIMA are comparable to singleplex quantitative real-time PCR. In addition, NAIMA amplification is faster since 20 min are sufficient to achieve full amplification. PMID:18710880

A nonenzymatic DNA nanomachine for biomolecular detection by target recycling of hairpin DNA cascade amplification.

PubMed

Zheng, Jiao; Li, Ningxing; Li, Chunrong; Wang, Xinxin; Liu, Yucheng; Mao, Guobin; Ji, Xinghu; He, Zhike

2018-06-01

Synthetic enzyme-free DNA nanomachine performs quasi-mechanical movements in response to external intervention, suggesting the promise of constructing sensitive and specific biosensors. Herein, a smart DNA nanomachine biosensor for biomolecule (such as nucleic acid, thrombin and adenosine) detection is developed by target-assisted enzyme-free hairpin DNA cascade amplifier. The whole DNA nanomachine system is constructed on gold nanoparticle which decorated with hundreds of locked hairpin substrate strands serving as DNA tracks, and the DNA nanomachine could be activated by target molecule toehold-mediated exchange on gold nanoparticle surface, resulted in the fluorescence recovery of fluorophore. The process is repeated so that each copy of the target can open multiplex fluorophore-labeled hairpin substrate strands, resulted in amplification of the fluorescence signal. Compared with the conventional biosensors of catalytic hairpin assembly (CHA) without substrate in solution, the DNA nanomachine could generate 2-3 orders of magnitude higher fluorescence signal. Furthermore, the DNA nanomachine could be used for nucleic acid, thrombin and adenosine highly sensitive specific detection based on isothermal, and homogeneous hairpin DNA cascade signal amplification in both buffer and a complicated biomatrix, and this kind of DNA nanomachine could be efficiently applied in the field of biomedical analysis. Copyright © 2018 Elsevier B.V. All rights reserved.

Detecting in situ copepod diet diversity using molecular technique: development of a copepod/symbiotic ciliate-excluding eukaryote-inclusive PCR protocol.

PubMed

Hu, Simin; Guo, Zhiling; Li, Tao; Carpenter, Edward J; Liu, Sheng; Lin, Senjie

2014-01-01

Knowledge of in situ copepod diet diversity is crucial for accurately describing pelagic food web structure but is challenging to achieve due to lack of an easily applicable methodology. To enable analysis with whole copepod-derived DNAs, we developed a copepod-excluding 18S rDNA-based PCR protocol. Although it is effective in depressing amplification of copepod 18S rDNA, its applicability to detect diverse eukaryotes in both mono- and mixed-species has not been demonstrated. Besides, the protocol suffers from the problem that sequences from symbiotic ciliates are overrepresented in the retrieved 18S rDNA libraries. In this study, we designed a blocking primer to make a combined primer set (copepod/symbiotic ciliate-excluding eukaryote-common: CEEC) to depress PCR amplification of symbiotic ciliate sequences while maximizing the range of eukaryotes amplified. We firstly examined the specificity and efficacy of CEEC by PCR-amplifying DNAs from 16 copepod species, 37 representative organisms that are potential prey of copepods and a natural microplankton sample, and then evaluated the efficiency in reconstructing diet composition by detecting the food of both lab-reared and field-collected copepods. Our results showed that the CEEC primer set can successfully amplify 18S rDNA from a wide range of isolated species and mixed-species samples while depressing amplification of that from copepod and targeted symbiotic ciliate, indicating the universality of CEEC in specifically detecting prey of copepods. All the predetermined food offered to copepods in the laboratory were successfully retrieved, suggesting that the CEEC-based protocol can accurately reconstruct the diets of copepods without interference of copepods and their associated ciliates present in the DNA samples. Our initial application to analyzing the food composition of field-collected copepods uncovered diverse prey species, including those currently known, and those that are unsuspected, as copepod prey. While testing is required, this protocol provides a useful strategy for depicting in situ dietary composition of copepods.

Attomolar quantitation of Mycobacterium tuberculosis by asymmetric helicase-dependent isothermal DNA-amplification and electrochemical detection.

PubMed

Barreda-García, Susana; González-Álvarez, María José; de-Los-Santos-Álvarez, Noemí; Palacios-Gutiérrez, Juan José; Miranda-Ordieres, Arturo J; Lobo-Castañón, María Jesús

2015-06-15

A highly sensitive and robust method for the quantification of specific DNA sequences based on coupling asymmetric helicase-dependent DNA amplification to electrochemical detection is described. This method relies on the entrapment of the amplified ssDNA sequences on magnetic beads followed by a post-amplification hybridization assay to provide an added degree of specificity. As a proof-of-concept a 84-bases long sequence specific of Mycobacterium tuberculosis is amplified at 65°C, providing 3×10(6) amplification after 90 min. Using this system 0.5 aM, corresponding to 15 copies of the target gene in 50 µL of sample, can be successfully detected and reliably quantified under isothermal conditions in less than 4h. The assay has been applied to the detection of M. tuberculosis in sputum, pleural fluid and urine samples. Besides this application, the proposed assays is a powerful and general tool for molecular diagnostic that can be applied to the detection of other specific DNA sequences, taking full advantage of the plethora of genomic information now available. Copyright © 2014 Elsevier B.V. All rights reserved.

Impacts of Sampling and Handling Procedures on DNA- and RNA-based Microbial Characterization and Quantification of Groundwater and Saturated Soil

DTIC Science & Technology

2012-07-01

use of molecular biological techniques (MBTs) has allowed microbial ecologists and environmental engineers to determine microbial community...metabolic genes). The most common approaches used in bioremediation research are those based on the polymerase chain reaction (PCR) amplification of... bioremediation . Because of its sensitivity compared to direct hybridization/probing, PCR is increasingly used to analyze groundwater samples and soil samples

[Estimation of time detection limit for human cytochrome b in females of Lutzomyia evansi].

PubMed

Vergara, José Gabriel; Verbel-Vergara, Daniel; Montesino, Ana Milena; Pérez-Doria, Alveiro; Bejarano, Eduar Elías

2017-03-29

Molecular biology techniques have allowed a better knowledge of sources of blood meals in vector insects. However, the usefulness of these techniques depends on both the quantity of ingested blood and the digestion process in the insect. To identify the time limit for detection of the human cytochrome b (Cyt b) gene in experimentally fed females of Lutzomyia evansi. Eight groups of L. evansi females were fed on human blood and sacrificed at intervals of 24 hours post-ingestion. Total DNA was extracted from each female and a segment of 358 bp of Cyt b was amplified. In order to eliminate false positives, amplification products were subjected to a restriction fragment length polymorphism (RFLP) analysis. The human Cyt b gene segment was detected in 86% (49/57) of the females of L. evansi, from 0 to 168 hours after blood ingestion. In 7% (4/57) of the individuals we amplified insect DNA, while in the remaining 7%, the band of interest was not amplified. We did not find any statistical differences between groups of females sacrificed at different times post-blood meal regarding the amplification of the human Cyt b gene segment or the number of samples amplified. The human Cyt b gene segment was detectable in L. evansi females up to 168 hours after blood ingestion.

Immunocapture loop-mediated isothermal amplification assays for the detection of canine parvovirus.

PubMed

Sun, Yu-Ling; Yen, Chon-Ho; Tu, Ching-Fu

2017-11-01

A loop-mediated isothermal amplification (LAMP) assay was used for rapid canine parvovirus (CPV) diagnosis. To reduce the time required and increase the sensitivity of the assay, an immunocapture (IC) technique was developed in this study to exclude the DNA extraction step in molecular diagnostic procedures for CPV. A polyclonal rabbit anti-CPV serum was produced against VP2-EpC that was cloned via DNA recombination. The polyclonal anti-VP2-EpC serum was used for virus capture to prepare microtubes. IC-LAMP was performed to amplify a specific CPV target gene sequence from the CPV viral particles that were captured on the microtubes, and the amplicons were analyzed using agarose electrophoresis or enzyme-linked immunosorbent assay (IC-LAMP-ELISA) and lateral-flow dipstick (IC-LAMP-LFD). The detection sensitivities of IC-LAMP, IC-LAMP-ELISA, and IC-LAMP-LFD were 10 -1 , 10 -1 , and 10 -1 TCID 50 /mL, respectively. Using the IC-LAMP-ELISA and IC-LAMP-LFD assays, the complete CPV diagnostic process can be achieved within 1.5h. Both of the developed IC-LAMP-based assays are simple, direct visual and efficient techniques that are applicable to the detection of CPV. Copyright © 2017 Elsevier B.V. All rights reserved.

Development of a rapid assay for the diagnosis of Myxobolus cerebralis in fish and oligochaetes using loop-mediated isothermal amplification.

PubMed

El-Matbouli, M; Soliman, H

2005-09-01

A loop-mediated isothermal amplification assay was developed for the rapid detection of Myxobolus cerebralis in both fish and oligochaete hosts. The assay was optimized to amplify parasitic DNA by incubation with Bst DNA polymerase and a set of six specially constructed primers at 65 degrees C for 60 min. The amplification products were detected visually using SYBR Green I dye which gave identical results to gel electrophoresis analysis. Parasite DNA was detected from infected oligochaetes, and from the anal fin, caudal fin, dorsal fin and operculum of clinically infected fish. This 'Myxo-LAMP' assay has a detection limit similar to that of a polymerase chain reaction assay (10(-6)), but is more rapid and only requires a water bath for amplification and is therefore practical for simple and rapid diagnosis of infected tissue.

Mitochondrial DNA Targets Increase Sensitivity of Malaria Detection Using Loop-Mediated Isothermal Amplification ▿

PubMed Central

Polley, Spencer D.; Mori, Yasuyoshi; Watson, Julie; Perkins, Mark D.; González, Iveth J.; Notomi, Tsugunori; Chiodini, Peter L.; Sutherland, Colin J.

2010-01-01

Loop-mediated isothermal amplification (LAMP) of DNA offers the ability to detect very small quantities of pathogen DNA following minimal tissue sample processing and is thus an attractive methodology for point-of-care diagnostics. Previous attempts to diagnose malaria by the use of blood samples and LAMP have targeted the parasite small-subunit rRNA gene, with a resultant sensitivity for Plasmodium falciparum of around 100 parasites per μl. Here we describe the use of mitochondrial targets for LAMP-based detection of any Plasmodium genus parasite and of P. falciparum specifically. These new targets allow routine amplification from samples containing as few as five parasites per μl of blood. Amplification is complete within 30 to 40 min and is assessed by real-time turbidimetry, thereby offering rapid diagnosis with greater sensitivity than is achieved by the most skilled microscopist or antigen detection using lateral flow immunoassays. PMID:20554824

Application of a molecular beacon based real-time isothermal amplification (MBRTIA) technology for simultaneous detection of Bacillus cereus and Staphylococcus aureus.

PubMed

Mandappa, I M; Joglekar, Prasanna; Manonmani, H K

2015-07-01

A multiplex real-time isothermal amplification assay was developed using molecular beacons for the detection of Bacillus cereus and Staphylococcus aureus by targeting four important virulence genes. A correlation between targeting highly accessible DNA sequences and isothermal amplification based molecular beacon efficiency and sensitivity was demonstrated using phi(Φ)29 DNA polymerase at a constant isothermal temperature of 30 °C. It was very selective and consistently detected down to 10(1) copies of DNA. The specificity and sensitivity of this assay, when tested with pure culture were high, surpassing those of currently used PCR assays for the detection of these organisms. The molecular beacon based real-time isothermal amplification (MBRTIA) assay could be carried out entirely in 96 well plates or well strips, enabling a rapid and high-throughput detection of food borne pathogens.

LAMP assay for rapid diagnosis of cow DNA in goat milk and meat samples.

PubMed

Deb, R; Sengar, G S; Singh, U; Kumar, S; Raja, T V; Alex, R; Alyethodi, R R; Prakash, B

2017-01-01

Animal species detection is one of the crucial steps for consumer's food analysis. In the present study we developed an in-house built loop-mediated isothermal amplification (LAMP) assay for rapid detection of adulterated cow DNA in goat milk/meat samples. The cow milk/tissue DNA in goat milk/meat samples were identified in the developed LAMP assay by either naked eye visualizing with SYBR Green I dyes or by detecting the typical ladder pattern on gel electrophoresis. This test can detect up to minimum 5% level of cow components admixed in goat milk/meat samples and can be completed within 1 h 40 min starting from DNA extraction from milk/meat samples and can be performed in a water bath. Developed LAMP methodology is simple; rapid and sensitive techniques that can detect adulterant like cow components in goat milk/meat are more accurate than other existing DNA based technologies.

Nucleotide-Specific Contrast for DNA Sequencing by Electron Spectroscopy.

PubMed

Mankos, Marian; Persson, Henrik H J; N'Diaye, Alpha T; Shadman, Khashayar; Schmid, Andreas K; Davis, Ronald W

2016-01-01

DNA sequencing by imaging in an electron microscope is an approach that holds promise to deliver long reads with low error rates and without the need for amplification. Earlier work using transmission electron microscopes, which use high electron energies on the order of 100 keV, has shown that low contrast and radiation damage necessitates the use of heavy atom labeling of individual nucleotides, which increases the read error rates. Other prior work using scattering electrons with much lower energy has shown to suppress beam damage on DNA. Here we explore possibilities to increase contrast by employing two methods, X-ray photoelectron and Auger electron spectroscopy. Using bulk DNA samples with monomers of each base, both methods are shown to provide contrast mechanisms that can distinguish individual nucleotides without labels. Both spectroscopic techniques can be readily implemented in a low energy electron microscope, which may enable label-free DNA sequencing by direct imaging.

Droplet Microfluidics for Compartmentalized Cell Lysis and Extension of DNA from Single-Cells

NASA Astrophysics Data System (ADS)

Zimny, Philip; Juncker, David; Reisner, Walter

Current single cell DNA analysis methods suffer from (i) bias introduced by the need for molecular amplification and (ii) limited ability to sequence repetitive elements, resulting in (iii) an inability to obtain information regarding long range genomic features. Recent efforts to circumvent these limitations rely on techniques for sensing single molecules of DNA extracted from single-cells. Here we demonstrate a droplet microfluidic approach for encapsulation and biochemical processing of single-cells inside alginate microparticles. In our approach, single-cells are first packaged inside the alginate microparticles followed by cell lysis, DNA purification, and labeling steps performed off-chip inside this microparticle system. The alginate microparticles are then introduced inside a micro/nanofluidic system where the alginate is broken down via a chelating buffer, releasing long DNA molecules which are then extended inside nanofluidic channels for analysis via standard mapping protocols.

LAMP assay for rapid diagnosis of cow DNA in goat milk and meat samples

PubMed Central

Deb, R.; Sengar, G. S.; Singh, U.; Kumar, S.; Raja, T. V.; Alex, R.; Alyethodi, R. R.; Prakash, B.

2017-01-01

Animal species detection is one of the crucial steps for consumer’s food analysis. In the present study we developed an in-house built loop-mediated isothermal amplification (LAMP) assay for rapid detection of adulterated cow DNA in goat milk/meat samples. The cow milk/tissue DNA in goat milk/meat samples were identified in the developed LAMP assay by either naked eye visualizing with SYBR Green I dyes or by detecting the typical ladder pattern on gel electrophoresis. This test can detect up to minimum 5% level of cow components admixed in goat milk/meat samples and can be completed within 1 h 40 min starting from DNA extraction from milk/meat samples and can be performed in a water bath. Developed LAMP methodology is simple; rapid and sensitive techniques that can detect adulterant like cow components in goat milk/meat are more accurate than other existing DNA based technologies. PMID:28775755

An Optimized DNA Analysis Workflow for the Sampling, Extraction, and Concentration of DNA obtained from Archived Latent Fingerprints.

PubMed

Solomon, April D; Hytinen, Madison E; McClain, Aryn M; Miller, Marilyn T; Dawson Cruz, Tracey

2018-01-01

DNA profiles have been obtained from fingerprints, but there is limited knowledge regarding DNA analysis from archived latent fingerprints-touch DNA "sandwiched" between adhesive and paper. Thus, this study sought to comparatively analyze a variety of collection and analytical methods in an effort to seek an optimized workflow for this specific sample type. Untreated and treated archived latent fingerprints were utilized to compare different biological sampling techniques, swab diluents, DNA extraction systems, DNA concentration practices, and post-amplification purification methods. Archived latent fingerprints disassembled and sampled via direct cutting, followed by DNA extracted using the QIAamp® DNA Investigator Kit, and concentration with Centri-Sep™ columns increased the odds of obtaining an STR profile. Using the recommended DNA workflow, 9 of the 10 samples provided STR profiles, which included 7-100% of the expected STR alleles and two full profiles. Thus, with carefully selected procedures, archived latent fingerprints can be a viable DNA source for criminal investigations including cold/postconviction cases. © 2017 American Academy of Forensic Sciences.

A novel magneto-DNA duplex probe for bacterial DNA detection based on exonuclease III-aided cycling amplification.

PubMed

Zeng, Yan; Wan, Yi; Zhang, Dun; Qi, Peng

2015-01-01

A novel magneto-DNA duplex probe for bacterial DNA detection based on exonuclease III (Exo-III) aided cycling amplification has been developed. This magneto-DNA duplex probe contains a partly hybrid fluorophore-modified capture probe and a fluorophore-modified signal probe with magnetic microparticle as carrier. In the presence of a perfectly matched target bacterial DNA, blunt 3'-terminus of the capture probe is formed, activating the Exo-III aided cycling amplification. Thus, Exo-III catalyzes the stepwise removal of mononucleotides from this terminus, releasing both fluorophore-modified signal probe, fluorescent dyes of the capture probe and target DNA. The released target DNA then starts a new cycle, while released fluorescent fragments are recovered with magnetic separation for fluorescence signal collection. This system exhibited sensitive detection of bacterial DNA, with a detection limit of 14 pM because of the unique cleavage function of Exo-III, high fluorescence intensity, and separating function of magneto-DNA duplex probes. Besides this sensitivity, this strategy exhibited excellent selectivity with mismatched bacterial DNA targets and other bacterial species targets and good applicability in real seawater samples, hence, this strategy could be potentially used for qualitative and quantitative analysis of bacteria. Copyright © 2014 Elsevier B.V. All rights reserved.

Reverse transcription strand invasion based amplification (RT-SIBA): a method for rapid detection of influenza A and B.

PubMed

Eboigbodin, Kevin; Filén, Sanna; Ojalehto, Tuomas; Brummer, Mirko; Elf, Sonja; Pousi, Kirsi; Hoser, Mark

2016-06-01

Rapid and accurate diagnosis of influenza viruses plays an important role in infection control, as well as in preventing the misuse of antibiotics. Isothermal nucleic acid amplification methods offer significant advantages over the polymerase chain reaction (PCR), since they are more rapid and do not require the sophisticated instruments needed for thermal cycling. We previously described a novel isothermal nucleic acid amplification method, 'Strand Invasion Based Amplification' (SIBA®), with high analytical sensitivity and specificity, for the detection of DNA. In this study, we describe the development of a variant of the SIBA method, namely, reverse transcription SIBA (RT-SIBA), for the rapid detection of viral RNA targets. The RT-SIBA method includes a reverse transcriptase enzyme that allows one-step reverse transcription of RNA to complementary DNA (cDNA) and simultaneous amplification and detection of the cDNA by SIBA under isothermal reaction conditions. The RT-SIBA method was found to be more sensitive than PCR for the detection of influenza A and B and could detect 100 copies of influenza RNA within 15 min. The development of RT-SIBA will enable rapid and accurate diagnosis of viral RNA targets within point-of-care or central laboratory settings.

Ultrasensitive electrochemical aptasensor for ochratoxin A based on two-level cascaded signal amplification strategy.

PubMed

Yang, Xingwang; Qian, Jing; Jiang, Ling; Yan, Yuting; Wang, Kan; Liu, Qian; Wang, Kun

2014-04-01

Ochratoxin A (OTA) has a number of toxic effects to both humans and animals, so developing sensitive detection method is of great importance. Herein, we describe an ultrasensitive electrochemical aptasensor for OTA based on the two-level cascaded signal amplification strategy with methylene blue (MB) as a redox indicator. In this method, capture DNA, aptamers, and reporter DNA functionalized-gold nanoparticles (GNPs) were immobilized on the electrode accordingly, where GNPs were used as the first-level signal enhancer. To receive the more sensitive response, a larger number of guanine (G)-rich DNA was bound to the GNPs' surface to provide abundant anchoring sites for MB to achieve the second-level signal amplification. By employing this novel strategy, an ~8.5 (±0.3) fold amplification in signal intensity was obtained. Afterward, OTA was added to force partial GNPs/G-rich DNA to release from the sensing interface and thus decreased the electrochemical response. An effective sensing range from 2.5pM to 2.5nM was received with an extremely low detection limit of 0.75 (±0.12) pM. This amplification strategy has the potential to be the main technology for aptamer-based electrochemical biosensor in a variety of fields. Copyright © 2013 Elsevier B.V. All rights reserved.

A novel restriction endonuclease GlaI for rapid and highly sensitive detection of DNA methylation coupled with isothermal exponential amplification reaction.

PubMed

Sun, Yueying; Sun, Yuanyuan; Tian, Weimin; Liu, Chenghui; Gao, Kejian; Li, Zhengping

2018-02-07

Sensitive and accurate detection of site-specific DNA methylation is of critical significance for early diagnosis of human diseases, especially cancers. Herein, for the first time we employ a novel methylation-dependent restriction endonuclease GlaI to detect site-specific DNA methylation in a highly specific and sensitive way by coupling with isothermal exponential amplification reaction (EXPAR). GlaI can only cut the methylated target site with excellent selectivity but leave the unmethylated DNA intact. Then the newly exposed end fragments of methylated DNA can trigger EXPAR for highly efficient signal amplification while the intact unmethylated DNA will not initiate EXPAR at all. As such, only the methylated DNA is quantitatively and faithfully reflected by the real-time fluorescence signal of the GlaI-EXPAR system, and the potential false positive interference from unmethylated DNA can be effectively eliminated. Therefore, by integrating the unique features of GlaI for highly specific methylation discrimination and EXPAR for rapid and powerful signal amplification, the elegant GlaI-EXPAR assay allows the direct quantification of methylated DNA with ultrahigh sensitivity and accuracy. The detection limit of methylated DNA target has been pushed down to the aM level and the whole detection process of GlaI-EXPAR can be accomplished within a short time of 2 h. More importantly, ultrahigh specificity is achieved and as low as 0.01% methylated DNA can be clearly identified in the presence of a large excess of unmethylated DNA. This GlaI-EXPAR is also demonstrated to be capable of determining site-specific DNA methylations in real genomic DNA samples. Sharing the distinct advantages of ultrahigh sensitivity, outstanding specificity and facile operation, this new GlaI-EXPAR strategy may provide a robust and reliable platform for the detection of site-specific DNA methylations with low abundances.

An Undergraduate Laboratory Experiment for Upper-Level Forensic Science, Biochemistry, or Molecular Biology Courses: Human DNA Amplification Using STR Single Locus Primers by Real-Time PCR with SYBR Green Detection

ERIC Educational Resources Information Center

Elkins, Kelly M.; Kadunc, Raelynn E.

2012-01-01

In this laboratory experiment, real-time polymerase chain reaction (real-time PCR) was conducted using published human TPOX single-locus DNA primers for validation and various student-designed short tandem repeat (STR) primers for Combined DNA Index System (CODIS) loci. SYBR Green was used to detect the amplification of the expected amplicons. The…

Centrosome Amplification: A Potential Marker of Breast Cancer Agressiveness

DTIC Science & Technology

2006-07-01

centrosome amplification. Introduction of DNA damage in the MCF-7 cell line by treatment with hydroxyurea (HU) or daunorubicin (DR) resulted in the...cycles of DNA synthesis and mitotic division in hydroxyurea - arrested Chinese hamster ovary cells. J Cell Biol, 130: 105-115, 1995. 23. D’Assoro, A. B...from cycles of DNA synthesis and mitotic division in hydroxyurea -arrested Chinese hamster ovary cells. J Cell Biol, 1995. 130(1): p. 105-15. 22

Single-Molecule Electrical Random Resequencing of DNA and RNA

NASA Astrophysics Data System (ADS)

Ohshiro, Takahito; Matsubara, Kazuki; Tsutsui, Makusu; Furuhashi, Masayuki; Taniguchi, Masateru; Kawai, Tomoji

2012-07-01

Two paradigm shifts in DNA sequencing technologies--from bulk to single molecules and from optical to electrical detection--are expected to realize label-free, low-cost DNA sequencing that does not require PCR amplification. It will lead to development of high-throughput third-generation sequencing technologies for personalized medicine. Although nanopore devices have been proposed as third-generation DNA-sequencing devices, a significant milestone in these technologies has been attained by demonstrating a novel technique for resequencing DNA using electrical signals. Here we report single-molecule electrical resequencing of DNA and RNA using a hybrid method of identifying single-base molecules via tunneling currents and random sequencing. Our method reads sequences of nine types of DNA oligomers. The complete sequence of 5'-UGAGGUA-3' from the let-7 microRNA family was also identified by creating a composite of overlapping fragment sequences, which was randomly determined using tunneling current conducted by single-base molecules as they passed between a pair of nanoelectrodes.

[Definition of appropriate temperature and storage conditions in the detection of Leishmania DNA with PCR in phlebotomine flies].

PubMed

Cabrera, Olga L; Munsterman, Leonard E; Cárdenas, Rocío; Gutiérrez, Reynaldo; Ferro, Cristina

2002-09-01

For epidemiological studies and control programs of leishmaniasis, taxonomic identification of the etiologic agent of the disease in the insect vector is of critical importance. The implementation of molecular techniques such as the polymerase chain reaction (PCR) has permitted great advances in the efficacy and sensitivity of parasite identification. Previously, these investigations involved labor-intensive dissections and required expert personnel. The present work evaluates the effects of storage methods of phlebotomine samples in the optimization of PCR identification of Leishmania. Females of Lutzomyia longipalpis, from the colony of the Instituto Nacional de Salud, were experimentally infected with Leishmania chagasi (= L. infantum), from the upper Magdalena Valley (Quipile, Cundinamarca, Colombia). The infected insects were preserved in three solutions: 100% ethanol, 70% ethanol, and TE; subsamples of each class were stored at -80 degrees C, -20 degrees C and room temperature. To determine infection rates, samples were dissected and screened microscopically. Chelex 100 was used for extraction of total Leishmania DNA. For PCR amplification, the kinetoplastic minicircle DNA primers OL1 and OL2 of Leishmania were used, and the products were visualized by electrophoresis in 1% agarose gels. For each of the 3 storage conditions, amplifications were successful, producing a approximately 120 base pair product unique to Leishmania. The results demonstrated the advantage of PCR as a routine screening method for detecting infected flies in endemic foci of visceral leishmaniasis. Since storage method did not affect PCR amplification success, the most cost effective method -70% ethanol at room temperature--is the option recommended for storing entomological samples in vector incrimination studies.

Ultrasensitive electrochemiluminescent aptasensor for ochratoxin A detection with the loop-mediated isothermal amplification.

PubMed

Yuan, Yali; Wei, Shiqiang; Liu, Guangpeng; Xie, Shunbi; Chai, Yaqin; Yuan, Ruo

2014-02-06

In this study, we for the first time presented an efficient, accurate, rapid, simple and ultrasensitive detection system for small molecule ochratoxin A (OTA) by using the integration of loop-mediated isothermal amplification (LAMP) technique and subsequently direct readout of LAMP amplicons with a signal-on electrochemiluminescent (ECL) system. Firstly, the dsDNA composed by OTA aptamer and its capture DNA were immobilized on the electrode. After the target recognition, the OTA aptamer bond with target OTA and subsequently left off the electrode, which effectively decreased the immobilization amount of OTA aptamer on electrode. Then, the remaining OTA aptamers on the electrode served as inner primer to initiate the LAMP reaction. Interestingly, the LAMP amplification was detected by monitoring the intercalation of DNA-binding Ru(phen)3(2+) ECL indictors into newly formed amplicons with a set of integrated electrodes. The ECL indictor Ru(phen)3(2+) binding to amplicons caused the reduction of the ECL intensity due to the slow diffusion of Ru(phen)3(2+)-amplicons complex to the electrode surface. Therefore, the presence of more OTA was expected to lead to the release of more OTA aptamer, which meant less OTA aptamer remained on electrode for producing LAMP amplicons, resulting in less Ru(phen)3(2+) interlaced into the formed amplicons within a fixed Ru(phen)3(2+) amount with an obviously increased ECL signal input. As a result, a detection limit as low as 10 fM for OTA was achieved. The aptasensor also has good reproducibility and stability. Copyright © 2013 Elsevier B.V. All rights reserved.

HPV-18 E6 mutants reveal p53 modulation of viral DNA amplification in organotypic cultures

PubMed Central

Kho, Eun-Young; Wang, Hsu-Kun; Banerjee, N. Sanjib; Broker, Thomas R.; Chow, Louise T.

2013-01-01

Human papillomaviruses (HPVs) amplify in differentiated strata of a squamous epithelium. The HPV E7 protein destabilizes the p130/retinoblastoma susceptibility protein family of tumor suppressors and reactivates S-phase reentry, thereby facilitating viral DNA amplification. The high-risk HPV E6 protein destabilizes the p53 tumor suppressor and many other host proteins. However, the critical E6 targets relevant to viral DNA amplification have not been identified, because functionally significant E6 mutants are not stably maintained in transfected cells. Using Cre-loxP recombination, which efficiently generates HPV genomic plasmids in transfected primary human keratinocytes, we have recapitulated a highly productive infection of HPV-18 in organotypic epithelial cultures. By using this system, we now report the characterization of four HPV-18 E6 mutations. An E6 null mutant accumulated high levels of p53 and amplified very poorly. p53 siRNA or ectopic WT E6 partially restored amplification, whereas three missense E6 mutations that did not effectively destabilize p53 complemented the null mutant poorly. Unexpectedly, in cis, two of the missense mutants amplified, albeit to a lower extent than the WT and only in cells with undetectable p53. These observations and others implicate p53 and additional host proteins in regulating viral DNA amplification and also suggest an inhibitory effect of E6 overexpression. We show that high levels of viral DNA amplification are critical for late protein expression and report several previously undescribed viral RNAs, including bicistronic transcripts predicted to encode E5 and L2 or an alternative form of E1^E4 and L1. PMID:23572574

Water-Soluble Conjugated Polymers: Self-Assembly and Biosensor Applications

NASA Astrophysics Data System (ADS)

Bazan, Guillermo

2005-03-01

Homogeneous assays can be designed which take advantage of the optical amplification of conjugated polymers and the self-assembly characteristic of aqueous polyelectrolytes. For example, a ssDNA sequence sensor comprises an aqueous solution containing a cationic water soluble conjugated polymer such as poly(9,9-bis(trimethylammonium)-hexyl)-fluorene phenylene) with a peptide nucleic acid (PNA) labeled with a dye (PNA-C*). Signal transduction is controlled by hybridization of the neutral PNA-C* probe and the negative ssDNA target, resulting in favorable electrostatic interactions between the hybrid complex and the cationic polymer. Distance requirements for Förster energy transfer are thus met only when ssDNA of complementary sequence to the PNA-C* probe is present. Signal amplification by the conjugated polymer provides fluorescein emission >25 times higher than that of the directly excited dye. Transduction by electrostatic interactions followed by energy transfer is a general strategy. Examples involving other biomolecular recognition events, such as DNA/DNA, RNA/protein and RNA/RNA, will also be provided. The mechanism of biosensing will be discussed, with special attention to the varying contributions of hydrophobic and electrostatic forces, polymer conformation, charge density, local concentration of C*s and tailored defect sites for aggregation-induced optical changes. Finally, the water solubility of these conjugated polymers opens possibilities for spin casting onto organic materials, without dissolving the underlying layers. This property is useful for fabricating multilayer organic optoelectronic devices by simple solution techniques.

Development of fluorescent methods for DNA methyltransferase assay

NASA Astrophysics Data System (ADS)

Li, Yueying; Zou, Xiaoran; Ma, Fei; Tang, Bo; Zhang, Chun-yang

2017-03-01

DNA methylation modified by DNA methyltransferase (MTase) plays an important role in regulating gene transcription, cell growth and proliferation. The aberrant DNA MTase activity may lead to a variety of human diseases including cancers. Therefore, accurate and sensitive detection of DNA MTase activity is crucial to biomedical research, clinical diagnostics and therapy. However, conventional DNA MTase assays often suffer from labor-intensive operations and time-consuming procedures. Alternatively, fluorescent methods have significant advantages of simplicity and high sensitivity, and have been widely applied for DNA MTase assay. In this review, we summarize the recent advances in the development of fluorescent methods for DNA MTase assay. These emerging methods include amplification-free and the amplification-assisted assays. Moreover, we discuss the challenges and future directions of this area.

Method of artificial DNA splicing by directed ligation (SDL).

PubMed Central

Lebedenko, E N; Birikh, K R; Plutalov, O V; Berlin YuA

1991-01-01

An approach to directed genetic recombination in vitro has been devised, which allows for joining together, in a predetermined way, a series of DNA segments to give a precisely spliced polynucleotide sequence (DNA splicing by directed ligation, SDL). The approach makes use of amplification, by means of several polymerase chain reactions (PCR), of a chosen set of DNA segments. Primers for the amplifications contain recognition sites of the class IIS restriction endonucleases, which transform blunt ends of the amplification products into protruding ends of unique primary structures, the ends to be used for joining segments together being mutually complementary. Ligation of the mixture of the segments so synthesized gives the desired sequence in an unambiguous way. The suggested approach has been exemplified by the synthesis of a totally processed (intronless) gene encoding human mature interleukin-1 alpha. Images PMID:1662363

Centrifugal LabTube platform for fully automated DNA purification and LAMP amplification based on an integrated, low-cost heating system.

PubMed

Hoehl, Melanie M; Weißert, Michael; Dannenberg, Arne; Nesch, Thomas; Paust, Nils; von Stetten, Felix; Zengerle, Roland; Slocum, Alexander H; Steigert, Juergen

2014-06-01

This paper introduces a disposable battery-driven heating system for loop-mediated isothermal DNA amplification (LAMP) inside a centrifugally-driven DNA purification platform (LabTube). We demonstrate LabTube-based fully automated DNA purification of as low as 100 cell-equivalents of verotoxin-producing Escherichia coli (VTEC) in water, milk and apple juice in a laboratory centrifuge, followed by integrated and automated LAMP amplification with a reduction of hands-on time from 45 to 1 min. The heating system consists of two parallel SMD thick film resistors and a NTC as heating and temperature sensing elements. They are driven by a 3 V battery and controlled by a microcontroller. The LAMP reagents are stored in the elution chamber and the amplification starts immediately after the eluate is purged into the chamber. The LabTube, including a microcontroller-based heating system, demonstrates contamination-free and automated sample-to-answer nucleic acid testing within a laboratory centrifuge. The heating system can be easily parallelized within one LabTube and it is deployable for a variety of heating and electrical applications.

Enhanced sequencing coverage with digital droplet multiple displacement amplification

PubMed Central

Sidore, Angus M.; Lan, Freeman; Lim, Shaun W.; Abate, Adam R.

2016-01-01

Sequencing small quantities of DNA is important for applications ranging from the assembly of uncultivable microbial genomes to the identification of cancer-associated mutations. To obtain sufficient quantities of DNA for sequencing, the small amount of starting material must be amplified significantly. However, existing methods often yield errors or non-uniform coverage, reducing sequencing data quality. Here, we describe digital droplet multiple displacement amplification, a method that enables massive amplification of low-input material while maintaining sequence accuracy and uniformity. The low-input material is compartmentalized as single molecules in millions of picoliter droplets. Because the molecules are isolated in compartments, they amplify to saturation without competing for resources; this yields uniform representation of all sequences in the final product and, in turn, enhances the quality of the sequence data. We demonstrate the ability to uniformly amplify the genomes of single Escherichia coli cells, comprising just 4.7 fg of starting DNA, and obtain sequencing coverage distributions that rival that of unamplified material. Digital droplet multiple displacement amplification provides a simple and effective method for amplifying minute amounts of DNA for accurate and uniform sequencing. PMID:26704978

Direct and quantitative detection of HIV-1 RNA in human plasma with a branched DNA signal amplification assay.

PubMed

Urdea, M S; Wilber, J C; Yeghiazarian, T; Todd, J A; Kern, D G; Fong, S J; Besemer, D; Hoo, B; Sheridan, P J; Kokka, R

1993-11-01

To determine the relative effect of sample matrix on the quantitation of HIV RNA in plasma. Two HIV-positive specimens were diluted into five and 10 different HIV-negative plasma samples, respectively. Branched DNA signal amplification technology and reverse-transcriptase polymerase chain reaction were used to measure the viral load. In one sample the viral load by polymerase chain reaction ranged from undetectable to 1.9 x 10(5) copies/ml, and the branched DNA results ranged from 2.6 x 10(4) to 4.2 x 10(4) HIV RNA equivalent/ml. In the other sample the corresponding figures were 6.3 x 10(4) to 5.5 x 10(5) copies/ml and 5.7 x 10(4) to 7.5 x 10(4) HIV RNA equivalents/ml. In contrast to reverse-transcriptase polymerase chain reaction the branched DNA signal amplification assay does not require a separate extraction step or enzymatic amplification of the target. Therefore this measurement is less affected by the sample matrix and the signal generated is directly proportional to the viral load.

Exponential isothermal amplification of nucleic acids and amplified assays for proteins, cells, and enzyme activities.

PubMed

Reid, Michael S; Le, X Chris; Zhang, Hongquan

2018-04-27

Isothermal exponential amplification techniques, such as strand-displacement amplification (SDA), rolling circle amplification (RCA), loop-mediated isothermal amplification (LAMP), nucleic acid sequence-based amplification (NASBA), helicase-dependent amplification (HDA), and recombinase polymerase amplification (RPA), have great potential for on-site, point-of-care, and in-situ assay applications. These amplification techniques eliminate the need for temperature cycling required for polymerase chain reaction (PCR) while achieving comparable amplification yield. We highlight here recent advances in exponential amplification reaction (EXPAR) for the detection of nucleic acids, proteins, enzyme activities, cells, and metal ions. We discuss design strategies, enzyme reactions, detection techniques, and key features. Incorporation of fluorescence, colorimetric, chemiluminescence, Raman, and electrochemical approaches enables highly sensitive detection of a variety of targets. Remaining issues, such as undesirable background amplification resulting from non-specific template interactions, must be addressed to further improve isothermal and exponential amplification techniques. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Molecular analysis of single oocyst of Eimeria by whole genome amplification (WGA) based nested PCR.

PubMed

Wang, Yunzhou; Tao, Geru; Cui, Yujuan; Lv, Qiyao; Xie, Li; Li, Yuan; Suo, Xun; Qin, Yinghe; Xiao, Lihua; Liu, Xianyong

2014-09-01

PCR-based molecular tools are widely used for the identification and characterization of protozoa. Here we report the molecular analysis of Eimeria species using combined methods of whole genome amplification (WGA) and nested PCR. Single oocyst of Eimeria stiedai or Eimeriamedia was directly used for random amplification of the genomic DNA with either primer extension preamplification (PEP) or multiple displacement amplification (MDA), and then the WGA product was used as template in nested PCR with species-specific primers for ITS-1, 18S rDNA and 23S rDNA of E. stiedai and E. media. WGA-based PCR was successful for the amplification of these genes from single oocyst. For the species identification of single oocyst isolated from mixed E. stiedai or E. media, the results from WGA-based PCR were exactly in accordance with those from morphological identification, suggesting the availability of this method in molecular analysis of eimerian parasites at the single oocyst level. WGA-based PCR method can also be applied for the identification and genetic characterization of other protists. Copyright © 2014 Elsevier Inc. All rights reserved.

Development of a Quantitative Recombinase Polymerase Amplification Assay with an Internal Positive Control

PubMed Central

Richards-Kortum, Rebecca

2015-01-01

It was recently demonstrated that recombinase polymerase amplification (RPA), an isothermal amplification platform for pathogen detection, may be used to quantify DNA sample concentration using a standard curve. In this manuscript, a detailed protocol for developing and implementing a real-time quantitative recombinase polymerase amplification assay (qRPA assay) is provided. Using HIV-1 DNA quantification as an example, the assembly of real-time RPA reactions, the design of an internal positive control (IPC) sequence, and co-amplification of the IPC and target of interest are all described. Instructions and data processing scripts for the construction of a standard curve using data from multiple experiments are provided, which may be used to predict the concentration of unknown samples or assess the performance of the assay. Finally, an alternative method for collecting real-time fluorescence data with a microscope and a stage heater as a step towards developing a point-of-care qRPA assay is described. The protocol and scripts provided may be used for the development of a qRPA assay for any DNA target of interest. PMID:25867513

Development of a quantitative recombinase polymerase amplification assay with an internal positive control.

PubMed

Crannell, Zachary A; Rohrman, Brittany; Richards-Kortum, Rebecca

2015-03-30

It was recently demonstrated that recombinase polymerase amplification (RPA), an isothermal amplification platform for pathogen detection, may be used to quantify DNA sample concentration using a standard curve. In this manuscript, a detailed protocol for developing and implementing a real-time quantitative recombinase polymerase amplification assay (qRPA assay) is provided. Using HIV-1 DNA quantification as an example, the assembly of real-time RPA reactions, the design of an internal positive control (IPC) sequence, and co-amplification of the IPC and target of interest are all described. Instructions and data processing scripts for the construction of a standard curve using data from multiple experiments are provided, which may be used to predict the concentration of unknown samples or assess the performance of the assay. Finally, an alternative method for collecting real-time fluorescence data with a microscope and a stage heater as a step towards developing a point-of-care qRPA assay is described. The protocol and scripts provided may be used for the development of a qRPA assay for any DNA target of interest.

A cascade autocatalytic strand displacement amplification and hybridization chain reaction event for label-free and ultrasensitive electrochemical nucleic acid biosensing.

PubMed

Chen, Zhiqiang; Liu, Ying; Xin, Chen; Zhao, Jikuan; Liu, Shufeng

2018-08-15

Herein, an autocatalytic strand displacement amplification (ASDA) strategy was proposed for the first time, which was further ingeniously coupled with hybridization chain reaction (HCR) event for the isothermal, label-free and multiple amplification toward nucleic acid detection. During the ASDA module, the target recognition opens the immobilized hairpin probe (IP) and initiates the annealing of the auxiliary DNA strand (AS) with the opened IP for the successive polymerization and nicking reaction in the presence of DNA polymerase and nicking endonuclease. This induces the target recycling and generation of a large amount of intermediate DNA sequences, which can be used as target analogy to execute the autocatalytic strand displacement amplification. Simultaneously, the introduced AS strand can propagate the HCR between two hairpins (H1 and H2) to form a linear DNA concatamer with cytosine (C)-rich loop region, which can facilitate the in-situ synthesis of silver nanoclusters (AgNCs) as electrochemical tags for further amplification toward target responses. With current cascade ASDA and HCR strategy, the detection of target DNA could be achieved with a low detection limit of about 0.16 fM and a good selectivity. The developed biosensor also exhibits the distinct advantages of flexibility and simplicity in probe design and biosensor fabrication, and label-free electrochemical detection, thus opens a promising avenue for the detection of nucleic acid with low abundance in bioanalysis and clinical biomedicine. Copyright © 2018 Elsevier B.V. All rights reserved.

Modular development of a prototype point of care molecular diagnostic platform for sexually transmitted infections.

PubMed

Branavan, Manoharanehru; Mackay, Ruth E; Craw, Pascal; Naveenathayalan, Angel; Ahern, Jeremy C; Sivanesan, Tulasi; Hudson, Chris; Stead, Thomas; Kremer, Jessica; Garg, Neha; Baker, Mark; Sadiq, Syed T; Balachandran, Wamadeva

2016-08-01

This paper presents the design of a modular point of care test platform that integrates a proprietary sample collection device directly with a microfluidic cartridge. Cell lysis, within the cartridge, is conducted using a chemical method and nucleic acid purification is done on an activated cellulose membrane. The microfluidic device incorporates passive mixing of the lysis-binding buffers and sample using a serpentine channel. Results have shown extraction efficiencies for this new membrane of 69% and 57% compared to the commercial Qiagen extraction method of 85% and 59.4% for 0.1ng/µL and 100ng/µL salmon sperm DNA respectively spiked in phosphate buffered solution. Extraction experiments using the serpentine passive mixer cartridges incorporating lysis and nucleic acid purification showed extraction efficiency around 80% of the commercial Qiagen kit. Isothermal amplification was conducted using thermophillic helicase dependant amplification and recombinase polymerase amplification. A low cost benchtop real-time isothermal amplification platform has been developed capable of running six amplifications simultaneously. Results show that the platform is capable of detecting 1.32×10(6) of sample DNA through thermophillic helicase dependant amplification and 1×10(5) copy numbers Chlamydia trachomatis genomic DNA within 10min through recombinase polymerase nucleic acid amplification tests. Copyright © 2016 The Author(s). Published by Elsevier Ltd.. All rights reserved.

Assessment of palindromes as platforms for DNA amplification in breast cancer

PubMed Central

Guenthoer, Jamie; Diede, Scott J.; Tanaka, Hisashi; Chai, Xiaoyu; Hsu, Li; Tapscott, Stephen J.; Porter, Peggy L.

2012-01-01

DNA amplification, particularly of chromosomes 8 and 11, occurs frequently in breast cancer and is a key factor in tumorigenesis, often associated with poor prognosis. The mechanisms involved in the amplification of these regions are not fully understood. Studies from model systems have demonstrated that palindrome formation can be an early step in DNA amplification, most notably seen in the breakage–fusion–bridge (BFB) cycle. Therefore, palindromes might be associated with gene amplicons in breast cancer. To address this possibility, we coupled high-resolution palindrome profiling by the Genome-wide Analysis of Palindrome Formation (GAPF) assay with genome-wide copy-number analyses on a set of breast cancer cell lines and primary tumors to spatially associate palindromes and copy-number gains. We identified GAPF-positive regions distributed nonrandomly throughout cell line and tumor genomes, often in clusters, and associated with copy-number gains. Commonly amplified regions in breast cancer, chromosomes 8q and 11q, had GAPF-positive regions flanking and throughout the copy-number gains. We also identified amplification-associated GAPF-positive regions at similar locations in subsets of breast cancers with similar characteristics (e.g., ERBB2 amplification). These shared positive regions offer the potential to evaluate the utility of palindromes as prognostic markers, particularly in premalignant breast lesions. Our results implicate palindrome formation in the amplification of regions with key roles in breast tumorigenesis, particularly in subsets of breast cancers. PMID:21752925

Pushing the limits for amplifying BrdU-labeled DNA encoding 16S rRNA: DNA polymerase as the determining factor.

PubMed

Roux-Michollet, Dad D; Schimel, Joshua P; Holden, Patricia A

2010-12-01

Identifying microorganisms that are active under specific conditions in ecosystems is a challenge in microbial ecology. Recently, the bromodeoxyuridine (BrdU) technique was developed to label actively growing cells. BrdU, a thymidine analog, is incorporated into newly synthesized DNA, and the BrdU-labeled DNA is then isolated from total extractable DNA by immunocapture using a BrdU-specific antibody. Analyzing the BrdU-labeled DNA allows for assessing the actively growing community, which can then be compared to the unlabeled DNA that represents the total community. However, applying the BrdU approach to study soils has been problematic due to low DNA amounts and soil contaminants. To address these challenges, we developed a protocol, optimizing specificity and reproducibility, to amplify BrdU-labeled gene fragments encoding 16S rRNA. We found that the determining factor was the DNA polymerase: among the 13 different polymerases we tested, only 3 provided adequate yields with minimal contamination, and only two of those three produced similar amplification patterns of community DNA. Copyright © 2010 Elsevier B.V. All rights reserved.

Design and Evaluation of PCR Primers for Analysis of Bacterial Populations in Wine by Denaturing Gradient Gel Electrophoresis

PubMed Central

Lopez, Isabel; Ruiz-Larrea, Fernanda; Cocolin, Luca; Orr, Erica; Phister, Trevor; Marshall, Megan; VanderGheynst, Jean; Mills, David A.

2003-01-01

Denaturing gradient gel electrophoresis (DGGE) of PCR-amplified ribosomal DNA (rDNA) is routinely used to compare levels of diversity of microbial communities and to monitor population dynamics. While using PCR-DGGE to examine the bacteria in wine fermentations, we noted that several commonly used PCR primers for amplifying bacterial 16S rDNA also coamplified yeast, fungal, or plant DNA present in samples. Unfortunately, amplification of nonbacterial DNA can result in a masking of bacterial populations in DGGE profiles. To surmount this problem, we developed two new primer sets for specific amplification of bacterial 16S rDNA in wine fermentation samples without amplification of eukaryotic DNA. One primer set, termed WLAB1 and WLAB2, amplified lactic acid bacteria, while another, termed WBAC1 and WBAC2, amplified both lactic acid bacterial and acetic acid bacterial populations found in wine. Primer specificity and efficacy were examined with DNA isolated from numerous bacterial, yeast, and fungal species commonly found in wine and must samples. Importantly, both primer sets effectively distinguished bacterial species in wine containing mixtures of yeast and bacteria. PMID:14602643

Optimization and evaluation of single-cell whole-genome multiple displacement amplification.

PubMed

Spits, C; Le Caignec, C; De Rycke, M; Van Haute, L; Van Steirteghem, A; Liebaers, I; Sermon, K

2006-05-01

The scarcity of genomic DNA can be a limiting factor in some fields of genetic research. One of the methods developed to overcome this difficulty is whole genome amplification (WGA). Recently, multiple displacement amplification (MDA) has proved very efficient in the WGA of small DNA samples and pools of cells, the reaction being catalyzed by the phi29 or the Bst DNA polymerases. The aim of the present study was to develop a reliable, efficient, and fast protocol for MDA at the single-cell level. We first compared the efficiency of phi29 and Bst polymerases on DNA samples and single cells. The phi29 polymerase generated accurately, in a short time and from a single cell, sufficient DNA for a large set of tests, whereas the Bst enzyme showed a low efficiency and a high error rate. A single-cell protocol was optimized using the phi29 polymerase and was evaluated on 60 single cells; the DNA obtained DNA was assessed by 22 locus-specific PCRs. This new protocol can be useful for many applications involving minute quantities of starting material, such as forensic DNA analysis, prenatal and preimplantation genetic diagnosis, or cancer research. (c) 2006 Wiley-Liss, Inc.

Highly sensitive DNA detection using cascade amplification strategy based on hybridization chain reaction and enzyme-induced metallization

PubMed Central

Yu, Xu; Zhang, Zhi-Ling; Zheng, Si-Yang

2014-01-01

A novel highly sensitive colorimetric assay for DNA detection using cascade amplification strategy based on hybridization chain reaction and enzyme-induced metallization was established. The DNA modified superparamagnetic beads were demonstrated to capture and enrich the target DNA in the hybridization buffer or human plasma. The hybridization chain reaction and enzyme-induced silver metallization on the gold nanoparticles were used as cascade signal amplification for the detection of target DNA. The metalization of silver on the gold nanoparticles induced a significant colour change from red to yellow until black depending on the concentration of the target DNA, which could be recognized by naked eyes. This method showed a good specificity for the target DNA detection, with the capabilty to discriminate single-base-pair mismatched DNA mutation (single nucleotide polymorphism). Meanwhile, this approach exhibited an excellent anti-interference capability with the convenience of the magentic seperation and washing, which enabled its usage in complex biological systems such as human blood plasma. As an added benefit, the utilization of hybridization chain reaction and enzyme-induced metallization improved detection sensitivity down to 10 pM, which is about 100-fold lower than that of traditional unamplified homogeneous assays. PMID:25500528

[Utilization of nylon membranes for specific isolation and characterization of verotoxin-producing Escherichia coli using DNA probes].

PubMed

Gallien, P; Klie, H; Perlberg, K W; Protz, D

1996-01-01

A method for specific isolation of VT(+)-strains in raw milk is given. DNA-hybridization technique with DIG-labeled PCR-amplificates as probes are the basis. No background is seen by using "DIG Easy Hyb" solution and nylon membranes for colony- and plaque-hybridization (Boehringer Mannheim GmbH). Marked colonies are visible on the membranes after detection. So it is possible to select these colonies from a masterplate. The results are available within one day (without enrichment and membrane preparation). After stripping the membranes can be used for a new hybridisation to detect another factor of virulence.

humpty dumpty is required for developmental DNA amplification and cell proliferation in Drosophila.

PubMed

Bandura, Jennifer L; Beall, Eileen L; Bell, Maren; Silver, Hannah R; Botchan, Michael R; Calvi, Brian R

2005-04-26

The full complement of proteins required for the proper regulation of genome duplication are yet to be described. We employ a genetic DNA-replication model system based on developmental amplification of Drosophila eggshell (chorion) genes [1]. Hypomorphic mutations in essential DNA replication genes result in a distinct thin-eggshell phenotype owing to reduced amplification [2]. Here, we molecularly identify the gene, which we have named humpty dumpty (hd), corresponding to the thin-eggshell mutant fs(3)272-9 [3]. We confirm that hd is essential for DNA amplification in the ovary and show that it also is required for cell proliferation during development. Mosaic analysis of hd mutant cells during development and RNAi in Kc cells reveal that depletion of Hd protein results in severe defects in genomic replication and DNA damage. Most Hd protein is found in nuclear foci, and some may traverse the nuclear envelope. Consistent with a role in DNA replication, expression of Hd protein peaks during late G1 and S phase, and it responds to the E2F1/Dp transcription factor. Hd protein sequence is conserved from plants to humans, and published microarrays indicate that expression of its putative human ortholog also peaks at G1/S [4]. Our data suggest that hd defines a new gene family likely required for cell proliferation in all multicellular eukaryotes.

Label-free thioflavin T/G-quadruplex-based real-time strand displacement amplification for biosensing applications.

PubMed

Du, Yi-Chen; Zhu, Li-Na; Kong, De-Ming

2016-12-15

To promote application of strand-displacement amplification (SDA) techniques in biosensing, a label-free, real-time monitoring strategy for isothermal nucleic acid amplification reactions was designed. G-quadruplex structures were introduced into SDA products using specific recognition of G-quadruplexes by the fluorogenic dye thioflavin T. Performance was good for real-time monitoring of traditional SDA by a linear-amplification mechanism and for exponential cross-triggered SDA amplification. The strategy worked on a commercial real-time PCR instrument, making it suitable for biosensing platforms. As examples, two highly sensitive and specific biosensors were designed for analysis of the activity of uracil-DNA glycosylase (UDG) and the restriction endonuclease EcoRI. Detection limits were 6×10(-5)U/mL for UDG and 0.016U/mL for EcoRI. Detection of corresponding targets in complex matrices such as cell lysates or human serum was also demonstrated. Compared to traditional end-point detection methods, real-time SDA-based approaches have the advantages of simple, fast operation; high sensitivity; low risk of carryover contamination; and very high throughput. The introduction of real-time monitoring strategies may promote application of SDA reactions in biosensor design. Copyright © 2016 Elsevier B.V. All rights reserved.

Recent development of nano-materials used in DNA biosensors.

PubMed

Xu, Kai; Huang, Junran; Ye, Zunzhong; Ying, Yibin; Li, Yanbin

2009-01-01

As knowledge of the structure and function of nucleic acid molecules has increased, sequence-specific DNA detection has gained increased importance. DNA biosensors based on nucleic acid hybridization have been actively developed because of their specificity, speed, portability, and low cost. Recently, there has been considerable interest in using nano-materials for DNA biosensors. Because of their high surface-to-volume ratios and excellent biological compatibilities, nano-materials could be used to increase the amount of DNA immobilization; moreover, DNA bound to nano-materials can maintain its biological activity. Alternatively, signal amplification by labeling a targeted analyte with nano-materials has also been reported for DNA biosensors in many papers. This review summarizes the applications of various nano-materials for DNA biosensors during past five years. We found that nano-materials of small sizes were advantageous as substrates for DNA attachment or as labels for signal amplification; and use of two or more types of nano-materials in the biosensors could improve their overall quality and to overcome the deficiencies of the individual nano-components. Most current DNA biosensors require the use of polymerase chain reaction (PCR) in their protocols. However, further development of nano-materials with smaller size and/or with improved biological and chemical properties would substantially enhance the accuracy, selectivity and sensitivity of DNA biosensors. Thus, DNA biosensors without PCR amplification may become a reality in the foreseeable future.

Recent Development of Nano-Materials Used in DNA Biosensors

PubMed Central

Xu, Kai; Huang, Junran; Ye, Zunzhong; Ying, Yibin; Li, Yanbin

2009-01-01

As knowledge of the structure and function of nucleic acid molecules has increased, sequence-specific DNA detection has gained increased importance. DNA biosensors based on nucleic acid hybridization have been actively developed because of their specificity, speed, portability, and low cost. Recently, there has been considerable interest in using nano-materials for DNA biosensors. Because of their high surface-to-volume ratios and excellent biological compatibilities, nano-materials could be used to increase the amount of DNA immobilization; moreover, DNA bound to nano-materials can maintain its biological activity. Alternatively, signal amplification by labeling a targeted analyte with nano-materials has also been reported for DNA biosensors in many papers. This review summarizes the applications of various nano-materials for DNA biosensors during past five years. We found that nano-materials of small sizes were advantageous as substrates for DNA attachment or as labels for signal amplification; and use of two or more types of nano-materials in the biosensors could improve their overall quality and to overcome the deficiencies of the individual nano-components. Most current DNA biosensors require the use of polymerase chain reaction (PCR) in their protocols. However, further development of nano-materials with smaller size and/or with improved biological and chemical properties would substantially enhance the accuracy, selectivity and sensitivity of DNA biosensors. Thus, DNA biosensors without PCR amplification may become a reality in the foreseeable future. PMID:22346713

Single-cell genomic sequencing using Multiple Displacement Amplification.

PubMed

Lasken, Roger S

2007-10-01

Single microbial cells can now be sequenced using DNA amplified by the Multiple Displacement Amplification (MDA) reaction. The few femtograms of DNA in a bacterium are amplified into micrograms of high molecular weight DNA suitable for DNA library construction and Sanger sequencing. The MDA-generated DNA also performs well when used directly as template for pyrosequencing by the 454 Life Sciences method. While MDA from single cells loses some of the genomic sequence, this approach will greatly accelerate the pace of sequencing from uncultured microbes. The genetically linked sequences from single cells are also a powerful tool to be used in guiding genomic assembly of shotgun sequences of multiple organisms from environmental DNA extracts (metagenomic sequences).

Accelerated isothermal nucleic acid amplification in betaine-free reaction.

PubMed

Ma, Cuiping; Wang, Yifan; Zhang, Pansong; Shi, Chao

2017-08-01

Betaine was used as a common additive to isothermal nucleic acid amplification reactions because of lowering the melting temperature (Tm) of DNA. Herein, we reported a novel finding that betaine was inhibiting the reaction efficiency of isothermal amplification reactions. In this work, we have verified this finding by classical loop-mediated isothermal amplification that the addition of 0.8 M betaine inhibited the efficiency of reaction dropping to approximately 1%. Additionally, we clarified the mechanism of betaine hindering isothermal amplification reactions with a molecular barrier to lower associate rate constant K1 for intermolecular hybridization. This finding would be very significant for studies on the interaction between small molecule substance and DNA, and the development of point-of-care testing because of simplifying reaction system and increasing reaction efficiency. Copyright © 2017 Elsevier Inc. All rights reserved.

Paternity testing in case of brother-sister incest.

PubMed

Macan, Marijana; Uvodić, Petra; Botica, Vladimir

2003-06-01

We performed a paternity test in a case of incest between brother and sister. DNA from blood samples of the alleged parents and their two children was obtained with Chelex DNA extraction method and quantified with Applied Biosystems QuantiBlot quantitation kit. Polymerase chain reaction (PCR) amplification of DNA samples was performed with AmpFlSTR SGM Plus PCR amplification kit and GenePrint PowerPlex PCR amplification kit. The amplified products were separated and detected by using the Perkin Elmer's ABI PRISM trade mark 310 Genetic Analyser. DNA and data analysis of 17 loci and Amelogenin confirmed the suspicion of brother-sister incest. Since both children had inherited all of the obligate alleles from the alleged father, we could confirm with certainty of 99.999999% that the oldest brother in the family was the biological father of both children. Calculated data showed that even in a case of brother-sister incest, paternity could be proved by the analysis of Amelogenin and 17 DNA loci.

Fluorescent Quantification of DNA Based on Core-Shell Fe3O4@SiO2@Au Nanocomposites and Multiplex Ligation-Dependent Probe Amplification.

PubMed

Fan, Jing; Yang, Haowen; Liu, Ming; Wu, Dan; Jiang, Hongrong; Zeng, Xin; Elingarami, Sauli; Ll, Zhiyang; Li, Song; Liu, Hongna; He, Nongyue

2015-02-01

In this research, a novel method for relative fluorescent quantification of DNA based on Fe3O4@SiO2@Au gold-coated magnetic nanocomposites (GMNPs) and multiplex ligation- dependent probe amplification (MLPA) has been developed. With the help of self-assembly, seed-mediated growth and chemical reduction method, core-shell Fe3O4@SiO2@Au GMNPs were synthesized. Through modified streptavidin on the GMNPs surface, we obtained a bead chip which can capture the biotinylated probes. Then we designed MLPA probes which were tagged with biotin or Cy3 and target DNA on the basis of human APP gene sequence. The products from the thermostable DNA ligase induced ligation reactions and PCR amplifications were incubated with SA-GMNPs. After washing, magnetic separation, spotting, the fluorescent scanning results showed our method can be used for the relative quantitative analysis of the target DNA in the concentration range of 03004~0.5 µM.

Self-reproduction of supramolecular giant vesicles combined with the amplification of encapsulated DNA.

PubMed

Kurihara, Kensuke; Tamura, Mieko; Shohda, Koh-Ichiroh; Toyota, Taro; Suzuki, Kentaro; Sugawara, Tadashi

2011-09-04

The construction of a protocell from a materials point of view is important in understanding the origin of life. Both self-reproduction of a compartment and self-replication of an informational substance have been studied extensively, but these processes have typically been carried out independently, rather than linked to one another. Here, we demonstrate the amplification of DNA (encapsulated guest) within a self-reproducible cationic giant vesicle (host). With the addition of a vesicular membrane precursor, we observe the growth and spontaneous division of the giant vesicles, accompanied by distribution of the DNA to the daughter giant vesicles. In particular, amplification of the DNA accelerated the division of the giant vesicles. This means that self-replication of an informational substance has been linked to self-reproduction of a compartment through the interplay between polyanionic DNA and the cationic vesicular membrane. Our self-reproducing giant vesicle system therefore represents a step forward in the construction of an advanced model protocell.

PCR amplification of up to 35-kb DNA with high fidelity and high yield from lambda bacteriophage templates.

PubMed Central

Barnes, W M

1994-01-01

A target length limitation to PCR amplification of DNA has been identified and addressed. Concomitantly, the base-pair fidelity, the ability to use PCR products as primers, and the maximum yield of target fragment were increased. These improvements were achieved by the combination of a high level of an exonuclease-free, N-terminal deletion mutant of Taq DNA polymerase, Klentaq1, with a very low level of a thermostable DNA polymerase exhibiting a 3'-exonuclease activity (Pfu, Vent, or Deep Vent). At least 35 kb can be amplified to high yields from 1 ng of lambda DNA template. Images PMID:8134376

A pure DNA hydrogel with stable catalytic ability produced by one-step rolling circle amplification.

PubMed

Huang, Yishun; Xu, Wanlin; Liu, Guoyuan; Tian, Leilei

2017-03-09

A rolling-circle-amplification method was developed to produce DNA hydrogels with horseradish-peroxidase-like catalytic capability. The catalytic hydrogel exhibits highly improved stability at elevated temperatures or during a long-term storage. Integrated with glucose oxidase, the complex hydrogel can be applied to the sensitive and reliable detection of glucose.

Limited efficiency of universal mini-barcode primers for DNA amplification from desert reptiles, birds and mammals.

PubMed

Arif, I A; Khan, H A; Al Sadoon, M; Shobrak, M

2011-10-31

In recent years, DNA barcoding has emerged as a powerful tool for species identification. We report an extended validation of a universal DNA mini-barcode for amplification of 130-bp COI segments from 23 specimens collected from a desert environment, including 11 reptiles, five mammals and seven birds. Besides the standard double-annealing protocol, we also tested a more stringent single-annealing protocol. The PCR success rate for the amplification of the mini-barcode region was: mammals (4/5), reptiles (5/11) and birds (4/7). These findings demonstrate the limited utility of universal primers for mini-barcoding, at least for these vertebrate taxa that we collected from the Saudi Arabian desert.

Enhanced DNA Profiling of the Semen Donor in Late Reported Sexual Assaults: Use of Y-Chromosome-Targeted Pre-amplification and Next Generation Y-STR Amplification Systems.

PubMed

Hanson, Erin K; Ballantyne, Jack

2016-01-01

In some cases of sexual assault the victim may not report the assault for several days after the incident due to various factors. The ability to obtain an autosomal STR profile of the semen donor from a living victim rapidly diminishes as the post-coital interval is extended due to the presence of only a small amount of male DNA amidst an overwhelming amount of female DNA. Previously, we have utilized various technological tools to overcome the limitations of male DNA profiling in extended interval post-coital samples including the use of Y-chromosome STR profiling, cervical sample, and post-PCR purification permitting the recovery of Y-STR profiles of the male DNA from samples collected 5-6 days after intercourse. Despite this success, the reproductive biology literature reports the presence of spermatozoa in the human cervix up to 7-10 days post-coitus. Therefore, novel and improved methods for recovery of male profiles in extended interval post-coital samples were required. Here, we describe enhanced strategies, including Y-chromosome-targeted pre-amplification and next generation Y-STR amplification kits, that have resulted in the ability to obtain probative male profiles from samples collected 6-9 days after intercourse.

[Evaluating the Stability of Loop-Mediated Isothermal Amplification Reagents at Irregular Storage Temperatures for On-Site Diagnosis].

PubMed

Inoshima, Yasuo; Ishiguro, Naotaka

2015-01-01

Temperature-stability of loop-mediated isothermal amplification (LAMP) reagents was determined for their use in on-site diagnosis, such as in farms/pastures. Bst and Csa DNA polymerases and the reagents that were stored at different temperatures (4 or 25°C) for 1, 2, or 4 days were used for the LAMP assay to detect orf virus DNA as a model. After storage at 4 and 25°C for 2 days, the enzymes and reagents were found to retain sufficient activity to carry out successful DNA amplification. Visual diagnosis was also possible with the reagents (Loopamp Fluorescent Detection Reagent or hydroxy naphthol blue, as well as DNA amplification checker, D-Quick) that were stored for 2 days at different temperatures. Although the time taken to obtain the positive/negative results were delayed, the enzymes and reagents, stored at 25°C for 4 days, were active and had the ability to efficiently amplify DNA in less than 50 min. These results indicate that LAMP assay can be successfully utilized for the diagnosis of infectious diseases under non-clinical settings such as for on-site diagnosis in farms/pastures, owing to the fact that the relevant enzymes and reagents does not require restricted temperature storage.

Electrochemical detection of Francisella tularensis genomic DNA using solid-phase recombinase polymerase amplification.

PubMed

del Río, Jonathan Sabaté; Yehia Adly, Nouran; Acero-Sánchez, Josep Lluis; Henry, Olivier Y F; O'Sullivan, Ciara K

2014-04-15

Solid-phase isothermal DNA amplification was performed exploiting the homology protein recombinase A (recA). The system was primarily tested on maleimide activated microtitre plates as a proof-of-concept and later translated to an electrochemical platform. In both cases, forward primer for Francisella tularensis holarctica genomic DNA was surface immobilised via a thiol or an amino moiety and then elongated during the recA mediated amplification, carried out in the presence of specific target sequence and reverse primers. The formation of the subsequent surface tethered amplicons was either colorimetrically or electrochemically monitored using a horseradish peroxidase (HRP)-labelled DNA secondary probe complementary to the elongated strand. The amplification time was optimised to amplify even low amounts of DNA copies in less than an hour at a constant temperature of 37°C, achieving a limit of detection of 1.3×10(-13) M (4×10(6) copies in 50 μL) for the colorimetric assay and 3.3×10(-14) M (2×10(5) copies in 10 μL) for the chronoamperometric assay. The system was demonstrated to be highly specific with negligible cross-reactivity with non-complementary targets or primers. © 2013 Elsevier B.V. All rights reserved.

Development of a recombinase polymerase amplification assay for the detection of pathogenic Leptospira.

PubMed

Ahmed, Ahmed; van der Linden, Hans; Hartskeerl, Rudy A

2014-05-08

Detection of leptospires based on DNA amplification techniques is essential for the early diagnosis of leptospirosis when anti-Leptospira antibodies are below the detection limit of most serological tests. In middle and low income countries where leptospirosis is endemic, routine implementation of real-time PCR is financially and technically challenging due to the requirement of expensive thermocycler equipment. In this study we report the development and evaluation of a novel isothermal recombinase polymerase amplification assay (RPA) for detection of pathogenic Leptospira based on TwistAmp chemistry. RPA enabled the detection of less than two genome copies per reaction. Retrospective evaluation revealed a high diagnostic accuracy (sensitivity and specificity of 94.7% and 97.7%, respectively) compared to culturing as the reference standard. RPA presents a powerful tool for the early diagnosis of leptospirosis in humans and in animals. Furthermore, it enables the detection of the causative agent in reservoirs and environment, and as such is a valuable adjunct to current tools for surveillance and early outbreak warning.

Development of a Recombinase Polymerase Amplification Assay for the Detection of Pathogenic Leptospira

PubMed Central

Ahmed, Ahmed; van der Linden, Hans; Hartskeerl, Rudy A.

2014-01-01

Detection of leptospires based on DNA amplification techniques is essential for the early diagnosis of leptospirosis when anti-Leptospira antibodies are below the detection limit of most serological tests. In middle and low income countries where leptospirosis is endemic, routine implementation of real-time PCR is financially and technically challenging due to the requirement of expensive thermocycler equipment. In this study we report the development and evaluation of a novel isothermal recombinase polymerase amplification assay (RPA) for detection of pathogenic Leptospira based on TwistAmp chemistry. RPA enabled the detection of less than two genome copies per reaction. Retrospective evaluation revealed a high diagnostic accuracy (sensitivity and specificity of 94.7% and 97.7%, respectively) compared to culturing as the reference standard. RPA presents a powerful tool for the early diagnosis of leptospirosis in humans and in animals. Furthermore, it enables the detection of the causative agent in reservoirs and environment, and as such is a valuable adjunct to current tools for surveillance and early outbreak warning. PMID:24814943

Ultrasensitive detection of nucleic acids by template enhanced hybridization followed by rolling circle amplification and catalytic hairpin assembly.

PubMed

Song, Weiling; Zhang, Qiao; Sun, Wenbo

2015-02-11

An ultrasensitive protocol for fluorescent detection of DNA is designed by combining the template enhanced hybridization process (TEHP) with Rolling Circle Amplification (RCA) and Catalytic Hairpin Assembly (CHA), showing a remarkable amplification efficiency.

Integrating DNA strand displacement circuitry to the nonlinear hybridization chain reaction.

PubMed

Zhang, Zhuo; Fan, Tsz Wing; Hsing, I-Ming

2017-02-23

Programmable and modular attributes of DNA molecules allow one to develop versatile sensing platforms that can be operated isothermally and enzyme-free. In this work, we present an approach to integrate upstream DNA strand displacement circuits that can be turned on by a sequence-specific microRNA analyte with a downstream nonlinear hybridization chain reaction for a cascading hyperbranched nucleic acid assembly. This system provides a two-step amplification strategy for highly sensitive detection of the miRNA analyte, conducive for multiplexed detection. Multiple miRNA analytes were tested with our integrated circuitry using the same downstream signal amplification setting, showing the decoupling of nonlinear self-assembly with the analyte sequence. Compared with the reported methods, our signal amplification approach provides an additional control module for higher-order DNA self-assembly and could be developed into a promising platform for the detection of critical nucleic-acid based biomarkers.

A microdissection approach to detect molecular markers during progression of prostate cancer.

PubMed Central

Berthon, P.; Dimitrov, T.; Stower, M.; Cussenot, O.; Maitland, N. J.

1995-01-01

To investigate the underlying mechanisms of carcinogenesis, we have developed a technique to determine the frequency of genetic changes in prostatic carcinoma tissue. We have demonstrated that at a ratio of between 1:4 and 1:9 mutant-normal alleles, the signal from a mutant TP53 allele is not apparent after polymerase chain reaction (PCR) amplification and further direct sequencing or single-strand conformation polymorphism (SSCP) analysis. To bypass this problem, which is inherent in the heterogeneity of the prostate tissue and of the tumour, we selected areas of graded prostate tumours (Gleason score) from cryosectioned preparations and microdissected these cells (20-100 cells). After anionic resin removal of proteins, PCR amplification of TP53 gene exons 5/6 and SSCP analysis, an abnormal SSCP band shift was observed in suspected tumour cells, compared with microdissected stromal cells used as an internal control, while (1) a crude preparation of tissue DNA carrying the tumour did not show any abnormality and (2) immunostaining by a set of monoclonal antibodies against TP53 protein remained negative. Nucleotide sequence analysis of the different bands confirmed the presence of a mutation in the TP53 gene exon 6 position 13,336 in an abnormal band for one specimen, while no mutation was detected in the normal SSCP band. By targeting recognised tumour cells we can find DNA mutations which are undetectable using the standard technique of whole-tissue DNA extraction, particularly in a heterogeneous tumour such as carcinoma of the prostate. Images Figure 1 Figure 2 Figure 3 Figure 4 PMID:7547246

Silver nanoclusters-assisted ion-exchange reaction with CdTe quantum dots for photoelectrochemical detection of adenosine by target-triggering multiple-cycle amplification strategy.

PubMed

Zhao, Yang; Tan, Lu; Gao, Xiaoshan; Jie, Guifen; Huang, Tingyu

2018-07-01

Herein, we successfully devised a novel photoelectrochemical (PEC) platform for ultrasensitive detection of adenosine by target-triggering cascade multiple cycle amplification based on the silver nanoparticles-assisted ion-exchange reaction with CdTe quantum dots (QDs). In the presence of target adenosine, DNA s1 is released from the aptamer and then hybridizes with hairpin DNA (HP1), which could initiate the cycling cleavage process under the reaction of nicking endonuclease. Then the product (DNA b) of cycle I could act as the "DNA trigger" of cycle II to further generate a large number of DNA s1, which again go back to cycle I, thus a cascade multiple DNA cycle amplification was carried out to produce abundant DNA c. These DNA c fragments with the cytosine (C)-rich loop were captured by magnetic beads, and numerous silver nanoclusters (Ag NCs) were synthesized by AgNO 3 and sodium borohydride. The dissolved AgNCs released numerous silver ions which could induce ion exchange reaction with the CdTe QDs, thus resulting in greatly amplified change of photocurrent for target detection. The detection linear range for adenosine was 1.0 fM ~10 nM with the detection limit of 0.5 fM. The present PEC strategy combining cascade multiple DNA cycle amplification and AgNCs-induced ion-exchange reaction with QDs provides new insight into rapid, and ultrasensitive PEC detection of different biomolecules, which showed great potential for detecting trace amounts in bioanalysis and clinical biomedicine. Copyright © 2018 Elsevier B.V. All rights reserved.

Primer Extension Mutagenesis Powered by Selective Rolling Circle Amplification

PubMed Central

Huovinen, Tuomas; Brockmann, Eeva-Christine; Akter, Sultana; Perez-Gamarra, Susan; Ylä-Pelto, Jani; Liu, Yuan; Lamminmäki, Urpo

2012-01-01

Primer extension mutagenesis is a popular tool to create libraries for in vitro evolution experiments. Here we describe a further improvement of the method described by T.A. Kunkel using uracil-containing single-stranded DNA as the template for the primer extension by additional uracil-DNA glycosylase treatment and rolling circle amplification (RCA) steps. It is shown that removal of uracil bases from the template leads to selective amplification of the nascently synthesized circular DNA strand carrying the desired mutations by phi29 DNA polymerase. Selective RCA (sRCA) of the DNA heteroduplex formed in Kunkel's mutagenesis increases the mutagenesis efficiency from 50% close to 100% and the number of transformants 300-fold without notable diversity bias. We also observed that both the mutated and the wild-type DNA were present in at least one third of the cells transformed directly with Kunkel's heteroduplex. In contrast, the cells transformed with sRCA product contained only mutated DNA. In sRCA, the complex cell-based selection for the mutant strand is replaced with the more controllable enzyme-based selection and less DNA is needed for library creation. Construction of a gene library of ten billion members is demonstrated with the described method with 240 nanograms of DNA as starting material. PMID:22355397

Extraction of high-quality DNA from ethanol-preserved tropical plant tissues.

PubMed

Bressan, Eduardo A; Rossi, Mônica L; Gerald, Lee T S; Figueira, Antonio

2014-04-24

Proper conservation of plant samples, especially during remote field collection, is essential to assure quality of extracted DNA. Tropical plant species contain considerable amounts of secondary compounds, such as polysaccharides, phenols, and latex, which affect DNA quality during extraction. The suitability of ethanol (96% v/v) as a preservative solution prior to DNA extraction was evaluated using leaves of Jatropha curcas and other tropical species. Total DNA extracted from leaf samples stored in liquid nitrogen or ethanol from J. curcas and other tropical species (Theobroma cacao, Coffea arabica, Ricinus communis, Saccharum spp., and Solanum lycopersicon) was similar in quality, with high-molecular-weight DNA visualized by gel electrophoresis. DNA quality was confirmed by digestion with EcoRI or HindIII and by amplification of the ribosomal gene internal transcribed spacer region. Leaf tissue of J. curcas was analyzed by light and transmission electron microscopy before and after exposure to ethanol. Our results indicate that leaf samples can be successfully preserved in ethanol for long periods (30 days) as a viable method for fixation and conservation of DNA from leaves. The success of this technique is likely due to reduction or inactivation of secondary metabolites that could contaminate or degrade genomic DNA. Tissue conservation in 96% ethanol represents an attractive low-cost alternative to commonly used methods for preservation of samples for DNA extraction. This technique yields DNA of equivalent quality to that obtained from fresh or frozen tissue.

Extraction of high-quality DNA from ethanol-preserved tropical plant tissues

PubMed Central

2014-01-01

Background Proper conservation of plant samples, especially during remote field collection, is essential to assure quality of extracted DNA. Tropical plant species contain considerable amounts of secondary compounds, such as polysaccharides, phenols, and latex, which affect DNA quality during extraction. The suitability of ethanol (96% v/v) as a preservative solution prior to DNA extraction was evaluated using leaves of Jatropha curcas and other tropical species. Results Total DNA extracted from leaf samples stored in liquid nitrogen or ethanol from J. curcas and other tropical species (Theobroma cacao, Coffea arabica, Ricinus communis, Saccharum spp., and Solanum lycopersicon) was similar in quality, with high-molecular-weight DNA visualized by gel electrophoresis. DNA quality was confirmed by digestion with EcoRI or HindIII and by amplification of the ribosomal gene internal transcribed spacer region. Leaf tissue of J. curcas was analyzed by light and transmission electron microscopy before and after exposure to ethanol. Our results indicate that leaf samples can be successfully preserved in ethanol for long periods (30 days) as a viable method for fixation and conservation of DNA from leaves. The success of this technique is likely due to reduction or inactivation of secondary metabolites that could contaminate or degrade genomic DNA. Conclusions Tissue conservation in 96% ethanol represents an attractive low-cost alternative to commonly used methods for preservation of samples for DNA extraction. This technique yields DNA of equivalent quality to that obtained from fresh or frozen tissue. PMID:24761774

Validation of an entirely in vitro approach for rapid prototyping of DNA regulatory elements for synthetic biology

PubMed Central

Chappell, James; Jensen, Kirsten; Freemont, Paul S.

2013-01-01

A bottleneck in our capacity to rationally and predictably engineer biological systems is the limited number of well-characterized genetic elements from which to build. Current characterization methods are tied to measurements in living systems, the transformation and culturing of which are inherently time-consuming. To address this, we have validated a completely in vitro approach for the characterization of DNA regulatory elements using Escherichia coli extract cell-free systems. Importantly, we demonstrate that characterization in cell-free systems correlates and is reflective of performance in vivo for the most frequently used DNA regulatory elements. Moreover, we devise a rapid and completely in vitro method to generate DNA templates for cell-free systems, bypassing the need for DNA template generation and amplification from living cells. This in vitro approach is significantly quicker than current characterization methods and is amenable to high-throughput techniques, providing a valuable tool for rapidly prototyping libraries of DNA regulatory elements for synthetic biology. PMID:23371936

DNA barcoding Indian freshwater fishes.

PubMed

Lakra, Wazir Singh; Singh, M; Goswami, Mukunda; Gopalakrishnan, A; Lal, K K; Mohindra, V; Sarkar, U K; Punia, P P; Singh, K V; Bhatt, J P; Ayyappan, S

2016-11-01

DNA barcoding is a promising technique for species identification using a short mitochondrial DNA sequence of cytochrome c oxidase I (COI) gene. In the present study, DNA barcodes were generated from 72 species of freshwater fish covering the Orders Cypriniformes, Siluriformes, Perciformes, Synbranchiformes, and Osteoglossiformes representing 50 genera and 19 families. All the samples were collected from diverse sites except the species endemic to a particular location. Species were represented by multiple specimens in the great majority of the barcoded species. A total of 284 COI sequences were generated. After amplification and sequencing of 700 base pair fragment of COI, primers were trimmed which invariably generated a 655 base pair barcode sequence. The average Kimura two-parameter (K2P) distances within-species, genera, families, and orders were 0.40%, 9.60%, 13.10%, and 17.16%, respectively. DNA barcode discriminated congeneric species without any confusion. The study strongly validated the efficiency of COI as an ideal marker for DNA barcoding of Indian freshwater fishes.

Comparison of loop-mediated isothermal amplification (LAMP) and nested-PCR assay targeting the RE and B1 gene for detection of Toxoplasma gondii in blood samples of children with leukaemia.

PubMed

Fallahi, Shirzad; Seyyed Tabaei, Seyyed Javad; Pournia, Yadollah; Zebardast, Nozhat; Kazemi, Bahram

2014-07-01

Toxoplasmosis diagnosis constitutes an important measure for disease prevention and control. In this paper, a newly described DNA amplification technique, loop-mediated isothermal amplification (LAMP), and nested-PCR targeting the repeated element (RE) and B1 gene, were compared to each other for the detection of Toxoplasma gondii DNA in blood samples of children with leukaemia. One hundred ten blood samples from these patients were analyzed by LAMP and nested-PCR. Out of 50 seropositive samples (IgM+, IgG+), positive results were obtained with 92% and 86% on RE, B1-LAMP and 82% and 68% on RE, B1-nested PCR analyses, respectively. Of the 50 seronegative samples, three, two and one samples were detected positive by RE-LAMP, B1-LAMP and RE-nested PCR assays, respectively, while none were detected positive by B1-nested PCR. None of the 10 IgM-, IgG+ samples was detected positive after testing LAMP and nested-PCR assays in duplicate. This is the first report of a study in which the LAMP method was applied with high sensitivity and efficacy for the diagnosis of T. gonii in blood samples of children with leukaemia. Copyright © 2014 Elsevier Inc. All rights reserved.

LAMP-PCR detection of ochratoxigenic Aspergillus species collected from peanut kernel.

PubMed

Al-Sheikh, H M

2015-01-30

Over the last decade, ochratoxin A (OTA) has been widely described and is ubiquitous in several agricultural products. Ochratoxins represent the second-most important mycotoxin group after aflatoxins. A total of 34 samples were surveyed from 3 locations, including Mecca, Madina, and Riyadh, Saudi Arabia, during 2012. Fungal contamination frequency was determined for surface-sterilized peanut seeds, which were seeded onto malt extract agar media. Aspergillus niger (35%), Aspergillus ochraceus (30%), and Aspergillus carbonarius (25%) were the most frequently observed Aspergillius species, while Aspergillus flavus and Aspergillus phoenicis isolates were only infrequently recovered and in small numbers (10%). OTA production was evaluated on yeast extract sucrose medium, which revealed that 57% of the isolates were A. niger and 60% of A. carbonarius isolates were OTA producers; 100% belonged to A. ochraceus. Only one isolate, morphologically identified as A. carbonarius, and 3 A. niger isolates unstably produced OTA. A polymerase chain reaction (PCR)-based identification and detection assay was used to identify A. ochraceus isolates. Using the primer sets OCRA1/OCRA2, 400-base pair PCR fragments were produced only when genomic DNA from A. ochraceus isolates was used. Recently, the loop-mediated isothermal amplification assay using recombinase polymerase amplification chemistry was used for A. carbonarius and A. niger DNA identification. As a non-gel-based technique, the amplification product was directly visualized in the reaction tube after adding calcein for naked-eye examination.

Effect of DNA Extraction Methods and Sampling Techniques on the Apparent Structure of Cow and Sheep Rumen Microbial Communities

PubMed Central

Henderson, Gemma; Cox, Faith; Kittelmann, Sandra; Miri, Vahideh Heidarian; Zethof, Michael; Noel, Samantha J.; Waghorn, Garry C.; Janssen, Peter H.

2013-01-01

Molecular microbial ecology techniques are widely used to study the composition of the rumen microbiota and to increase understanding of the roles they play. Therefore, sampling and DNA extraction methods that result in adequate yields of microbial DNA that also accurately represents the microbial community are crucial. Fifteen different methods were used to extract DNA from cow and sheep rumen samples. The DNA yield and quality, and its suitability for downstream PCR amplifications varied considerably, depending on the DNA extraction method used. DNA extracts from nine extraction methods that passed these first quality criteria were evaluated further by quantitative PCR enumeration of microbial marker loci. Absolute microbial numbers, determined on the same rumen samples, differed by more than 100-fold, depending on the DNA extraction method used. The apparent compositions of the archaeal, bacterial, ciliate protozoal, and fungal communities in identical rumen samples were assessed using 454 Titanium pyrosequencing. Significant differences in microbial community composition were observed between extraction methods, for example in the relative abundances of members of the phyla Bacteroidetes and Firmicutes. Microbial communities in parallel samples collected from cows by oral stomach-tubing or through a rumen fistula, and in liquid and solid rumen digesta fractions, were compared using one of the DNA extraction methods. Community representations were generally similar, regardless of the rumen sampling technique used, but significant differences in the abundances of some microbial taxa such as the Clostridiales and the Methanobrevibacter ruminantium clade were observed. The apparent microbial community composition differed between rumen sample fractions, and Prevotellaceae were most abundant in the liquid fraction. DNA extraction methods that involved phenol-chloroform extraction and mechanical lysis steps tended to be more comparable. However, comparison of data from studies in which different sampling techniques, different rumen sample fractions or different DNA extraction methods were used should be avoided. PMID:24040342

Effect of DNA extraction methods and sampling techniques on the apparent structure of cow and sheep rumen microbial communities.

PubMed

Henderson, Gemma; Cox, Faith; Kittelmann, Sandra; Miri, Vahideh Heidarian; Zethof, Michael; Noel, Samantha J; Waghorn, Garry C; Janssen, Peter H

2013-01-01

Molecular microbial ecology techniques are widely used to study the composition of the rumen microbiota and to increase understanding of the roles they play. Therefore, sampling and DNA extraction methods that result in adequate yields of microbial DNA that also accurately represents the microbial community are crucial. Fifteen different methods were used to extract DNA from cow and sheep rumen samples. The DNA yield and quality, and its suitability for downstream PCR amplifications varied considerably, depending on the DNA extraction method used. DNA extracts from nine extraction methods that passed these first quality criteria were evaluated further by quantitative PCR enumeration of microbial marker loci. Absolute microbial numbers, determined on the same rumen samples, differed by more than 100-fold, depending on the DNA extraction method used. The apparent compositions of the archaeal, bacterial, ciliate protozoal, and fungal communities in identical rumen samples were assessed using 454 Titanium pyrosequencing. Significant differences in microbial community composition were observed between extraction methods, for example in the relative abundances of members of the phyla Bacteroidetes and Firmicutes. Microbial communities in parallel samples collected from cows by oral stomach-tubing or through a rumen fistula, and in liquid and solid rumen digesta fractions, were compared using one of the DNA extraction methods. Community representations were generally similar, regardless of the rumen sampling technique used, but significant differences in the abundances of some microbial taxa such as the Clostridiales and the Methanobrevibacter ruminantium clade were observed. The apparent microbial community composition differed between rumen sample fractions, and Prevotellaceae were most abundant in the liquid fraction. DNA extraction methods that involved phenol-chloroform extraction and mechanical lysis steps tended to be more comparable. However, comparison of data from studies in which different sampling techniques, different rumen sample fractions or different DNA extraction methods were used should be avoided.

Robust detection of EGFR copy number changes and EGFR variant III: technical aspects and relevance for glioma diagnostics.

PubMed

Jeuken, Judith; Sijben, Angelique; Alenda, Cristina; Rijntjes, Jos; Dekkers, Marieke; Boots-Sprenger, Sandra; McLendon, Roger; Wesseling, Pieter

2009-10-01

Epidermal growth factor receptor (EGFR) is commonly affected in cancer, generally in the form of an increase in DNA copy number and/or as mutation variants [e.g., EGFR variant III (EGFRvIII), an in-frame deletion of exons 2-7]. While detection of EGFR aberrations can be expected to be relevant for glioma patients, such analysis has not yet been implemented in a routine setting, also because feasible and robust assays were lacking. We evaluated multiplex ligation-dependent probe amplification (MLPA) for detection of EGFR amplification and EGFRvIII in DNA of a spectrum of 216 diffuse gliomas. EGFRvIII detection was verified at the protein level by immunohistochemistry and at the RNA level using the conventionally used endpoint RT-PCR as well as a newly developed quantitative RT-PCR. Compared to these techniques, the DNA-based MLPA assay for EGFR/EGFRvIII analysis tested showed 100% sensitivity and specificity. We conclude that MLPA is a robust assay for detection of EGFR/EGFRvIII aberrations. While the exact diagnostic, prognostic and predictive value of such EGFR testing remains to be seen, MLPA has great potential as it can reliably and relatively easily be performed on routinely processed (formalin-fixed, paraffin-embedded) tumor tissue in combination with testing for other relevant glioma markers.

Enzyme-free and isothermal detection of microRNA based on click-chemical ligation-assisted hybridization coupled with hybridization chain reaction signal amplification.

PubMed

Oishi, Motoi

2015-05-01

An enzyme-free and isothermal microRNA (miRNA) detection method has been developed based on click-chemical ligation-assisted hybridization coupled with hybridization chain reaction (HCR) on magnetic beads (MBs). The click-chemical ligation between an azide-modified probe DNA and a dibenzocyclooctyne-modified probe DNA occurred through the hybridization of target miRNA (miR-141). HCR on MBs was performed by the addition of DNA hairpin monomers (H1 and H2). After magnetic separation and denaturation/rehybridization of HCR products ([H1/H2] n ), the resulting HCR products were analyzed by the fluorescence emitted from an intercalative dye, allowing amplification of the fluorescent signal. The proposed assay had a limit of detection of 0.55 fmol, which was 230-fold more sensitive than that of the HCR on the MBs coupled with a conventional sandwich hybridization assay (without click-chemical ligation) (limit of detection 127 fmol). Additionally, the proposed assay could discriminate between miR-141 and other miR-200 family members. In contrast to quantitative reverse transcription polymerase chain reaction techniques using enzymes and thermal cycling, this is an enzyme-free assay that can be conducted under isothermal conditions and can specifically detect miR-141 in fetal bovine serum.

A cascade signal amplification strategy for surface enhanced Raman spectroscopy detection of thrombin based on DNAzyme assistant DNA recycling and rolling circle amplification.

PubMed

Gao, Fenglei; Du, Lili; Tang, Daoquan; Lu, Yao; Zhang, Yanzhuo; Zhang, Lixian

2015-04-15

A sensitive protocol for surface enhanced Raman spectroscopy (SERS) detection of thrombin is designed with R6G-Ag NPs as a signal tag by combining DNAzyme assistant DNA recycling and rolling circle amplification (RCA). Molecular beacon (MB) as recognition probe immobilizes on the glass slides and performs the amplification procedure. After thrombin-induced structure-switching DNA hairpins of probe 1, the DNAzyme is liberated from the caged structure, which hybridizes with the MB for cleavage of the MB in the presence of cofactor Zn(2+) and initiates the DNA recycling process, leading to the cleavage of a large number of MB and the generation of numerous primers for triggering RCA reaction. The long amplified RCA product which contained hundreds of tandem-repeat sequences, which can bind with oligonucleotide functionalized Ag NPs reporters. The attached signal tags can be easily read out by SERS. Because of the cascade signal amplification, these newly designed protocols provides a sensitive SERS detection of thrombin down to the femolar level (2.3fM) with a linear range of 5 orders of magnitude (from 10(-14) to 10(-9)M) and have high selectivity toward its target protein. The proposed method is expected to be a good clinical tool for the diagnosis of a thrombotic disease. Copyright © 2014 Elsevier B.V. All rights reserved.

Boronic Acid Functionalized Au Nanoparticles for Selective MicroRNA Signal Amplification in Fiber-Optic Surface Plasmon Resonance Sensing System.

PubMed

Qian, Siyu; Lin, Ming; Ji, Wei; Yuan, Huizhen; Zhang, Yang; Jing, Zhenguo; Zhao, Jianzhang; Masson, Jean-François; Peng, Wei

2018-05-25

MicroRNA (miRNA) regulates gene expression and plays a fundamental role in multiple biological processes. However, if both single-stranded RNA and DNA can bind with capture DNA on the sensing surface, selectively amplifying the complementary RNA signal is still challenging for researchers. Fiber-optic surface plasmon resonance (SPR) sensors are small, accurate, and convenient tools for monitoring biological interaction. In this paper, we present a high sensitivity microRNA detection technique using phenylboronic acid functionalized Au nanoparticles (PBA-AuNPs) in fiber-optic SPR sensing systems. Due to the inherent difficulty directly detecting the hybridized RNA on the sensing surface, the PBA-AuNPs were used to selectively amplify the signal of target miRNA. The result shows that the method has high selectivity and sensitivity for miRNA, with a detection limit at 2.7 × 10 -13 M (0.27 pM). This PBA-AuNPs amplification strategy is universally applicable for RNA detection with various sensing technologies, such as surface-enhanced Raman spectroscopy and electrochemistry, among others.

A Paper and Plastic Device for Performing Recombinase Polymerase Amplification of HIV DNA

PubMed Central

Rohrman, Brittany A.; Richards-Kortum, Rebecca R.

2013-01-01

Despite the importance of early diagnosis and treatment of HIV, only a small fraction of HIV-exposed infants in low- and middle-income countries are tested for the disease. The gold standard for early infant diagnosis, DNA PCR, requires resources that are unavailable in poor settings, and no point-of-care HIV DNA test is currently available. We have developed a device constructed of layers of paper, glass fiber, and plastic that is capable of performing isothermal, enzymatic amplification of HIV DNA. The device is inexpensive, small, light-weight, and easy to assemble. The device stores lyophilized enzymes, facilitates mixing of reaction components, and supports recombinase polymerase amplification in five steps of operation. Using commercially available lateral flow strips as a detection method, we demonstrate the ability of our device to amplify 10 copies of HIV DNA to detectable levels in 15 minutes. Our results suggest that our device, which is designed to be used after DNA extraction from dried-blood spots, may serve in conjunction with lateral flow strips as part of a point-of-care HIV DNA test to be used in low resource settings. PMID:22733333

A paper and plastic device for performing recombinase polymerase amplification of HIV DNA.

PubMed

Rohrman, Brittany A; Richards-Kortum, Rebecca R

2012-09-07

Despite the importance of early diagnosis and treatment of HIV, only a small fraction of HIV-exposed infants in low- and middle-income countries are tested for the disease. The gold standard for early infant diagnosis, DNA PCR, requires resources that are unavailable in poor settings, and no point-of-care HIV DNA test is currently available. We have developed a device constructed of layers of paper, glass fiber, and plastic that is capable of performing isothermal, enzymatic amplification of HIV DNA. The device is inexpensive, small, light-weight, and easy to assemble. The device stores lyophilized enzymes, facilitates mixing of reaction components, and supports recombinase polymerase amplification in five steps of operation. Using commercially available lateral flow strips as a detection method, we demonstrate the ability of our device to amplify 10 copies of HIV DNA to detectable levels in 15 min. Our results suggest that our device, which is designed to be used after DNA extraction from dried-blood spots, may serve in conjunction with lateral flow strips as part of a point-of-care HIV DNA test to be used in low resource settings.

Helicase-dependent amplification of nucleic acids.

PubMed

Cao, Yun; Kim, Hyun-Jin; Li, Ying; Kong, Huimin; Lemieux, Bertrand

2013-10-11

Helicase-dependent amplification (HDA) is a novel method for the isothermal in vitro amplification of nucleic acids. The HDA reaction selectively amplifies a target sequence by extension of two oligonucleotide primers. Unlike the polymerase chain reaction (PCR), HDA uses a helicase enzyme to separate the deoxyribonucleic acid (DNA) strands, rather than heat denaturation. This allows DNA amplification without the need for thermal cycling. The helicase used in HDA is a helicase super family II protein obtained from a thermophilic organism, Thermoanaerobacter tengcongensis (TteUvrD). This thermostable helicase is capable of unwinding blunt-end nucleic acid substrates at elevated temperatures (60° to 65°C). The HDA reaction can also be coupled with reverse transcription for ribonucleic acid (RNA) amplification. The products of this reaction can be detected during the reaction using fluorescent probes when incubations are conducted in a fluorimeter. Alternatively, products can be detected after amplification using a disposable amplicon containment device that contains an embedded lateral flow strip. Copyright © 2013 John Wiley & Sons, Inc.

Inhibition of recombinase polymerase amplification by background DNA: a lateral flow-based method for enriching target DNA.

PubMed

Rohrman, Brittany; Richards-Kortum, Rebecca

2015-02-03

Recombinase polymerase amplification (RPA) may be used to detect a variety of pathogens, often after minimal sample preparation. However, previous work has shown that whole blood inhibits RPA. In this paper, we show that the concentrations of background DNA found in whole blood prevent the amplification of target DNA by RPA. First, using an HIV-1 RPA assay with known concentrations of nonspecific background DNA, we show that RPA tolerates more background DNA when higher HIV-1 target concentrations are present. Then, using three additional assays, we demonstrate that the maximum amount of background DNA that may be tolerated in RPA reactions depends on the DNA sequences used in the assay. We also show that changing the RPA reaction conditions, such as incubation time and primer concentration, has little effect on the ability of RPA to function when high concentrations of background DNA are present. Finally, we develop and characterize a lateral flow-based method for enriching the target DNA concentration relative to the background DNA concentration. This sample processing method enables RPA of 10(4) copies of HIV-1 DNA in a background of 0-14 μg of background DNA. Without lateral flow sample enrichment, the maximum amount of background DNA tolerated is 2 μg when 10(6) copies of HIV-1 DNA are present. This method requires no heating or other external equipment, may be integrated with upstream DNA extraction and purification processes, is compatible with the components of lysed blood, and has the potential to detect HIV-1 DNA in infant whole blood with high proviral loads.

Sensitive SERS detection of DNA methyltransferase by target triggering primer generation-based multiple signal amplification strategy.

PubMed

Li, Ying; Yu, Chuanfeng; Han, Huixia; Zhao, Caisheng; Zhang, Xiaoru

2016-07-15

A novel and sensitive surface-enhanced Raman scattering (SERS) method is proposed for the assay of DNA methyltransferase (MTase) activity and evaluation of inhibitors by developing a target triggering primer generation-based multiple signal amplification strategy. By using of a duplex substrate for Dam MTase, two hairpin templates and a Raman probe, multiple signal amplification mode is achieved. Once recognized by Dam MTase, the duplex substrate can be cleaved by Dpn I endonuclease and two primers are released for triggering the multiple signal amplification reaction. Consequently, a wide dynamic range and remarkably high sensitivity are obtained under isothermal conditions. The detection limit is 2.57×10(-4)UmL(-1). This assay exhibits an excellent selectivity and is successfully applied in the screening of inhibitors for Dam MTase. In addition, this novel sensing system is potentially universal as the recognition element can be conveniently designed for other target analytes by changing the substrate of DNA MTase. Copyright © 2016 Elsevier B.V. All rights reserved.

Genome amplification of single sperm using multiple displacement amplification.

PubMed

Jiang, Zhengwen; Zhang, Xingqi; Deka, Ranjan; Jin, Li

2005-06-07

Sperm typing is an effective way to study recombination rate on a fine scale in regions of interest. There are two strategies for the amplification of single meiotic recombinants: repulsion-phase allele-specific PCR and whole genome amplification (WGA). The former can selectively amplify single recombinant molecules from a batch of sperm but is not scalable for high-throughput operation. Currently, primer extension pre-amplification is the only method used in WGA of single sperm, whereas it has limited capacity to produce high-coverage products enough for the analysis of local recombination rate in multiple large regions. Here, we applied for the first time a recently developed WGA method, multiple displacement amplification (MDA), to amplify single sperm DNA, and demonstrated its great potential for producing high-yield and high-coverage products. In a 50 mul reaction, 76 or 93% of loci can be amplified at least 2500- or 250-fold, respectively, from single sperm DNA, and second-round MDA can further offer >200-fold amplification. The MDA products are usable for a variety of genetic applications, including sequencing and microsatellite marker and single nucleotide polymorphism (SNP) analysis. The use of MDA in single sperm amplification may open a new era for studies on local recombination rates.

Prediction of autosomal STR typing success in ancient and Second World War bone samples.

PubMed

Zupanič Pajnič, Irena; Zupanc, Tomaž; Balažic, Jože; Geršak, Živa Miriam; Stojković, Oliver; Skadrić, Ivan; Črešnar, Matija

2017-03-01

Human-specific quantitative PCR (qPCR) has been developed for forensic use in the last 10 years and is the preferred DNA quantification technique since it is very accurate, sensitive, objective, time-effective and automatable. The amount of information that can be gleaned from a single quantification reaction using commercially available quantification kits has increased from the quantity of nuclear DNA to the amount of male DNA, presence of inhibitors and, most recently, to the degree of DNA degradation. In skeletal remains samples from disaster victims, missing persons and war conflict victims, the DNA is usually degraded. Therefore the new commercial qPCR kits able to assess the degree of degradation are potentially able to predict the success of downstream short tandem repeat (STR) typing. The goal of this study was to verify the quantification step using the PowerQuant kit with regard to its suitability as a screening method for autosomal STR typing success on ancient and Second World War (WWII) skeletal remains. We analysed 60 skeletons excavated from five archaeological sites and four WWII mass graves from Slovenia. The bones were cleaned, surface contamination was removed and the bones ground to a powder. Genomic DNA was obtained from 0.5g of bone powder after total demineralization. The DNA was purified using a Biorobot EZ1 device. Following PowerQuant quantification, DNA samples were subjected to autosomal STR amplification using the NGM kit. Up to 2.51ng DNA/g of powder were extracted. No inhibition was detected in any of bones analysed. 82% of the WWII bones gave full profiles while 73% of the ancient bones gave profiles not suitable for interpretation. Four bone extracts yielded no detectable amplification or zero quantification results and no profiles were obtained from any of them. Full or useful partial profiles were produced only from bone extracts where short autosomal (Auto) and long degradation (Deg) PowerQuant targets were detected. It is concluded that STR typing of old bones after quantification with the PowerQuant should be performed only when both Auto and Deg targets are detected simultaneously with no respect to [Auto]/[Deg] ratio. Prediction of STR typing success could be made according to successful amplification of Deg fragment. The PowerQuant kit is capable of identifying bone DNA samples that will not yield useful STR profiles using the NGM kit, and it can be used as a predictor of autosomal STR typing success of bone extracts obtained from ancient and WWII skeletal remains. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Identification of Genetic Elements Associated with EPSPS Gene Amplification

PubMed Central

Gaines, Todd A.; Wright, Alice A.; Molin, William T.; Lorentz, Lothar; Riggins, Chance W.; Tranel, Patrick J.; Beffa, Roland; Westra, Philip; Powles, Stephen B.

2013-01-01

Weed populations can have high genetic plasticity and rapid responses to environmental selection pressures. For example, 100-fold amplification of the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene evolved in the weed species Amaranthus palmeri to confer resistance to glyphosate, the world’s most important herbicide. However, the gene amplification mechanism is unknown. We sequenced the EPSPS gene and genomic regions flanking EPSPS loci in A. palmeri, and searched for mobile genetic elements or repetitive sequences. The EPSPS gene was 10,229 bp, containing 8 exons and 7 introns. The gene amplification likely proceeded through a DNA-mediated mechanism, as introns exist in the amplified gene copies and the entire amplified sequence is at least 30 kb in length. Our data support the presence of two EPSPS loci in susceptible (S) A. palmeri, and that only one of these was amplified in glyphosate-resistant (R) A. palmeri. The EPSPS gene amplification event likely occurred recently, as no sequence polymorphisms were found within introns of amplified EPSPS copies from R individuals. Sequences with homology to miniature inverted-repeat transposable elements (MITEs) were identified next to EPSPS gene copies only in R individuals. Additionally, a putative Activator (Ac) transposase and a repetitive sequence region were associated with amplified EPSPS genes. The mechanism controlling this DNA-mediated amplification remains unknown. Further investigation is necessary to determine if the gene amplification may have proceeded via DNA transposon-mediated replication, and/or unequal recombination between different genomic regions resulting in replication of the EPSPS gene. PMID:23762434

Signal-on electrochemical detection of antibiotics at zeptomole level based on target-aptamer binding triggered multiple recycling amplification.

PubMed

Wang, Hongzhi; Wang, Yu; Liu, Su; Yu, Jinghua; Guo, Yuna; Xu, Ying; Huang, Jiadong

2016-06-15

In the work, a signal-on electrochemical DNA sensor based on multiple amplification for ultrasensitive detection of antibiotics has been reported. In the presence of target, the ingeniously designed hairpin probe (HP1) is opened and the polymerase-assisted target recycling amplification is triggered, resulting in autonomous generation of secondary target. It is worth noting that the produced secondary target could not only hybridize with other HP1, but also displace the Helper from the electrode. Consequently, methylene blue labeled HP2 forms a "close" probe structure, and the increase of signal is monitored. The increasing current provides an ultrasensitive electrochemical detection for antibiotics down to 1.3 fM. To our best knowledge, such work is the first report about multiple recycling amplification combing with signal-on sensing strategy, which has been utilized for quantitative determination of antibiotics. It would be further used as a general strategy associated with more analytical techniques toward the detection of a wide spectrum of analytes. Thus, it holds great potential for the development of ultrasensitive biosensing platform for the applications in bioanalysis, disease diagnostics, and clinical biomedicine. Copyright © 2016 Elsevier B.V. All rights reserved.

DNA barcoding the floras of biodiversity hotspots.

PubMed

Lahaye, Renaud; van der Bank, Michelle; Bogarin, Diego; Warner, Jorge; Pupulin, Franco; Gigot, Guillaume; Maurin, Olivier; Duthoit, Sylvie; Barraclough, Timothy G; Savolainen, Vincent

2008-02-26

DNA barcoding is a technique in which species identification is performed by using DNA sequences from a small fragment of the genome, with the aim of contributing to a wide range of ecological and conservation studies in which traditional taxonomic identification is not practical. DNA barcoding is well established in animals, but there is not yet any universally accepted barcode for plants. Here, we undertook intensive field collections in two biodiversity hotspots (Mesoamerica and southern Africa). Using >1,600 samples, we compared eight potential barcodes. Going beyond previous plant studies, we assessed to what extent a "DNA barcoding gap" is present between intra- and interspecific variations, using multiple accessions per species. Given its adequate rate of variation, easy amplification, and alignment, we identified a portion of the plastid matK gene as a universal DNA barcode for flowering plants. Critically, we further demonstrate the applicability of DNA barcoding for biodiversity inventories. In addition, analyzing >1,000 species of Mesoamerican orchids, DNA barcoding with matK alone reveals cryptic species and proves useful in identifying species listed in Convention on International Trade of Endangered Species (CITES) appendixes.

DNA barcoding the floras of biodiversity hotspots

PubMed Central

Lahaye, Renaud; van der Bank, Michelle; Bogarin, Diego; Warner, Jorge; Pupulin, Franco; Gigot, Guillaume; Maurin, Olivier; Duthoit, Sylvie; Barraclough, Timothy G.; Savolainen, Vincent

2008-01-01

DNA barcoding is a technique in which species identification is performed by using DNA sequences from a small fragment of the genome, with the aim of contributing to a wide range of ecological and conservation studies in which traditional taxonomic identification is not practical. DNA barcoding is well established in animals, but there is not yet any universally accepted barcode for plants. Here, we undertook intensive field collections in two biodiversity hotspots (Mesoamerica and southern Africa). Using >1,600 samples, we compared eight potential barcodes. Going beyond previous plant studies, we assessed to what extent a “DNA barcoding gap” is present between intra- and interspecific variations, using multiple accessions per species. Given its adequate rate of variation, easy amplification, and alignment, we identified a portion of the plastid matK gene as a universal DNA barcode for flowering plants. Critically, we further demonstrate the applicability of DNA barcoding for biodiversity inventories. In addition, analyzing >1,000 species of Mesoamerican orchids, DNA barcoding with matK alone reveals cryptic species and proves useful in identifying species listed in Convention on International Trade of Endangered Species (CITES) appendixes. PMID:18258745

Functional integration of PCR amplification and capillary eletrophoresis in a microfabricated DNA analysis device

DOE Office of Scientific and Technical Information (OSTI.GOV)

Woolley, A.T.; deMello, A.J.; Mathies, R.A.

Microfabricated silicon PCR reactors and glass capillary electrophoresis (CE) chips have been successfully coupled to form an integrated DNA analysis system. This construct combines the rapid thermal cycling capabilities of microfabricated PCR devices (10{degree}C/s heating, 2.5{degree}C/s cooling) with the high-speed (<120 s) DNA separations provided by microfabricated CE chips. The PCR chamber and the CE chip were directly linked through a photolithographically fabricated channel filled with hydroxyethylcellulose sieving matrix. Electrophoretic injection directly from the PCR chamber through the cross injection channel was used as an `electrophoretic valve` to couple the PCR and CE devices on-chip. To demonstrate the functionality ofmore » this system, a 15 min PCR amplification of a {Beta}-globin target cloned in m13 was immediately followed by high-speed CE chip separation in under 120 s, providing a rapid PCR-CE analysis in under 20 min. A rapid assay for genomic Salmonella DNA was performed in under 45 min, demonstrating that challenging amplifications of diagnostically interesting targets can also be performed. Real-time monitoring of PCR target amplification in these integrated PCR-CE devices is also feasible. 33 refs., 6 figs.« less

Opposing effects of pericentrin and microcephalin on the pericentriolar material regulate CHK1 activation in the DNA damage response.

PubMed

Antonczak, A K; Mullee, L I; Wang, Y; Comartin, D; Inoue, T; Pelletier, L; Morrison, C G

2016-04-14

Genotoxic stresses lead to centrosome amplification, a frequently-observed feature in cancer that may contribute to genome instability and to tumour cell invasion. Here we have explored how the centrosome controls DNA damage responses. For most of the cell cycle, centrosomes consist of two centrioles embedded in the proteinaceous pericentriolar material (PCM). Recent data indicate that the PCM is not an amorphous assembly of proteins, but actually a highly organised scaffold around the centrioles. The large coiled-coil protein, pericentrin, participates in PCM assembly and has been implicated in the control of DNA damage responses (DDRs) through its interactions with checkpoint kinase 1 (CHK1) and microcephalin (MCPH1). CHK1 is required for DNA damage-induced centrosome amplification, whereas MCPH1 deficiency greatly increases the amplification seen after DNA damage. We found that the PCM showed a marked expansion in volume and a noticeable change in higher-order organisation after ionising radiation treatment. PCM expansion was dependent on CHK1 kinase activity and was potentiated by MCPH1 deficiency. Furthermore, pericentrin deficiency or mutation of a separase cleavage site blocked DNA damage-induced PCM expansion. The extent of nuclear CHK1 activation after DNA damage reflected the level of PCM expansion, with a reduction in pericentrin-deficient or separase cleavage site mutant-expressing cells, and an increase in MCPH1-deficient cells that was suppressed by the loss of pericentrin. Deletion of the nuclear export signal of CHK1 led to its hyperphosphorylation after irradiation and reduced centrosome amplification. Deletion of the nuclear localisation signal led to low CHK1 activation and low centrosome amplification. From these data, we propose a feedback loop from the PCM to the nuclear DDR in which CHK1 regulates pericentrin-dependent PCM expansion to control its own activation.

Dual phase multiplex polymerase chain reaction

DOEpatents

Pemov, Alexander [Charlottesville, VA; Bavykin, Sergei [Darien, IL

2008-10-07

Highly specific and sensitive methods were developed for multiplex amplification of nucleic acids on supports such as microarrays. Based on a specific primer design, methods include five types of amplification that proceed in a reaction chamber simultaneously. These relate to four types of multiplex amplification of a target DNA on a solid support, directed by forward and reverse complex primers immobilized to the support and a fifth type--pseudo-monoplex polymerase chain reaction (PCR) of multiple targets in solution, directed by a single pair of unbound universal primers. The addition of the universal primers in the reaction mixture increases the yield over the traditional "bridge" amplification on a solid support by approximately ten times. Methods that provide multitarget amplification and detection of as little as 0.45-4.5.times.10.sup.-12 g (equivalent to 10.sup.2-10.sup.3 genomes) of a bacterial genomic DNA are disclosed.

[Applying competitive polymerase chain reaction to the detection of hepatitis B virus DNA].

PubMed

Wang, Ling; Yang, Peng; Li, Shuang-qing; Xu, Shu-hui; Cao, Gui-qun; Zhang, Fa-qiang; Zhang, Mei-xia; Chen, Qing-ying; Xia, Qing-jie; Liu, Kai; Tang, Fang; Zhang, Yuan-zheng

2004-11-01

To reduce the rate of accidental false negative result in the HBV DNA PCR test on clinical serum samples. A competitive polymerase chain reaction (C-PCR) was used to decrease the false negative ratio. In the C-PCR, a constructed inner control DNA was added for co-amplification with the HBV target DNA. In a 20 microl C-PCR system, about 60 to 200 copies of inner control DNA could give apparent co-amplification signal band after electrophoresis on a 2% agarose gel. Five of 120 samples of clinical serum (4.2%) could not be amplified. C-PCR has the advantage of yielding information on false negative in the HBV DNA PCR assay of clinical serum samples.

DNA extraction from formalin-fixed, paraffin-embedded tissues: protein digestion as a limiting step for retrieval of high-quality DNA.

PubMed

Díaz-Cano, S J; Brady, S P

1997-12-01

Several DNA extraction methods have been used for formalin-fixed, paraffin-embedded tissues, with variable results being reported regarding the suitability of DNA obtained from such sources to serve as template in polymerase chain reaction (PCR)-based genetic analyses. We present a method routinely used for archival material in our laboratory that reliably yields DNA of sufficient quality for PCR studies. This method is based on extended proteinase K digestion (250 micrograms/ml in an EDTA-free calcium-containing buffer supplemented with mussel glycogen) followed by phenol-chloroform extraction. Agarose gel electrophoresis of both digestion buffer aliquots and PCR amplification of the beta-globin gene tested the suitability of the retrieved DNA for PCR amplification.

A novel nonenzymatic cascade amplification for ultrasensitive photoelectrochemical DNA sensing based on target driven to initiate cyclic assembly of hairpins.

PubMed

Wen, Guangming; Dong, Wenxia; Liu, Bin; Li, Zhongping; Fan, Lifang

2018-05-29

A novel cascade photoelectrochemical (PEC) signal amplification biosensing tactics was developed for DNA detection based on a target-driven DNA association to induce cyclic hairpin assembly. In the circulatory system there are two ssDNA (A and B) and two hairpins (C and D). The hybridization of these ssDNA led to the formation of an A-target-B structure. The close proximity of their toehold and branch-migration regions was able to induce the cyclic hairpin assembly. Afterwards, the assembly result further causes the separation of a double-stranded probe DNA (Q:F) to switch the PEC signal via toehold-mediated strand replacement. As such, the signal stranded DNA-CdS QDs (F) as the signal tag was released in the presence of the target DNA. The signal DNA-CdS QDs was then coated to F-doped tin oxide (FTO) electrode leading to the "signal-on" PEC signal. The designed biosensing strategy showed a low detection limit of 21.3 pM for target DNA and a broad linear range from 50 pM to 100 nM. This signal amplification PEC sensing method exhibited a potential application to detect protein molecules, RNA or metal ions via changing the sequence of A and B recognition. Copyright © 2018 Elsevier B.V. All rights reserved.

An electrochemical biosensor for double-stranded Wnt7B gene detection based on enzymatic isothermal amplification.

PubMed

Li, Junlong; Chen, Zhongping; Xiang, Yu; Zhou, Lili; Wang, Ting; Zhang, Zhang; Sun, Kexin; Yin, Dan; Li, Yi; Xie, Guoming

2016-12-15

Wnt7B gene plays an important role in the development and progression of breast cancer, gastric cancer, esophageal cancer and pancreatic cancer. While, the natural state of DNA is double stranded, which makes it difficult to be directly detected. Here, we develop an electrochemical biosensor method for Wnt7B gene detection without the need to denature the target. This method firstly used nicking enzyme for exploiting in the double-stranded DNA (dsDNA). Then, long single-stranded DNA (ssDNA) was generated from the cutting site through polymerase extension reaction. Whereafter, the long ssDNA triggered a hairpin self-assembly recycling reaction, which gave rise to another isothermal amplification reaction. Last, short ssDNA was formed after the this amplification process, which could hybridize with the capture probe immobilized on Au electrode and result in signal variation. This method showed excellent analytical performance for dsDNA, of which the linear range was 2fM to 500pM and the detection limit was 1.6fM (S/N=3). It also showed an good results when applied to the real sample of Wnt7B gene detection. Copyright © 2016 Elsevier B.V. All rights reserved.

Improving efficiency and reliability of environmental DNA analysis for silver carp

USGS Publications Warehouse

Amberg, Jon J.; McCalla, S. Grace; Monroe, Emy; Lance, Richard; Baerwaldt, Kelly; Gaikowski, Mark P.

2015-01-01

Natural resource agencies have established surveillance programs which use environmental DNA (eDNA) for the early detection of bighead carp Hypophthalmichthys nobilis and silver carp Hypophthalmichthys molitrix before they establish populations within the Great Lakes. This molecular monitoring technique must be highly accurate and precise for confident interpretation and also efficient, both in detection threshold and cost. Therefore, we compared two DNA extraction techniques and compared a new quantitative PCR (qPCR) assay with the conventional PCR (cPCR) assay used by monitoring programs. Both the qPCR and cPCR assays were able to amplify the DNA of silver carp present in environmental samples taken from locations where mixed populations of bigheaded carps existed. However, the qPCR assay had substantially fewer PCR positive samples which were subsequently determined not to contain DNA of bigheaded carps than the cPCR assay. Additionally, the qPCR assay was able to amplify the DNA of bigheaded carps even in the presence of inhibitors that blocked amplification with cPCR. Also, the selection of an appropriate DNA extraction method can significantly alter the efficiency of eDNA surveillance programs by lowering detection limits and by decreasing costs associated with sample processing. The results reported herein are presently being incorporated into eDNA surveillance programs to decrease the costs, increase DNA yield and increase the confidence that assays are amplifying the target DNA. These results are critical to enhancing our ability to accurately and confidently interpret the results reported from monitoring programs using eDNA for early detection of invasive species.

Preliminary study on applicability of microsatellite DNA primers from parasite protozoa Trypanosoma cruzi in free-living protozoa

NASA Astrophysics Data System (ADS)

Zhang, Wenjing; Yu, Yuhe; Shen, Yunfen; Miao, Wei; Feng, Weisong

2004-04-01

In this paper, we took the lead in studying on specificity of the microsatellite DNA loci and applicability of microsatellite DNA primers in protozoa. In order to study characters of microsatellites in free-living protozoa, eight microsatellite loci primers developed from Trypanosoma cruzi (MCLE01, SCLE10, MCLE08, SCLE11, MCLF10, MCLG10, MCL03, MCL05) were employed to amplify microsatellite in four free-living protozoa, including Bodo designis, Euglena gracilis FACHB848, Paramecium bruzise and Tetrahymena thermophila BF1. In the amplification systems of P. bruzise, four loci (SCLE10, SCLE11, MCLF10, MCL03) were amplified successfully, and four amplification fragments were in proper size. In genome of E. gracilis FACHB848, five of eight primers brought five clear amplification bands. In B. designis, three (No.4, 5 and 7) of eight loci produced clear and sharp products without stutter bands, whereas no bands appeared in T. thermophila BF1. Further, eight 300 500 bp amplification fragments were cloned and sequenced. Nevertheless, all sequenced products did not contain corresponding microsatellite sequence, although Bodo is in the same order and has the nearest phylogenetic relation with Trypanosoma among these four species. Thus, the microsatellite DNA primers can not be applied among order or more far taxa, and the specificity of microsatellite DNA is very high in protozoa. The results of this study will contribute to our understanding of microsatellite DNA in protozoa.

A signal-on electrochemical aptasensor for ultrasensitive detection of endotoxin using three-way DNA junction-aided enzymatic recycling and graphene nanohybrid for amplification

NASA Astrophysics Data System (ADS)

Bai, Lijuan; Chai, Yaqin; Pu, Xiaoyun; Yuan, Ruo

2014-02-01

Endotoxin, also known as lipopolysaccharide (LPS), is able to induce a strong immune response on its internalization into mammalian cells. To date, aptamer-based biosensors for LPS detection have been rarely reported. This work describes a new signal-on electrochemical aptasensor for the ultrasensitive detection of LPS by combining the three-way DNA hybridization process and nanotechnology-based amplification. With the help of DNA1 (associated with the concentration of target LPS), the capture probe hybridizes with DNA1 and the assistant probe to open its hairpin structure and form a ternary ``Y'' junction structure. The DNA1 can be released from the structure in the presence of nicking endonuclease to initiate the next hybridization process. Then a great deal of cleaved capture probe produced in the cyclic process can bind with DNA2-nanocomposite, which contains the electroactive toluidine blue (Tb) with the amplification materials graphene (Gra) and gold nanoparticles (AuNPs). Thus, an enhanced electrochemical signal can be easily read out. With the cascade signal amplification, this newly designed protocol provides an ultrasensitive electrochemical detection of LPS down to the femtogram level (8.7 fg mL-1) with a linear range of 6 orders of magnitude (from 10 fg mL-1 to 50 ng mL-1). Moreover, the high sensitivity and specificity make this method versatile for the detection of other biomolecules by changing the corresponding sequences of the capture probe and the assistant probe.

An electrochemical biosensor for microRNA-196a detection based on cyclic enzymatic signal amplification and template-free DNA extension reaction with the adsorption of methylene blue.

PubMed

Guo, Jing; Yuan, Changjing; Yan, Qi; Duan, Qiuyue; Li, Xiaolu; Yi, Gang

2018-05-15

A simple and sensitive electrochemical biosensor was developed for microRNA-196a detection, which is of important diagnostic significance for pancreatic cancer. It was based on cyclic enzymatic signal amplification (CESA) and template-free DNA extension reaction. In the presence of microRNA-196a, duplex-specific nuclease (DSN) catalyzed the digestion of the 3'-PO 4 terminated capture probe (CP), resulting in the target recycling amplification. Meanwhile, the 3'-OH terminal of CP was exposed. Then, template-free DNA extension reaction was triggered by terminal deoxynucleotidyl transferase (TdT), producing amounts of single-stranded DNA (ssDNA). After ssDNA absorbed numerous methylene blue (MB), an ultrasensitive electrochemical readout was obtained. Based on this dual amplification mechanism, the proposed biosensor exhibited a high sensitivity for detection of microRNA-196a down to 15 aM with a linear range from 0.05 fM to 50 pM. This biosensor displayed high specificity, which could discriminate target microRNAs from one base mismatched microRNAs. It also showed good reproducibility and stability. Furthermore, it was successfully applied to the determination of microRNA-196a in plasma samples. In conclusion, with the excellent analytical performance, this biosensor might have the potential for application in clinical diagnostics of pancreatic cancer. Copyright © 2018 Elsevier B.V. All rights reserved.

Excision Repair-Initiated Enzyme-Assisted Bicyclic Cascade Signal Amplification for Ultrasensitive Detection of Uracil-DNA Glycosylase.

PubMed

Wang, Li-Juan; Ren, Ming; Zhang, Qianyi; Tang, Bo; Zhang, Chun-Yang

2017-04-18

Uracil-DNA glycosylase (UDG) is an important base excision repair (BER) enzyme responsible for the repair of uracil-induced DNA lesion and the maintenance of genomic integrity, while the aberrant expression of UDG is associated with a variety of cancers. Thus, the accurate detection of UDG activity is essential to biomedical research and clinical diagnosis. Here, we develop a fluorescent method for ultrasensitive detection of UDG activity using excision repair-initiated enzyme-assisted bicyclic cascade signal amplification. This assay involves (1) UDG-actuated uracil-excision repair, (2) excision repair-initiated nicking enzyme-mediated isothermal exponential amplification, (3) ribonuclease H (RNase H)-induced hydrolysis of signal probes for generating fluorescence signal. The presence of UDG enables the removal of uracil from U·A pairs and generates an apurinic/apyrimidinic (AP) site. Endonuclease IV (Endo IV) subsequently cleaves the AP site, resulting in the break of DNA substrate. The cleaved DNA substrate functions as both a primer and a template to initiate isothermal exponential amplification, producing a large number of triggers. The resultant trigger may selectively hybridize with the signal probe which is modified with FAM and BHQ1, forming a RNA-DNA heterogeneous duplex. The subsequent hydrolysis of RNA-DNA duplex by RNase H leads to the generation of fluorescence signal. This assay exhibits ultrahigh sensitivity with a detection limit of 0.0001 U/mL, and it can even measure UDG activity at the single-cell level. Moreover, this method can be applied for the measurement of kinetic parameters and the screening of inhibitors, thereby providing a powerful tool for DNA repair enzyme-related biomedical research and clinical diagnosis.

Hairpin DNA Switch for Ultrasensitive Spectrophotometric Detection of DNA Hybridization Based on Gold Nanoparticles and Enzyme Signal Amplification

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Youyu; Tang, Zhiwen; Wang, Jun

2010-08-01

A novel DNA detection platform based on a hairpin-DNA switch, nanoparticles, and enzyme signal amplification for ultrasensitive detection of DNA hybridization has been developed in this work. In this DNA assay, a “stem-loop” DNA probe dually labeled with a thiol at its 5’ end and a biotin at its 3’ end, respectively, was used. This probe was immobilized on the gold nanoparticles (AuNPs) anchored by a protein, globulin, on a 96-well microplate. In the absence of target DNA, the immobilized probe with the stem-loop structure shields the biotin from being approached by a bulky horseradish peroxidase linked-avidin (avidin-HRP) conjugate duemore » to the steric hindrance. However, in the presence of target DNA, the hybridization between the hairpin DNA probe and the target DNA causes significant conformational change of the probe, which forces biotin away from the surface of AuNPs. As a result, the biotin becomes accessible by the avidin-HRP, and the target hybridization event can be sensitively detected via the HRP catalyzed substrate 3, 3', 5, 5'-tetramethylbenzidine using spectrophometric method. Some experimental parameters governing the performance of the assay have been optimized. At optimal conditions, this DNA assay can detect DNA at the concentration of femtomolar level by means of a signal amplification strategy based on the combination of enzymes and nanoparticles. This approach also has shown excellent specificity to distinguish single-base mismatches of DNA targets because of the intrinsic high selectivity of the hairpin DNA probe.« less

Complete sequence analysis of 18S rDNA based on genomic DNA extraction from individual Demodex mites (Acari: Demodicidae).

PubMed

Zhao, Ya-E; Xu, Ji-Ru; Hu, Li; Wu, Li-Ping; Wang, Zheng-Hang

2012-05-01

The study for the first time attempted to accomplish 18S ribosomal DNA (rDNA) complete sequence amplification and analysis for three Demodex species (Demodex folliculorum, Demodex brevis and Demodex canis) based on gDNA extraction from individual mites. The mites were treated by DNA Release Additive and Hot Start II DNA Polymerase so as to promote mite disruption and increase PCR specificity. Determination of D. folliculorum gDNA showed that the gDNA yield reached the highest at 1 mite, tending to descend with the increase of mite number. The individual mite gDNA was successfully used for 18S rDNA fragment (about 900 bp) amplification examination. The alignments of 18S rDNA complete sequences of individual mite samples and those of pooled mite samples ( ≥ 1000mites/sample) showed over 97% identities for each species, indicating that the gDNA extracted from a single individual mite was as satisfactory as that from pooled mites for PCR amplification. Further pairwise sequence analyses showed that average divergence, genetic distance, transition/transversion or phylogenetic tree could not effectively identify the three Demodex species, largely due to the differentiation in the D. canis isolates. It can be concluded that the individual Demodex mite gDNA can satisfy the molecular study of Demodex. 18S rDNA complete sequence is suitable for interfamily identification in Cheyletoidea, but whether it is suitable for intrafamily identification cannot be confirmed until the ascertainment of the types of Demodex mites parasitizing in dogs. Copyright © 2012 Elsevier Inc. All rights reserved.

Citrus huanglongbing in São Paulo State, Brazil: PCR detection of the 'Candidatus' Liberibacter species associated with the disease.

PubMed

do Carmo Teixeira, Diva; Luc Danet, Jean; Eveillard, Sandrine; Cristina Martins, Elaine; de Jesus Junior, Waldir Cintra; Takao Yamamoto, Pedro; Aparecido Lopes, Silvio; Beozzo Bassanezi, Renato; Juliano Ayres, Antonio; Saillard, Colette; Bové, Joseph Marie

2005-06-01

Symptoms of huanglongbing (HLB), one of the most serious diseases of citrus in Asia and Africa, have been noticed in March 2004 in the Araraquara region of São Paulo State, Brazil. HLB has not been reported previously from America. The causal HLB bacteria, Candidatus Liberibacter africanus in Africa and Candidatus Liberibacter asiaticus in Asia, can be detected in symptomatic citrus leaves by PCR amplification of their 16S rDNA with previously described primers. When this technique was applied to 43 symptomatic leaf samples from the Araraquara region, all PCR reactions were negative. This suggested that a new pathogen, not detected by the above primers, could be involved in HLB in the State of São Paulo. Indeed, by using universal primers for amplification of bacterial 16S rDNA, a new liberibacter species, Candidatus Liberibacter americanus, has recently been identified. Specific primers for PCR amplification of the 16S rDNA of Ca. L. americanus have been selected. Using these primers, the new liberibacter could be detected in 214 symptomatic leaf samples tested. The leaves of two additional samples were infected with Candidatus Liberibacter asiaticus, and two further samples contained both Ca. L. americanus and Ca. L. asiaticus. The samples came from 47 farms in 35 municipalities. The psyllid vector of Ca. L. asiaticus, Diaphorina citri, is established in South, Central, and North America (Florida and Texas). Ca. L. americanus could be detected by PCR in several batches of D. citri psyllids collected on symptomatic sweet orange trees infected with Ca. L. americanus, strongly suggesting that D. citri is the vector of Ca. L. americanus. The results reported here confirm the presence of HLB in the State of São Paulo. Ca. L. americanus is the most widely distributed pathogen.

Genetic screening of spinal muscular atrophy using a real-time modified COP-PCR technique with dried blood-spot DNA.

PubMed

Ar Rochmah, Mawaddah; Harahap, Nur Imma Fatimah; Niba, Emma Tabe Eko; Nakanishi, Kenta; Awano, Hiroyuki; Morioka, Ichiro; Iijima, Kazumoto; Saito, Toshio; Saito, Kayoko; Lai, Poh San; Takeshima, Yasuhiro; Takeuchi, Atsuko; Bouike, Yoshihiro; Okamoto, Maya; Nishio, Hisahide; Shinohara, Masakazu

2017-10-01

Spinal muscular atrophy (SMA) is a common neuromuscular disorder caused by mutations in SMN1. More than 95% of SMA patients carry homozygous SMN1 deletion. SMA is the leading genetic cause of infant death, and has been considered an incurable disease. However, a recent clinical trial with an antisense oligonucleotide drug has shown encouraging clinical efficacy. Thus, early and accurate detection of SMN1 deletion may improve prognosis of many infantile SMA patients. A total of 88 DNA samples (37 SMA patients, 12 carriers and 39 controls) from dried blood spots (DBS) on filter paper were analyzed. All participants had previously been screened for SMN genes by PCR restriction fragment length polymorphism (PCR-RFLP) using DNA extracted from freshly collected blood. DNA was extracted from DBS that had been stored at room temperature (20-25°C) for 1week to 5years. To ensure sufficient quality and quantity of DNA samples, target sequences were pre-amplified by conventional PCR. Real-time modified competitive oligonucleotide priming-PCR (mCOP-PCR) with the pre-amplified PCR products was performed for the gene-specific amplification of SMN1 and SMN2 exon 7. Compared with PCR-RFLP using DNA from freshly collected blood, results from real-time mCOP-PCR using DBS-DNA for detection of SMN1 exon 7 deletion showed a sensitivity of 1.00 (CI [0.87, 1.00])] and specificity of 1.00 (CI [0.90, 1.00]), respectively. We combined DNA extraction from DBS on filter paper, pre-amplification of target DNA, and real-time mCOP-PCR to specifically detect SMN1 and SMN2 genes, thereby establishing a rapid, accurate, and high-throughput system for detecting SMN1-deletion with practical applications for newborn screening. Copyright © 2017 The Japanese Society of Child Neurology. Published by Elsevier B.V. All rights reserved.

Comparison of PCR primer-based strategies for characterization of ammonia oxidizer communities in environmental samples.

PubMed

Mahmood, Shahid; Freitag, Thomas E; Prosser, James I

2006-06-01

PCR-based techniques are commonly used to characterize microbial communities, but are subject to bias that is difficult to assess. This study aimed to evaluate bias of several PCR primer-based strategies used to study diversity of autotrophic ammonia oxidizers. 16S rRNA genes from soil- or sediment-DNA were amplified using primers considered either selective or specific for betaproteobacterial ammonia oxidizers. Five approaches were assessed: (a) amplification with primers betaAMO143f-betaAMO1315r; (b) amplification with primers CTO189f-CTO654r; (c) nested amplification with betaAMO143f-betaAMO1315r followed by CTO189f-CTO654r primers; (d) nested amplification with betaAMO143f-betaAMO1315r and CTO189f-Pf1053r primers; (e) nested amplification with 27f-1492r and CTO189f-CTO654r primers. Amplification products were characterized by denaturing gradient gel electrophoresis (DGGE) analysis after further amplification with 357f-GC-518r primers. DGGE profiles of soil communities were heterogeneous and depended on the approach followed. Ammonia oxidizer diversity was higher using approaches (b), (c) and (e) than using (a) and (d), where sequences of the most prominent bands showed similarities to nonammonia oxidizers. Profiles from marine sediments were more consistent, regardless of the approach adopted, and sequence analysis of excised bands indicated that these consisted of ammonia oxidizers only. The study demonstrates the importance of choice of primer, of screening for sequences of nontarget organisms and use of several approaches when characterizing microbial communities in natural environments.

Dual-primer self-generation SERS signal amplification assay for PDGF-BB using label-free aptamer.

PubMed

Ye, SuJuan; Zhai, XiaoMo; Wu, YanYing; Kuang, ShaoPing

2016-05-15

Highly sensitive detection of proteins, especially those associated with cancers, is essential to biomedical research as well as clinical diagnosis. In this work, a simple and novel one-two-three signal amplification surface-enhanced Raman scattering (SERS) method for the detection of protein is fabricated by using label-free aptamer and dual-primer self-generation. Platelet-derived growth factor B-chain (PDGF-BB) is selected as the model protein. The one-two-three cascade DNA amplification means one target-aptamer binding event, two hairpin DNA switches and three DNA amplification reactions. This strategy possesses some remarkable features compared to conventional signal amplification methods: (i) A smart probe including a label-free aptamer is fabricated, for suitable hybridization without hindering the affinity of the aptamer toward its target. (ii) Using the unique structure switch of the aptamer and cooperator, a one-two-three working mode is developed to amplify the SERS signal. The amplification efficiency is enhanced. Given the unique and attractive characteristics, a simple and universal strategy is designed to accomplish ultrasensitive detection of proteins. The detection limit of PDGF-BB via SERS detection is 0.42 pM, with the linear range from 1.0×10(-12)M to 10(-8)M. It is potentially universal because the aptamer can be easily designed for biomolecules whose aptamers undergo similar conformational changes. Copyright © 2015 Elsevier B.V. All rights reserved.

Structured oligonucleotides for target indexing to allow single-vessel PCR amplification and solid support microarray hybridization

PubMed Central

Girard, Laurie D.; Boissinot, Karel; Peytavi, Régis; Boissinot, Maurice; Bergeron, Michel G.

2014-01-01

The combination of molecular diagnostic technologies is increasingly used to overcome limitations on sensitivity, specificity or multiplexing capabilities, and provide efficient lab-on-chip devices. Two such techniques, PCR amplification and microarray hybridization are used serially to take advantage of the high sensitivity and specificity of the former combined with high multiplexing capacities of the latter. These methods are usually performed in different buffers and reaction chambers. However, these elaborate methods have a high complexity cost related to reagent requirements, liquid storage and the number of reaction chambers to integrate into automated devices. Furthermore, microarray hybridizations have a sequence dependent efficiency not always predictable. In this work, we have developed the concept of a structured oligonucleotide probe which is activated by cleavage from polymerase exonuclease activity. This technology is called SCISSOHR for Structured Cleavage Induced Single-Stranded Oligonucleotide Hybridization Reaction. The SCISSOHR probes enable indexing the target sequence to a tag sequence. The SCISSOHR technology also allows the combination of nucleic acid amplification and microarray hybridization in a single vessel in presence of the PCR buffer only. The SCISSOHR technology uses an amplification probe that is irreversibly modified in presence of the target, releasing a single-stranded DNA tag for microarray hybridization. Each tag is composed of a 3-nucleotidesequence-dependent segment and a unique “target sequence-independent” 14-nucleotide segment allowing for optimal hybridization with minimal cross-hybridization. We evaluated the performance of five (5) PCR buffers to support microarray hybridization, compared to a conventional hybridization buffer. Finally, as a proof of concept, we developed a multiplexed assay for the amplification, detection, and identification of three (3) DNA targets. This new technology will facilitate the design of lab-on-chip microfluidic devices, while also reducing consumable costs. At term, it will allow the cost-effective automation of highly multiplexed assays for detection and identification of genetic targets. PMID:25489607

Structured oligonucleotides for target indexing to allow single-vessel PCR amplification and solid support microarray hybridization.

PubMed

Girard, Laurie D; Boissinot, Karel; Peytavi, Régis; Boissinot, Maurice; Bergeron, Michel G

2015-02-07

The combination of molecular diagnostic technologies is increasingly used to overcome limitations on sensitivity, specificity or multiplexing capabilities, and provide efficient lab-on-chip devices. Two such techniques, PCR amplification and microarray hybridization are used serially to take advantage of the high sensitivity and specificity of the former combined with high multiplexing capacities of the latter. These methods are usually performed in different buffers and reaction chambers. However, these elaborate methods have high complexity and cost related to reagent requirements, liquid storage and the number of reaction chambers to integrate into automated devices. Furthermore, microarray hybridizations have a sequence dependent efficiency not always predictable. In this work, we have developed the concept of a structured oligonucleotide probe which is activated by cleavage from polymerase exonuclease activity. This technology is called SCISSOHR for Structured Cleavage Induced Single-Stranded Oligonucleotide Hybridization Reaction. The SCISSOHR probes enable indexing the target sequence to a tag sequence. The SCISSOHR technology also allows the combination of nucleic acid amplification and microarray hybridization in a single vessel in presence of the PCR buffer only. The SCISSOHR technology uses an amplification probe that is irreversibly modified in presence of the target, releasing a single-stranded DNA tag for microarray hybridization. Each tag is composed of a 3-nucleotide sequence-dependent segment and a unique "target sequence-independent" 14-nucleotide segment allowing for optimal hybridization with minimal cross-hybridization. We evaluated the performance of five (5) PCR buffers to support microarray hybridization, compared to a conventional hybridization buffer. Finally, as a proof of concept, we developed a multiplexed assay for the amplification, detection, and identification of three (3) DNA targets. This new technology will facilitate the design of lab-on-chip microfluidic devices, while also reducing consumable costs. At term, it will allow the cost-effective automation of highly multiplexed assays for detection and identification of genetic targets.

Techniques for investigation of an apparent outbreak of infections with Candida glabrata.

PubMed Central

Arif, S; Barkham, T; Power, E G; Howell, S A

1996-01-01

A cluster of Candida glabrata isolates recovered from seven patients in an intensive care unit over a 10-week period were compared with a collection of isolates from six epidemiologically distinct outpatients and a reference strain by several DNA typing methods. Restriction enzyme analysis with HinII distinguished 13 strains from the 14 sources and was the method of choice. Pulsed-field gel electrophoresis and random amplification of polymorphic DNA both detected nine types from the 14 sources; however, the results of these two methods did not always correlate. These methods demonstrated that five of the seven patients had distinguishable strains and that cross-infection was unlikely. PMID:8862586

The significance of gtf genes in caries expression: a rapid identification of Streptococcus mutans from dental plaque of child patients.

PubMed

Mishra, Apurva; Pandey, Ramesh K; Manickam, Natesan

2015-01-01

Rapid phylogenetic and functional gene (gtfB) identification of S. mutans from the dental plaque derived from children. Dental plaque collected from fifteen patients of age group 7-12 underwent centrifugation followed by genomic DNA extraction for S. mutans. Genomic DNA was processed with S. mutans specific primers in suitable PCR condtions for phylogenetic and functional gene (gtfB) identification. The yield and results were confirmed by agarose gel electrophoresis. 1% agarose gel electrophoresis depicts the positive PCR amplification at 1,485 bp when compared with standard 1 kbp indicating the presence of S. mutans in the test sample. Another PCR reaction was set using gtfB primers specific for S. mutans for functional gene identification. 1.2% agarose gel electrophoresis was done and a positive amplication was observed at 192 bp when compared to 100 bp standards. With the advancement in molecular biology techniques, PCR based identification and quantification of the bacterial load can be done within hours using species-specific primers and DNA probes. Thus, this technique may reduce the laboratory time spend in conventional culture methods, reduces the possibility of colony identification errors and is more sensitive to culture techniques.

Successful isolation and PCR amplification of DNA from National Institute of Standards and Technology herbal dietary supplement standard reference material powders and extracts.

PubMed

Cimino, Matthew T

2010-03-01

Twenty-four herbal dietary supplement powder and extract reference standards provided by the National Institute of Standards and Technology (NIST) were investigated using three different commercially available DNA extraction kits to evaluate DNA availability for downstream nucleotide-based applications. The material included samples of Camellia, Citrus, Ephedra, Ginkgo, Hypericum, Serenoa, And Vaccinium. Protocols from Qiagen, MoBio, and Phytopure were used to isolate and purify DNA from the NIST standards. The resulting DNA concentration was quantified using SYBR Green fluorometry. Each of the 24 samples yielded DNA, though the concentration of DNA from each approach was notably different. The Phytopure method consistently yielded more DNA. The average yield ratio was 22 : 3 : 1 (ng/microL; Phytopure : Qiagen : MoBio). Amplification of the internal transcribed spacer II region using PCR was ultimately successful in 22 of the 24 samples. Direct sequencing chromatograms of the amplified material suggested that most of the samples were comprised of mixtures. However, the sequencing chromatograms of 12 of the 24 samples were sufficient to confirm the identity of the target material. The successful extraction, amplification, and sequencing of DNA from these herbal dietary supplement extracts and powders supports a continued effort to explore nucleotide sequence-based tools for the authentication and identification of plants in dietary supplements. (c) Georg Thieme Verlag KG Stuttgart . New York.

DNA extraction in Echinococcus granulosus and Taenia spp. eggs in dogs stool samples applying thermal shock.

PubMed

Hidalgo, Alejandro; Melo, Angélica; Romero, Fernando; Hidalgo, Víctor; Villanueva, José; Fonseca-Salamanca, Flery

2018-03-01

The extraction of DNA in taeniid eggs shows complications attached to the composition of stool samples and the high resistance of eggs to degradation. The objective of this study was to test a method of DNA extraction in taeniid eggs by applying a thermal shock to facilitate the chemical-enzymatic degradation of these elements. A group of six tubes containing 1 ml of dog stool sample was spiked with eggs of Echinococcus granulosus and another group of six with Taenia pisiformis. Samples were floated with supersaturated sugar solution and centrifuged. The upper portion of each tube (500 μl) was aspirated and deposited in 1.5 ml tubes. Three tubes from each group were incubated at -20 °C and then at 90 °C, the remaining three from each group, incubated at room temperature. Proteinase K and lysis buffer were added to each tube and incubated for 12 h at 58 °C. The lysis effect was evaluated by microscopy at 3, 6 and 12 h and integrity by electrophoresis in 1% agarose gels. With the same experimental scheme, the thermal shock effect was evaluated in extractions of 1, 2, 3 and 4 eggs of each species and the DNA was quantified. Additionally, the protocol was applied in samples of 4 dogs diagnosed with natural infection by Taeniidae worms. Finally, all the extractions were tested by PCR amplification. Both E. granulosus and T. pisiformis eggs showed a similar response in the tests. In samples without treatment, the lysis effect was poor and showed no differences over time, but in those subjected to thermal shock, eggs degradation increased with time. In both treatments, there was no DNA loss integrity. The protocol applied to limited amounts of eggs yielded PCR products in 100% of the samples exposed to thermal shock, allowing PCR amplifications up to 1 egg. In non-exposed samples, the results were not replicable. However, DNA quantification showed low values in both treatments. In turn, DNA extractions with thermal shock in infected dog samples finally yielded PCR amplifications in 100%. It was concluded that thermal shock facilitates the DNA extraction for molecular analysis in taeniid eggs. The technique is effective extracting DNA even from a single egg and also to analyze natural infections samples with a relatively simple implementation. Published by Elsevier Inc.

Improving enrichment of circulating fetal DNA for genetic testing: size fractionation followed by whole gene amplification.

PubMed

Jorgez, Carolina J; Bischoff, Farideh Z

2009-01-01

Among the pitfalls of using cell-free fetal DNA in plasma for prenatal diagnosis is quality of the recovered DNA fragments and concomitant presence of maternal DNA (>95%). Our objective is to provide alternative methods for achieving enrichment and high-quality fetal DNA from plasma. Cell-free DNA from 31 pregnant women and 18 controls (10 males and 8 females) were size separated using agarose gel electrophoresis. DNA fragments of 100-300, 500-700 and 1,500-2,000 bp were excised and extracted, followed by whole genome amplification (WGA) of recovered fragments. Levels of beta-globin and DYS1 were measured. Distribution of beta-globin size fragments was similar among pregnant women and controls. Among control male cases, distribution of size fragments was the same for both beta-globin and DYS1. Among maternal cases confirmed to be male, the smallest size fragment (100-300 bp) accounted for nearly 50% (39.76 +/- 17.55%) of the recovered DYS1-DNA (fetal) and only 10% (10.40 +/- 6.49%) of beta-globin (total) DNA. After WGA of plasma fragments from pregnant women, DYS1 sequence amplification was best observed when using the 100-300 bp fragments as template. Combination of electrophoresis for size separation and WGA led to enriched fetal DNA from plasma. This novel combination of strategies is more likely to permit universal clinical applications of cell-free fetal DNA. Copyright 2009 S. Karger AG, Basel.

Cross-subtype Detection of HIV-1 Using Reverse Transcription and Recombinase Polymerase Amplification

PubMed Central

Lillis, Lorraine; Lehman, Dara A.; Siverson, Joshua B.; Weis, Julie; Cantera, Jason; Parker, Mathew; Piepenburg, Olaf; Overbaugh, Julie; Boyle, David S.

2016-01-01

A low complexity diagnostic test that rapidly and reliably detects HIV infection in infants at the point of care could facilitate early treatment, improving outcomes. However, many infant HIV diagnostics can only be performed in laboratory settings. Recombinase polymerase amplification (RPA) is an isothermal amplification technology that can rapidly amplify proviral DNA from multiple subtypes of HIV-1 in under twenty minutes without complex equipment. In this study we added reverse transcription (RT) to RPA to allow detection of both HIV-1 RNA and DNA. We show that this RT-RPA HIV-1 assay has a limit of detection of 10 to 30 copies of an exact sequence matched DNA or RNA, respectively. In addition, at 100 copies of RNA or DNA, the assay detected 171 of 175 (97.7 %) sequence variants that represent all the major subtypes and recombinant forms of HIV-1 Groups M and O. This data suggests that the application of RT-RPA for the combined detection of HIV-1 viral RNA and proviral DNA may prove a highly sensitive tool for rapid and accurate diagnosis of infant HIV. PMID:26821087

Development of loop-mediated isothermal amplification (LAMP) assay for the rapid detection of Penicillium nordicum in dry-cured meat products.

PubMed

Ferrara, M; Perrone, G; Gallo, A; Epifani, F; Visconti, A; Susca, A

2015-06-02

The need of powerful diagnostic tools for rapid, simple, and cost-effective detection of food-borne fungi has become very important in the area of food safety. Currently, several isothermal nucleic acid amplification methods have been developed as an alternative to PCR-based analyses. Loop-mediated isothermal amplification (LAMP) is one of these innovative methods; it requires neither gel electrophoresis to separate and visualize the products nor expensive laboratory equipment and it has been applied already for detection of pathogenic organisms. In the current study, we developed a LAMP assay for the specific detection of Penicillium nordicum, the major causative agent of ochratoxin A contamination in protein-rich food, especially dry-cured meat products. The assay was based on targeting otapksPN gene, a key gene in the biosynthesis of ochratoxin A (OTA) in P. nordicum. Amplification of DNA during the reaction was detected directly in-tube by color transition of hydroxynaphthol blue from violet to sky blue, visible to the naked eye, avoiding further post amplification analyses. Only DNAs isolated from several P. nordicum strains led to positive results and no amplification was observed from non-target OTA and non OTA-producing strains. The assay was able to detect down to 100 fg of purified targeted genomic DNA or 10(2) conidia/reaction within 60 min. The LAMP assay for detection and identification of P. nordicum was combined with a rapid DNA extraction method set up on serially diluted conidia, providing an alternative rapid, specific and sensitive DNA-based method suitable for application directly "on-site", notably in key steps of dry-cured meat production. Copyright © 2015 Elsevier B.V. All rights reserved.

An enzyme-free flow cytometric bead assay for the sensitive detection of microRNAs based on click nucleic acid ligation-mediated signal amplification.

PubMed

Qi, Yan; Qiu, Liying; Fan, Wenjiao; Liu, Chenghui; Li, Zhengping

2017-08-07

A versatile flow cytometric bead assay (FCBA) coupled with a completely enzyme-free signal amplification mechanism is developed for the sensitive detection of microRNAs (miRNAs). This new strategy integrates click chemistry-mediated ligation chain reaction (CLCR) with hybridization chain reaction (HCR) for enzyme-free signal amplification on magnetic beads (MBs), and a flow cytometer for the robust fluorescence readout of the MBs. Firstly, target miRNA can initiate CLCR on the surface of MBs based on the click chemical ligation between dibenzocyclooctyne (DBCO)- and azide-modified single-stranded DNA (ssDNA) probes, and the amount of ligated ssDNA sequences on the MBs will be proportional to the dosage of target miRNA. Afterward, each of the ligated ssDNA products can trigger a cascade chain reaction of hybridization events between two alternating fluorophore-tagged hairpin probes, resulting in another signal amplification pathway with an amplified accumulation of fluorophores on the MBs. Finally, the fluorophore-anchored MBs are directly and rapidly analyzed by using a flow cytometer without any separation or elution processes. Herein, the click nucleic acid ligation only occurs on the surface of MBs, so the nonspecific ligations are greatly inhibited compared with that of ligation reaction performed in homogeneous solution. Furthermore, the signal amplification by CLCR-HCR is highly efficient but totally enzyme-free, which may overcome the potential drawbacks of conventional enzyme-catalyzed signal amplification protocols and lead to a high sensitivity. The CLCR-HCR-based FCBA has pushed the detection limit of let-7a miRNA down to the femtomolar (fM) level, showing great potential in miRNA-related biological studies and disease diagnosis.

Detection of Giardia in environmental waters by immuno-PCR amplification methods.

PubMed

Mahbubani, M H; Schaefer, F W; Jones, D D; Bej, A K

1998-02-01

Genomic DNA was extracted either directly from Giardia muris cysts seeded into environmental surface waters or from cysts isolated by immunomagnetic beads (IMB). A 0.171-kbp segment of the giardin gene was PCR-amplified following "direct extraction" of Giardia DNA from seeded Cahaba river water concentrate with moderate turbidity (780 JTU's), but DNA purified from seeded Colorado river water concentrates with high turbidity (2 x 10(5) JTUs) failed to amplify. However, if the cysts were first separated by the IMB approach from seeded Cahaba or Colorado river waters, and the DNA released by a freeze-boil Chelex(R)100 treatment, detection of G. muris by PCR amplification could be achieved at a sensitivity of 3 x 10(0) or 3 x 10(1) cysts/ml, respectively. If, however, the G. muris cysts used to seed even moderately turbid river waters (780 JTUs) were formalin treated (which is conventionally used for microscopic examination), neither direct extraction nor IMB purification methods yielded amplifiable DNA. Use of immunomagnetic beads to separate Giardia cysts from complex matrices of environmental surface waters followed by DNA release and PCR amplification of the target giardin gene improved the reliability of detection of this pathogen with the required sensitivity.

A simple and inexpensive molecular method for sexing and identification of the forensic samples of elephant origin.

PubMed

Gupta, Sandeep K; Thangaraj, Kumarasamy; Singh, Lalji

2006-07-01

The population of the Asian elephant is being dramatically reduced due to poaching of the ivory from the male. As poaching occurs in remote forests, it often takes weeks or longer for it to be discovered and it is therefore often very difficult to determine the sex of the decomposed body. Data suggest that in the recent past, over 2000 male elephants have been poached in South India. We have developed a technique based on molecular markers to determine that the carcass is an elephant and that it is a male. Using DNA sequence information from Genbank, we have developed two primer pairs: one for the mitochondrial DNA (mtDNA) and the other for the sex-determining region of Y chromosome (SRY) gene of the Indian elephant. After PCR amplification of known elephant DNA, we found that the mtDNA was common in both males and females, whereas the SRY-specific amplicon was observed only in the male.

Nucleotide-Specific Contrast for DNA Sequencing by Electron Spectroscopy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mankos, Marian; Persson, Henrik H. J.; N’Diaye, Alpha T.

DNA sequencing by imaging in an electron microscope is an approach that holds promise to deliver long reads with low error rates and without the need for amplification. Earlier work using transmission electron microscopes, which use high electron energies on the order of 100 keV, has shown that low contrast and radiation damage necessitates the use of heavy atom labeling of individual nucleotides, which increases the read error rates. Other prior work using scattering electrons with much lower energy has shown to suppress beam damage on DNA. Here we explore possibilities to increase contrast by employing two methods, X-ray photoelectronmore » and Auger electron spectroscopy. Using bulk DNA samples with monomers of each base, both methods are shown to provide contrast mechanisms that can distinguish individual nucleotides without labels. In conclusion, both spectroscopic techniques can be readily implemented in a low energy electron microscope, which may enable label-free DNA sequencing by direct imaging.« less

Nucleotide-Specific Contrast for DNA Sequencing by Electron Spectroscopy

DOE PAGES

Mankos, Marian; Persson, Henrik H. J.; N’Diaye, Alpha T.; ...

2016-05-05

DNA sequencing by imaging in an electron microscope is an approach that holds promise to deliver long reads with low error rates and without the need for amplification. Earlier work using transmission electron microscopes, which use high electron energies on the order of 100 keV, has shown that low contrast and radiation damage necessitates the use of heavy atom labeling of individual nucleotides, which increases the read error rates. Other prior work using scattering electrons with much lower energy has shown to suppress beam damage on DNA. Here we explore possibilities to increase contrast by employing two methods, X-ray photoelectronmore » and Auger electron spectroscopy. Using bulk DNA samples with monomers of each base, both methods are shown to provide contrast mechanisms that can distinguish individual nucleotides without labels. In conclusion, both spectroscopic techniques can be readily implemented in a low energy electron microscope, which may enable label-free DNA sequencing by direct imaging.« less

Nucleic acid detection system and method for detecting influenza

DOEpatents

Cai, Hong; Song, Jian

2015-03-17

The invention provides a rapid, sensitive and specific nucleic acid detection system which utilizes isothermal nucleic acid amplification in combination with a lateral flow chromatographic device, or DNA dipstick, for DNA-hybridization detection. The system of the invention requires no complex instrumentation or electronic hardware, and provides a low cost nucleic acid detection system suitable for highly sensitive pathogen detection. Hybridization to single-stranded DNA amplification products using the system of the invention provides a sensitive and specific means by which assays can be multiplexed for the detection of multiple target sequences.

Digital quantification of DNA via isothermal amplification on a self-driven microfluidic chip featuring hydrophilic film-coated polydimethylsiloxane.

PubMed

Ma, Yu-Dong; Chang, Wen-Hsin; Luo, Kang; Wang, Chih-Hung; Liu, Shih-Yuan; Yen, Wen-Hsiang; Lee, Gwo-Bin

2018-01-15

Loop-mediated isothermal amplification (LAMP) is a DNA amplification approach characterized by high sensitivity and specificity. In "digital LAMP", small quantities of both template DNA and reagents are encapsulated within a droplet or microwell, allowing for analysis of precious nucleic acid samples in shorter amounts of time relative to traditional DNA amplification protocols (e.g., PCR) with an improved limit of detection. In this study, an integrated, self-driven microfluidic chip was designed to carry out digital LAMP. The entire quantification process could be automatically performed on this chip via capillary forces enabled through microwells comprised of polydimethylsiloxane (PDMS) surfaces coated with a hydrophilic film; no external pumps were required. Moreover, digitized droplets could be separated from each other by normally-closed microvalves. The contact angle of the hydrophilic film-coated PDMS surface was only 14.3°. This is the first time that a rapid (30min) and simple method has been used to create hydrophilic PDMS surfaces that allow for digital LAMP to be performed in a self-driven microfluidic device. As a proof of concept, amplification of a gene specific to a vancomycin-resistant Enterococcus strain was performed on the developed microfluidic chip within 30min, and the limit of detection was only 11 copies with a volume of 30μL. This device may therefore become a promising tool for clinical diagnosis and point-of-care applications. Copyright © 2017 Elsevier B.V. All rights reserved.

Magnetic nanobeads present during enzymatic amplification and labeling for a simplified DNA detection protocol based on AC susceptometry

NASA Astrophysics Data System (ADS)

Bejhed, Rebecca S.; Strømme, Maria; Svedlindh, Peter; Ahlford, Annika; Strömberg, Mattias

2015-12-01

Magnetic biosensors are promising candidates for low-cost point-of-care biodiagnostic devices. For optimal efficiency it is crucial to minimize the time and complexity of the assay protocol including target recognition, amplification, labeling and read-out. In this work, possibilities for protocol simplifications for a DNA biodetection principle relying on hybridization of magnetic nanobeads to rolling circle amplification (RCA) products are investigated. The target DNA is recognized through a padlock ligation assay resulting in DNA circles serving as templates for the RCA process. It is found that beads can be present during amplification without noticeably interfering with the enzyme used for RCA (phi29 polymerase). As a result, the bead-coil hybridization can be performed immediately after amplification in a one-step manner at elevated temperature within a few minutes prior to read-out in an AC susceptometer setup, i.e. a combined protocol approach. Moreover, by recording the phase angle ξ = arctan(χ″/χ'), where χ and χ″ are the in-phase and out-of-phase components of the AC susceptibility, respectively, at one single frequency the total assay time for the optimized combined protocol would be no more than 1.5 hours, often a relevant time frame for diagnosis of cancer and infectious disease. Also, applying the phase angle method normalization of AC susceptibility data is not needed. These findings are useful for the development of point-of-care biodiagnostic devices relying on bead-coil binding and magnetic AC susceptometry.

Inhibition mechanisms of hemoglobin, immunoglobulin G, and whole blood in digital and real-time PCR.

PubMed

Sidstedt, Maja; Hedman, Johannes; Romsos, Erica L; Waitara, Leticia; Wadsö, Lars; Steffen, Carolyn R; Vallone, Peter M; Rådström, Peter

2018-04-01

Blood samples are widely used for PCR-based DNA analysis in fields such as diagnosis of infectious diseases, cancer diagnostics, and forensic genetics. In this study, the mechanisms behind blood-induced PCR inhibition were evaluated by use of whole blood as well as known PCR-inhibitory molecules in both digital PCR and real-time PCR. Also, electrophoretic mobility shift assay was applied to investigate interactions between inhibitory proteins and DNA, and isothermal titration calorimetry was used to directly measure effects on DNA polymerase activity. Whole blood caused a decrease in the number of positive digital PCR reactions, lowered amplification efficiency, and caused severe quenching of the fluorescence of the passive reference dye 6-carboxy-X-rhodamine as well as the double-stranded DNA binding dye EvaGreen. Immunoglobulin G was found to bind to single-stranded genomic DNA, leading to increased quantification cycle values. Hemoglobin affected the DNA polymerase activity and thus lowered the amplification efficiency. Hemoglobin and hematin were shown to be the molecules in blood responsible for the fluorescence quenching. In conclusion, hemoglobin and immunoglobulin G are the two major PCR inhibitors in blood, where the first affects amplification through a direct effect on the DNA polymerase activity and quenches the fluorescence of free dye molecules, and the latter binds to single-stranded genomic DNA, hindering DNA polymerization in the first few PCR cycles. Graphical abstract PCR inhibition mechanisms of hemoglobin and immunoglobulin G (IgG). Cq quantification cycle, dsDNA double-stranded DNA, ssDNA single-stranded DNA.

A dual amplification strategy for DNA detection combining bio-barcode assay and metal-enhanced fluorescence modality.

PubMed

Zhou, Zhenpeng; Li, Tian; Huang, Hongduan; Chen, Yang; Liu, Feng; Huang, Chengzhi; Li, Na

2014-11-11

Silver-enhanced fluorescence was coupled with a bio-barcode assay to facilitate a dual amplification assay to demonstrate a non-enzymatic approach for simple and sensitive detection of DNA. In the assay design, magnetic nanoparticles seeded with silver nanoparticles were modified with the capture DNA, and silver nanoparticles were modified with the binding of ssDNA and the fluorescently labeled barcode dsDNA. Upon introduction of the target DNA, a sandwich structure was formed because of the hybridization reaction. By simple magnetic separation, silver-enhanced fluorescence of barcode DNAs could be readily measured without the need of a further step to liberate barcode DNAs from silver nanoparticles, endowing the method with simplicity and high sensitivity with a detection limit of 1 pM.

[Application of repair enzymes to improve the quality of degraded DNA templates for PCR amplification].

PubMed

Dovgerd, A P; Zharkov, D O

2014-01-01

PCR amplification of severely degraded DNA, including ancient DNA, forensic samples, and preparations from deeply processed foodstuffs, is a serious problem. Living organisms have a set of enzymes to repair lesions in their DNA. In this work, we have developed and characterized model systems of degraded high-molecular-weight DNA with a predominance of different types of damage. It was shown that depurination and oxidation of the model plasmid DNA template led to a decrease in the PCR efficiency. A set of enzymes performing a full cycle of excision repair of some lesions was determined. The treatment of model-damaged substrates with this set of enzymes resulted in an increased PCR product yield as compared with that of the unrepaired samples.

Rolling Circle Amplification For Spatially Directed Synthesis Of A Solid Phase Anchored Single-Stranded DNA Molecule

NASA Astrophysics Data System (ADS)

Reiß, Edda; Hölzel, Ralph; von Nickisch-Rosenegk, Markus; Bier, Frank F.

2006-09-01

In this article the usefulness of the enzyme phi29 DNA polymerase and the principle of rolling circle amplification (RCA) for creating single-stranded DNA (ssDNA) nanostructures is described. Currently we are working on the spatial orientation of a growing ssDNA molecule during its RCA-based synthesis by the application of a hydrodynamic force. Starting at an immobilized primer at single molecule level, the aim is to construct a nanostructure of known location and orientation, providing multiple repeating binding sites that can be addressed via complementary base-pairing. Proof-of-principle experiments demonstrate the potential of the enzymatic reaction. ssDNA molecules of more than 20 μm length were created at an immobilized primer and detected by means of fluorescence microscopy.

Point of care diagnosis of multiple schistosome parasites: Species-specific DNA detection in urine by loop-mediated isothermal amplification (LAMP).

PubMed

Lodh, Nilanjan; Mikita, Kei; Bosompem, Kwabena M; Anyan, William K; Quartey, Joseph K; Otchere, Joseph; Shiff, Clive J

2017-09-01

Schistosomes are easily transmitted and high chance of repeat infection, so if control strategies based on targeted mass drug administration (MDA) are to succeed it is essential to have a test that is sensitive, accurate and simple to use. It is known and regularly demonstrated that praziquantel does not always eliminate an infection so in spite of the successes of control programs a residual of the reservoir survives to re-infect snails. The issue of diagnostic sensitivity becomes more critical in the assessment of program effectiveness. While serology, such as antigen capture tests might improve sensitivity, it has been shown that the presence of species-specific DNA fragments will indicate, most effectively, the presence of active parasites. Polymerase chain reaction (PCR) can amplify and detect DNA from urine residue captured on Whatman No. 3 filter paper that is dried after filtration. Previously we have detected S. mansoni and S. haematobium parasite-specific small repeat DNA fragment from filtered urine on filter paper by PCR. In the current study, we assessed the efficacy of detection of 86 urine samples for either or both schistosome parasites by PCR and loop-mediated isothermal amplification (LAMP) that were collected from a low to moderate transmission area in Ghana. Two different DNA extraction methods, standard extraction kit and field usable LAMP-PURE kit were also evaluated by PCR and LAMP amplification. With S. haematobium LAMP amplification for both extractions showed similar sensitivity and specificity when compared with PCR amplification (100%) verified by gel electrophoresis. For S. mansoni sensitivity was highest for LAMP amplification (100%) for standard extraction than PCR and LAMP with LAMP-PURE (99% and 94%). The LAMP-PURE extraction produced false negatives, which require further investigation for this field usable extraction kit. Overall high positive and negative predictive values (90% - 100%) for both species demonstrated a highly robust approach. The LAMP approach is close to point of care use and equally sensitive and specific to detection of species-specific DNA by PCR. LAMP can be an effective means to detect low intensity infection due to its simplicity and minimal DNA extraction requirement. This will enhance the effectiveness of surveillance and MDA control programs of schistosomiasis. Copyright © 2017 Elsevier B.V. All rights reserved.

Detection and quantitation of HER-2/neu gene amplification in human breast cancer archival material using fluorescence in situ hybridization.

PubMed

Pauletti, G; Godolphin, W; Press, M F; Slamon, D J

1996-07-04

Amplification and overexpression of the HER-2/neu gene occurs in 25-30% of human breast cancers. This genetic alteration is associated with a poor clinical prognosis in women with either node negative or node positive breast cancers. The initial studies testing this association were somewhat controversial and this controversy was due in large part to significant heterogeneity in both the methods and/or reagents used in testing archival material for the presence of the alteration. These methods included a number of solid matrix blotting techniques for DNA, RNA and protein as well as immunohistochemistry. Fluorescence in situ hybridization (FISH) represents the newest methodologic approach for testing for this genetic alteration. In this study, FISH is compared to Southern, Northern and Western blot analyses as well as immunohistochemistry in a large cohort of archival human breast cancer specimens. FISH was found to be superior to all other methodologies tested in assessing formalin fixed, paraffin embedded material for HER-2/neu amplification. The results from this study also confirm that overexpression of HER-2/neu rarely occurs in the absence of gene amplification in breast cancer (approximately 3% of cases). This method of analysis is rapid, reproducible and extremely reliable in detecting presence of HER-2/neu gene amplification and should have clinical utility.

Ultrasensitive electrochemical detection of nucleic acids by template enhanced hybridization followed with rolling circle amplification.

PubMed

Ji, Hanxu; Yan, Feng; Lei, Jianping; Ju, Huangxian

2012-08-21

An ultrasensitive protocol for electrochemical detection of DNA is designed with quantum dots (QDs) as a signal tag by combining the template enhanced hybridization process (TEHP) and rolling circle amplification (RCA). Upon the recognition of the molecular beacon (MB) to target DNA, the MB hybridizes with assistants and target DNA to form a ternary ''Y-junction''. The target DNA can be dissociated from the structure under the reaction of nicking endonuclease to initiate the next hybridization process. The template enhanced MB fragments further act as the primers of the RCA reaction to produce thousands of repeated oligonucleotide sequences, which can bind with oligonucleotide functionalized QDs. The attached signal tags can be easily read out by square-wave voltammetry after dissolving with acid. Because of the cascade signal amplification and the specific TEHP and RCA reaction, this newly designed protocol provides an ultrasensitive electrochemical detection of DNA down to the attomolar level (11 aM) with a linear range of 6 orders of magnitude (from 1 × 10(-17) to 1 × 10(-11) M) and can discriminate mismatched DNA from perfect matched target DNA with high selectivity. The high sensitivity and specificity make this method a great potential for early diagnosis in gene-related diseases.

Rapid ABO genotyping by high-speed droplet allele-specific PCR using crude samples.

PubMed

Taira, Chiaki; Matsuda, Kazuyuki; Takeichi, Naoya; Furukawa, Satomi; Sugano, Mitsutoshi; Uehara, Takeshi; Okumura, Nobuo; Honda, Takayuki

2018-01-01

ABO genotyping has common tools for personal identification of forensic and transplantation field. We developed a new method based on a droplet allele-specific PCR (droplet-AS-PCR) that enabled rapid PCR amplification. We attempted rapid ABO genotyping using crude DNA isolated from dried blood and buccal cells. We designed allele-specific primers for three SNPs (at nucleotides 261, 526, and 803) in exons 6 and 7 of the ABO gene. We pretreated dried blood and buccal cells with proteinase K, and obtained crude DNAs without DNA purification. Droplet-AS-PCR allowed specific amplification of the SNPs at the three loci using crude DNA, with results similar to those for DNA extracted from fresh peripheral blood. The sensitivity of the methods was 5%-10%. The genotyping of extracted DNA and crude DNA were completed within 8 and 9 minutes, respectively. The genotypes determined by the droplet-AS-PCR method were always consistent with those obtained by direct sequencing. The droplet-AS-PCR method enabled rapid and specific amplification of three SNPs of the ABO gene from crude DNA treated with proteinase K. ABO genotyping by the droplet-AS-PCR has the potential to be applied to various fields including a forensic medicine and transplantation medical care. © 2017 Wiley Periodicals, Inc.

Enhanced detection of intracellular organism of swine proliferative enteritis, ileal symbiont intracellularis, in feces by polymerase chain reaction.

PubMed Central

Jones, G F; Ward, G E; Murtaugh, M P; Lin, G; Gebhart, C J

1993-01-01

A sensitive assay based on amplification of a 319-bp DNA fragment of the intracellular bacterium of swine proliferative enteritis was developed for the detection of the organism in the feces of swine. A vernacular name, ileal symbiont intracellularis (IS-intracellularis), has recently been published for the intracellular bacterium, which was formerly known as a Campylobacter-like organism (C.J. Gebhart, S.M. Barnes, S. McOrist, G.F. Lin, and G.H.K. Larson, Int. J. Syst. Bacteriol. 43:533-538, 1993). As few as 10(1) IS-intracellularis organisms purified from intestinal mucosa, or 10(3) IS-intracellularis per g of feces, were detected. No amplification product was produced from a polymerase chain reaction performed on DNA extracted from the feces of healthy pigs. A 319-bp DNA fragment specific for IS-intracellularis was produced on amplification of DNA from the feces of pigs with experimental and naturally occurring proliferative enteritis. Images PMID:8253956

Prognostic significance of MYCN gene amplification and protein expression in primary brain tumors: Astrocytoma and meningioma.

PubMed

Estiar, Mehrdad Asghari; Javan, Firouzeh; Zekri, Ali; Mehrazin, Masoud; Mehdipour, Parvin

2017-07-04

Astrocytoma and meningioma are the most common primary brain tumors. MYCN as a member of MYC proto-oncogenes has recently appeared as an attractive therapeutic target. Functions of MYCN are critical for growth of nervous system and neural differentiation. We examined MYCN amplification and protein expression in astrocytoma and meningioma cases. In this study, we used real-time PCR, FISH assay and flowcytometry to analyze DNA amplification and protein expression of MYCN. Among 30 samples of brain tumor, 14 cases (46.6%) revealed MYCN amplification. High-protein expression of MYCN was also observed in 43.3% of patients. There was a significant correlation between MYCN gene amplification and protein expression (r= 0.523; p= 0.003), interestingly five case showed discrepancy between the gene amplification and protein expression. Although MYCN amplification fails to show correlation with poor prognosis (p= 0.305), protein high-expression of MYCN significantly reduce disease-free survival (p= 0.019). Our results challenge the concept of the neural specificity of MYCN by demonstrating contribution of MYCN in meningioma. Moreover, this study highlights the importance of research at both level of DNA and protein, to determine the biological functions and medical impacts of MYCN.

Disintegration of cruciform and G-quadruplex structures during the course of helicase-dependent amplification (HDA).

PubMed

Li, Dawei; Lv, Bei; Zhang, Hao; Lee, Jasmine Yiqin; Li, Tianhu

2015-04-15

Unlike chemical damages on DNA, physical alterations of B-form of DNA occur commonly in organisms that serve as signals for specified cellular events. Although the modes of action for repairing of chemically damaged DNA have been well studied nowadays, the repairing mechanisms for physically altered DNA structures have not yet been understood. Our current in vitro studies show that both breakdown of stable non-B DNA structures and resumption of canonical B-conformation of DNA can take place during the courses of isothermal helicase-dependent amplification (HDA). The pathway that makes the non-B DNA structures repairable is presumably the relieving of the accumulated torsional stress that was caused by the positive supercoiling. Our new findings suggest that living organisms might have evolved this distinct and economical pathway for repairing their physically altered DNA structures. Copyright © 2015 Elsevier Ltd. All rights reserved.

Photoelectrochemical DNA Biosensor Based on Dual-Signal Amplification Strategy Integrating Inorganic-Organic Nanocomposites Sensitization with λ-Exonuclease-Assisted Target Recycling.

PubMed

Shi, Xiao-Mei; Fan, Gao-Chao; Shen, Qingming; Zhu, Jun-Jie

2016-12-28

Sensitive and accurate analysis of DNA is crucial to better understanding of DNA functions and early diagnosis of fatal disease. Herein, an enhanced photoelectrochemical (PEC) DNA biosensor was proposed based on dual-signal amplification via coupling inorganic-organic nanocomposites sensitization with λ-exonuclease (λ-Exo)-assisted target recycling. The short DNA sequence about chronic myelogenous leukemia (CML, type b3a2) was selected as target DNA (tDNA). ZnO nanoplates were deposited with CdS nanocrystals to form ZnO/CdS hetero-nanostructure, and it was used as PEC substrate for immobilizing hairpin DNA (hDNA). CdTe quantum dots (QDs) covalently linked with meso-tetra(4-carboxyphenyl)porphine (TCPP) to form CdTe/TCPP inorganic-organic nanocomposites, which were utilized as sensitization agents labeling at the terminal of probe DNA (pDNA). When the hDNA-modified sensing electrode was incubated with tDNA and λ-Exo, hDNA hybridized with tDNA, and meanwhile it could be recognized and cleaved by λ-Exo, resulting in the release of tDNA. The rest of nonhybridized hDNA would continuously hybridize with the released tDNA, cleave by λ-Exo, and set free the tDNA again. After λ-Exo-assisted tDNA recycling, more amounts of short DNA (sDNA) fragments coming from digestion of hDNA produced on the electrode and hybridized with CdTe/TCPP-labeled pDNA (pDNA-CdTe/TCPP conjugates). In this case, the sensitization of CdTe/TCPP inorganic-organic nanocomposites occurred, which evidently extend the absorption range and strengthened the absorption intensity of light energy, and accordingly the photocurrent signal significantly promoted. Through introducing the dual-signal amplification tactics, the developed PEC assay allowed a low calculated detection limit of 25.6 aM with a wide detection scope from 0.1 fM to 5 pM for sensitive and selective determination of tDNA.

Development of RAPD-SCAR markers for different Ganoderma species authentication by improved RAPD amplification and molecular cloning.

PubMed

Fu, J J; Mei, Z Q; Tania, M; Yang, L Q; Cheng, J L; Khan, M A

2015-05-25

The sequence-characterized amplified region (SCAR) is a valuable molecular technique for the genetic identification of any species. This method is mainly derived from the molecular cloning of the amplified DNA fragments achieved from the random amplified polymorphic DNA (RAPD). In this study, we collected DNA from 10 species of Ganoderma mushroom and amplified the DNA using an improved RAPD technique. The amplified fragments were then cloned into a T-vector, and positive clones were screened, indentified, and sequenced for the development of SCAR markers. After designing PCR primers and optimizing PCR conditions, 4 SCAR markers, named LZ1-4, LZ2-2, LZ8-2, and LZ9-15, were developed, which were specific to Ganoderma gibbosum (LZ1-4 and LZ8-2), Ganoderma sinense (LZ2-2 and LZ8-2), Ganoderma tropicum (LZ8-2), and Ganoderma lucidum HG (LZ9-15). These 4 novel SCAR markers were deposited into GenBank with the accession Nos. KM391935, KM391936, KM391937, and KM391938, respectively. Thus, in this study we developed specific SCAR markers for the identification and authentication of different Ganoderma species.

Compaction of rolling circle amplification products increases signal integrity and signal-to-noise ratio

PubMed Central

Clausson, Carl-Magnus; Arngården, Linda; Ishaq, Omer; Klaesson, Axel; Kühnemund, Malte; Grannas, Karin; Koos, Björn; Qian, Xiaoyan; Ranefall, Petter; Krzywkowski, Tomasz; Brismar, Hjalmar; Nilsson, Mats; Wählby, Carolina; Söderberg, Ola

2015-01-01

Rolling circle amplification (RCA) for generation of distinct fluorescent signals in situ relies upon the self-collapsing properties of single-stranded DNA in commonly used RCA-based methods. By introducing a cross-hybridizing DNA oligonucleotide during rolling circle amplification, we demonstrate that the fluorophore-labeled RCA products (RCPs) become smaller. The reduced size of RCPs increases the local concentration of fluorophores and as a result, the signal intensity increases together with the signal-to-noise ratio. Furthermore, we have found that RCPs sometimes tend to disintegrate and may be recorded as several RCPs, a trait that is prevented with our cross-hybridizing DNA oligonucleotide. These effects generated by compaction of RCPs improve accuracy of visual as well as automated in situ analysis for RCA based methods, such as proximity ligation assays (PLA) and padlock probes. PMID:26202090

Multiple Cross Displacement Amplification Combined with Gold Nanoparticle-Based Lateral Flow Biosensor for Detection of Vibrio parahaemolyticus

PubMed Central

Wang, Yi; Li, Hui; Li, Dongxun; Li, Kewei; Wang, Yan; Xu, Jianguo; Ye, Changyun

2016-01-01

Vibrio parahaemolyticus (V. parahaemolyticus) is a marine seafood-borne pathogen causing severe illnesses in humans and aquatic animals. In the present study, multiple cross displacement amplification was combined with a lateral flow biosensor (MCDA-LFB) to detect the toxR gene of V. parahaemolyticus in DNA extracts from pure cultures and spiked oyster homogenates. Amplification was carried out at a constant temperature (62°C) for only 30 min, and amplification products were directly applied to the biosensor. The entire process, including oyster homogenate processing (30 min), isothermal amplification (30 min) and results indicating (∼2 min), could be completed within 65 min. Amplification product was detectable from as little as 10 fg of pure V. parahaemolyticus DNA and from approximately 4.2 × 102 CFU in 1 mL of oyster homogenate. No cross-reaction with other Vibrio species and with non-Vibrio species was observed. Therefore, the MCDA-LFB method established in the current report is suitable for the rapid screening of V. parahaemolyticus in clinical, food, and environmental samples. PMID:28066368

SYBR Green Real-Time PCR Method To Detect Clostridium botulinum Type A▿

PubMed Central

Fenicia, Lucia; Anniballi, Fabrizio; De Medici, Dario; Delibato, Elisabetta; Aureli, Paolo

2007-01-01

Botulinum toxins (BoNTs) are classically produced by Clostridium botulinum but rarely also from neurotoxigenic strains of Clostridium baratii and Clostridium butyricum. BoNT type A (BoNT/A), BoNT/B, BoNT/E, and very rarely BoNT/F are mainly responsible for human botulism. Standard microbiological methods take into consideration only the detection of C. botulinum. The presumptive identification of the toxigenic strains together with the typing of BoNT has to be performed by mouse bioassay. The development of PCR-based methods for the detection and typing of BoNT-producing clostridia would be an ideal alternative to the mouse bioassay. The objective of this study was to develop a rapid and robust real-time PCR method for detecting C. botulinum type A. Four different techniques for the extraction and purification of DNA from cultured samples were initially compared. Of the techniques used, Chelex 100, DNeasy tissue kit, InstaGene matrix DNA, and boiling, the boiling technique was significantly less efficient than the other three. These did not give statistically different results, and Chelex 100 was chosen because it was less expensive than the others. In order to eliminate any false-negative results, an internal amplification control was synthesized and included in the amplification mixture according to ISO 22174. The specificity of the method was tested against 75 strains of C. botulinum type A, 4 strains of C. botulinum type Ab, and 101 nontarget strains. The detection limit of the reaction was less than 6 × 101 copies of C. botulinum type A DNA. The robustness of the method was confirmed using naturally contaminated stool specimens to evaluate the tolerance of inhibitor substances. SYBR green real-time PCR showed very high specificity for the detection of C. botulinum types A and Ab (inclusivity and exclusivity, 100%). PMID:17369349

DNA nanomechanics allows direct digital detection of complementary DNA and microRNA targets.

PubMed

Husale, Sudhir; Persson, Henrik H J; Sahin, Ozgur

2009-12-24

Techniques to detect and quantify DNA and RNA molecules in biological samples have had a central role in genomics research. Over the past decade, several techniques have been developed to improve detection performance and reduce the cost of genetic analysis. In particular, significant advances in label-free methods have been reported. Yet detection of DNA molecules at concentrations below the femtomolar level requires amplified detection schemes. Here we report a unique nanomechanical response of hybridized DNA and RNA molecules that serves as an intrinsic molecular label. Nanomechanical measurements on a microarray surface have sufficient background signal rejection to allow direct detection and counting of hybridized molecules. The digital response of the sensor provides a large dynamic range that is critical for gene expression profiling. We have measured differential expressions of microRNAs in tumour samples; such measurements have been shown to help discriminate between the tissue origins of metastatic tumours. Two hundred picograms of total RNA is found to be sufficient for this analysis. In addition, the limit of detection in pure samples is found to be one attomolar. These results suggest that nanomechanical read-out of microarrays promises attomolar-level sensitivity and large dynamic range for the analysis of gene expression, while eliminating biochemical manipulations, amplification and labelling.

Preconcentration of DNA using magnetic ionic liquids that are compatible with real-time PCR for rapid nucleic acid quantification.

PubMed

Emaus, Miranda N; Clark, Kevin D; Hinners, Paige; Anderson, Jared L

2018-04-28

Nucleic acid extraction and purification represents a major bottleneck in DNA analysis. Traditional methods for DNA purification often require reagents that may inhibit quantitative polymerase chain reaction (qPCR) if not sufficiently removed from the sample. Approaches that employ magnetic beads may exhibit lower extraction efficiencies due to sedimentation and aggregation. In this study, four hydrophobic magnetic ionic liquids (MILs) were investigated as DNA extraction solvents with the goal of improving DNA enrichment factors and compatibility with downstream bioanalytical techniques. By designing custom qPCR buffers, we directly incorporated DNA-enriched MILs including trihexyl(tetradecyl)phosphonium tris(hexafluoroacetylaceto)nickelate(II) ([P 6,6,6,14 + ][Ni(hfacac) 3 - ]), [P 6,6,6,14 + ] tris(hexafluoroacetylaceto)colbaltate(II) ([Co(hfacac) 3 - ]), [P 6,6,6,14 + ] tris(hexafluoroacetylaceto)manganate(II) ([Mn(hfacac) 3 - ]), or [P 6,6,6,14 + ] tetrakis(hexafluoroacetylaceto)dysprosate(III) ([Dy(hfacac) 4 - ]) into reaction systems, thereby circumventing the need for time-consuming DNA recovery steps. Incorporating MILs into the reaction buffer did not significantly impact the amplification efficiency of the reaction (91.1%). High enrichment factors were achieved using the [P 6,6,6,14 + ][Ni(hfacac) 3 - ] MIL for the extraction of single-stranded and double-stranded DNA with extraction times as short as 2 min. When compared to a commercial magnetic bead-based platform, the [P 6,6,6,14 + ][Ni(hfacac) 3 - ] MIL was capable of producing higher enrichment factors for single-stranded DNA and similar enrichment factors for double-stranded DNA. The MIL-based method was applied for the extraction and direct qPCR amplification of mutation prone-KRAS oncogene fragment in plasma samples. Graphical abstract Magnetic ionic liquid solvents are shown to preconcentrate sufficient KRAS DNA template from an aqueous solution in as short as 2 min without using chaotropic salts or toxic organic solvents. By using custom-designed qPCR buffers, DNA can be directly amplified and quantified from four MILs examined in this study.

Simultaneous identification and DNA barcoding of six Eimeria species infecting turkeys using PCR primers targeting the mitochondrial cytochrome c oxidase subunit I (mtCOI) locus.

PubMed

Hafeez, Mian A; Shivaramaiah, Srichaitanya; Dorsey, Kristi Moore; Ogedengbe, Mosun E; El-Sherry, Shiem; Whale, Julia; Cobean, Julie; Barta, John R

2015-05-01

Species-specific PCR primers targeting the mitochondrial cytochrome c oxidase subunit I (mtCOI) locus were generated that allow for the specific identification of the most common Eimeria species infecting turkeys (i.e., Eimeria adenoeides, Eimeria meleagrimitis, Eimeria gallopavonis, Eimeria meleagridis, Eimeria dispersa, and Eimeria innocua). PCR reaction chemistries were optimized with respect to divalent cation (MgCl2) and dNTP concentrations, as well as PCR cycling conditions (particularly anneal temperature for primers). Genomic DNA samples from single oocyst-derived lines of six Eimeria species were tested to establish specificity and sensitivity of these newly designed primer pairs. A mixed 60-ng total DNA sample containing 10 ng of each of the six Eimeria species was used as DNA template to demonstrate specific amplification of the correct product using each of the species-specific primer pairs. Ten nanograms of each of the five non-target Eimeria species was pooled to provide a non-target, control DNA sample suitable to test the specificity of each primer pair. The amplifications of the COI region with species-specific primer pairs from pooled samples yielded products of expected sizes (209 to 1,012 bp) and no amplification of non-target Eimeria sp. DNA was detected using the non-target, control DNA samples. These primer pairs specific for Eimeria spp. of turkeys did not amplify any of the seven Eimeria species infecting chickens. The newly developed PCR primers can be used as a diagnostic tool capable of specifically identifying six turkey Eimeria species; additionally, sequencing of the PCR amplification products yields sequence-based genotyping data suitable for identification and molecular phylogenetics.

Target-regulated proximity hybridization with three-way DNA junction for in situ enhanced electronic detection of marine biotoxin based on isothermal cycling signal amplification strategy.

PubMed

Liu, Bingqian; Chen, Jinfeng; Wei, Qiaohua; Zhang, Bing; Zhang, Lan; Tang, Dianping

2015-07-15

A new signal amplification strategy based on target-regulated DNA proximity hybridization (TRPH) reaction accompanying formation of three-way DNA junction was designed for electronic detection of Microcystin-LR (MC-LR used in this case), coupling with junction-induced isothermal cycling signal amplification. Initially, a sandwiched-type immunoreaction was carried out in a low-cost PCR tube between anti-MC-LR mAb1 antibody-labeled DNA1 (mAb1-DNA1) and anti-MC-LR mAb2-labeled DNA2 (mAb2-DNA2) in the presence of target to form a three-way DNA junction. Then, the junction could undergo an unbiased strand displacement reaction on an h-like DNA nanostructure-modified electrode (labeled with methylene blue redox tag on the short DNA strand), thereby resulting in the dissociation of methylene blue-labeled signal DNA from the electrode. The newly formed double-stranded DNA could be cleaved again by exonuclease III, and the released three-way DNA junction retriggered the strand-displacement reaction with h-like DNA nanostructures for junction recycling. During the strand-displacement reaction, numerous methylene blue-labeled DNA strands were far away from the electrode, thus decreasing the detectable electrochemical signal within the applied potentials. Under optimal conditions, the TRPH-based immunosensing system exhibited good electrochemical responses for detecting target MC-LR at a concentration as low as 1.0ngkg(-1) (1.0ppt). Additionally, the precision, reproducibility, specificity and method accuracy were also investigated with acceptable results. Copyright © 2015 Elsevier B.V. All rights reserved.

An advanced uracil DNA glycosylase-supplemented loop-mediated isothermal amplification (UDG-LAMP) technique used in the sensitive and specific detection of Cryptosporidium parvum, Cryptosporidium hominis, and Cryptosporidium meleagridis in AIDS patients.

PubMed

Fallahi, Shirzad; Moosavi, Seyedeh Fatemeh; Karimi, Azadeh; Chegeni, Ali Sharafi; Saki, Mohammad; Namdari, Parsa; Rashno, Mohammad Menati; Varzi, Ali Mohamad; Tarrahi, Mohammad Javad; Almasian, Mohammad

2018-05-01

The rapid and accurate detection of Cryptosporidium spp. is critically important for the prevention and timely treatment of cryptosporidiosis in AIDS patients (APs). This study was conducted to examine a UDG-LAMP technique for the first time to diagnose cryptosporidiosis in APs. After collecting demographic and clinical data, three stool samples were collected from the participants (120 volunteering APs). The microscopic examination of stained smears using the acid-fast method and the UDG-LAMP assay were performed for each sample. 10% of APs were infected with Cryptosporidium spp. The number of detected cryptosporidiosis cases using the acid-fast staining and UDG-LAMP methods were significantly different (P < 0.001). Diarrhea and weight loss were found to be significantly associated with cryptosporidiosis in patients (P < 0.05). The pretreatment of LAMP reagents with UDG successfully eliminated the likelihood of product re-amplification remaining from previous reactions. The UDG-LAMP technique could detect cryptosporidiosis in APs with high sensitivity and rapidity without carryover contamination. Copyright © 2017 Elsevier Inc. All rights reserved.

Label-free and ultrasensitive fluorescence detection of cocaine based on a strategy that utilizes DNA-templated silver nanoclusters and the nicking endonuclease-assisted signal amplification method.

PubMed

Zhang, Kai; Wang, Ke; Zhu, Xue; Zhang, Jue; Xu, Lan; Huang, Biao; Xie, Minhao

2014-01-07

A general and reliable strategy for the detection of cocaine was proposed utilizing DNA-templated silver nanoclusters as signal indicators and the nicking endonuclease-assisted signal amplification method. This strategy can detect cocaine specifically with a detection limit as low as 2 nM by using a small volume of 5 μL.

Isothermal multiple displacement amplification: a methodical approach enhancing molecular routine diagnostics of microcarcinomas and small biopsies.

PubMed

Mairinger, Fabian D; Walter, Robert Fh; Vollbrecht, Claudia; Hager, Thomas; Worm, Karl; Ting, Saskia; Wohlschläger, Jeremias; Zarogoulidis, Paul; Zarogoulidis, Konstantinos; Schmid, Kurt W

2014-01-01

Isothermal multiple displacement amplification (IMDA) can be a powerful tool in molecular routine diagnostics for homogeneous and sequence-independent whole-genome amplification of notably small tumor samples, eg, microcarcinomas and biopsies containing a small amount of tumor. Currently, this method is not well established in pathology laboratories. We designed a study to confirm the feasibility and convenience of this method for routine diagnostics with formalin-fixed, paraffin-embedded samples prepared by laser-capture microdissection. A total of 250 μg DNA (concentration 5 μg/μL) was generated by amplification over a period of 8 hours with a material input of approximately 25 cells, approximately equivalent to 175 pg of genomic DNA. In the generated DNA, a representation of all chromosomes could be shown and the presence of elected genes relevant for diagnosis in clinical samples could be proven. Mutational analysis of clinical samples could be performed without any difficulty and showed concordance with earlier diagnostic findings. We established the feasibility and convenience of IMDA for routine diagnostics. We also showed that small amounts of DNA, which were not analyzable with current molecular methods, could be sufficient for a wide field of applications in molecular routine diagnostics when they are preamplified with IMDA.

Amplification volume reduction on DNA database samples using FTA™ Classic Cards.

PubMed

Wong, Hang Yee; Lim, Eng Seng Simon; Tan-Siew, Wai Fun

2012-03-01

The DNA forensic community always strives towards improvements in aspects such as sensitivity, robustness, and efficacy balanced with cost efficiency. Therefore our laboratory decided to study the feasibility of PCR amplification volume reduction using DNA entrapped in FTA™ Classic Card and to bring cost savings to the laboratory. There were a few concerns the laboratory needed to address. First, the kinetics of the amplification reaction could be significantly altered. Second, an increase in sensitivity might affect interpretation due to increased stochastic effects even though they were pristine samples. Third, statics might cause FTA punches to jump out of its allocated well into another thus causing sample-to-sample contamination. Fourth, the size of the punches might be too small for visual inspection. Last, there would be a limit to the extent of volume reduction due to evaporation and the possible need of re-injection of samples for capillary electrophoresis. The laboratory had successfully optimized a reduced amplification volume of 10 μL for FTA samples. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

Optically Controlled Signal Amplification for DNA Computation.

PubMed

Prokup, Alexander; Hemphill, James; Liu, Qingyang; Deiters, Alexander

2015-10-16

The hybridization chain reaction (HCR) and fuel-catalyst cycles have been applied to address the problem of signal amplification in DNA-based computation circuits. While they function efficiently, these signal amplifiers cannot be switched ON or OFF quickly and noninvasively. To overcome these limitations, a light-activated initiator strand for the HCR, which enabled fast optical OFF → ON switching, was developed. Similarly, when a light-activated version of the catalyst strand or the inhibitor strand of a fuel-catalyst cycle was applied, the cycle could be optically switched from OFF → ON or ON → OFF, respectively. To move the capabilities of these devices beyond solution-based operations, the components were embedded in agarose gels. Irradiation with customizable light patterns and at different time points demonstrated both spatial and temporal control. The addition of a translator gate enabled a spatially activated signal to travel along a predefined path, akin to a chemical wire. Overall, the addition of small light-cleavable photocaging groups to DNA signal amplification circuits enabled conditional control as well as fast photocontrol of signal amplification.

Design and application of cotranscriptional non-enzymatic RNA circuits and signal transducers

PubMed Central

Bhadra, Sanchita; Ellington, Andrew D.

2014-01-01

Nucleic acid circuits are finding increasing real-life applications in diagnostics and synthetic biology. Although DNA has been the main operator in most nucleic acid circuits, transcriptionally produced RNA circuits could provide powerful alternatives for reagent production and their use in cells. Towards these goals, we have implemented a particular nucleic acid circuit, catalytic hairpin assembly, using RNA for both information storage and processing. Our results demonstrated that the design principles developed for DNA circuits could be readily translated to engineering RNA circuits that operated with similar kinetics and sensitivities of detection. Not only could purified RNA hairpins perform amplification reactions but RNA hairpins transcribed in vitro also mediated amplification, even without purification. Moreover, we could read the results of the non-enzymatic amplification reactions using a fluorescent RNA aptamer ‘Spinach’ that was engineered to undergo sequence-specific conformational changes. These advances were applied to the end-point and real-time detection of the isothermal strand displacement amplification reaction that produces single-stranded DNAs as part of its amplification cycle. We were also able to readily engineer gate structures with RNA similar to those that have previously formed the basis of DNA circuit computations. Taken together, these results validate an entirely new chemistry for the implementation of nucleic acid circuits. PMID:24493736

Optimization of multiplexed PCR on an integrated microfluidic forensic platform for rapid DNA analysis.

PubMed

Estes, Matthew D; Yang, Jianing; Duane, Brett; Smith, Stan; Brooks, Carla; Nordquist, Alan; Zenhausern, Frederic

2012-12-07

This study reports the design, prototyping, and assay development of multiplexed polymerase chain reaction (PCR) on a plastic microfluidic device. Amplification of 17 DNA loci is carried out directly on-chip as part of a system for continuous workflow processing from sample preparation (SP) to capillary electrophoresis (CE). For enhanced performance of on-chip PCR amplification, improved control systems have been developed making use of customized Peltier assemblies, valve actuators, software, and amplification chemistry protocols. Multiple enhancements to the microfluidic chip design have been enacted to improve the reliability of sample delivery through the various on-chip modules. This work has been enabled by the encapsulation of PCR reagents into a solid phase material through an optimized Solid Phase Encapsulating Assay Mix (SPEAM) bead-based hydrogel fabrication process. SPEAM bead technology is reliably coupled with precise microfluidic metering and dispensing for efficient amplification and subsequent DNA short tandem repeat (STR) fragment analysis. This provides a means of on-chip reagent storage suitable for microfluidic automation, with the long shelf-life necessary for point-of-care (POC) or field deployable applications. This paper reports the first high quality 17-plex forensic STR amplification from a reference sample in a microfluidic chip with preloaded solid phase reagents, that is designed for integration with up and downstream processing.

New Fpg probe chemistry for direct detection of recombinase polymerase amplification on lateral flow strips.

PubMed

Powell, Michael L; Bowler, Frank R; Martinez, Aurore J; Greenwood, Catherine J; Armes, Niall; Piepenburg, Olaf

2018-02-15

Rapid, cost-effective and sensitive detection of nucleic acids has the ability to improve upon current practices employed for pathogen detection in diagnosis of infectious disease and food testing. Furthermore, if assay complexity can be reduced, nucleic acid amplification tests could be deployed in resource-limited and home use scenarios. In this study, we developed a novel Fpg (Formamidopyrimidine DNA glycosylase) probe chemistry, which allows lateral flow detection of amplification in undiluted recombinase polymerase amplification (RPA) reactions. The prototype nucleic acid lateral flow chemistry was applied to a human genomic target (rs1207445), Campylobacter jejuni 16S rDNA and two genetic markers of the important food pathogen E. coli O157:H7. All four assays have an analytical sensitivity between 10 and 100 copies DNA per amplification. Furthermore, the assay is performed with fewer hands-on steps than using the current RPA Nfo lateral flow method as dilution of amplicon is not required for lateral flow analysis. Due to the simplicity of the workflow, we believe that the lateral flow chemistry for direct detection could be readily adapted to a cost-effective single-use consumable, ideal for use in non-laboratory settings. Copyright © 2017. Published by Elsevier Inc.

3' rapid amplification of cDNA ends (RACE) walking for rapid structural analysis of large transcripts.

PubMed

Ozawa, Tatsuhiko; Kondo, Masato; Isobe, Masaharu

2004-01-01

The 3' rapid amplification of cDNA ends (3' RACE) is widely used to isolate the cDNA of unknown 3' flanking sequences. However, the conventional 3' RACE often fails to amplify cDNA from a large transcript if there is a long distance between the 5' gene-specific primer and poly(A) stretch, since the conventional 3' RACE utilizes 3' oligo-dT-containing primer complementary to the poly(A) tail of mRNA at the first strand cDNA synthesis. To overcome this problem, we have developed an improved 3' RACE method suitable for the isolation of cDNA derived from very large transcripts. By using the oligonucleotide-containing random 9mer together with the GC-rich sequence for the suppression PCR technology at the first strand of cDNA synthesis, we have been able to amplify the cDNA from a very large transcript, such as the microtubule-actin crosslinking factor 1 (MACF1) gene, which codes a transcript of 20 kb in size. When there is no splicing variant, our highly specific amplification allows us to perform the direct sequencing of 3' RACE products without requiring cloning in bacterial hosts. Thus, this stepwise 3' RACE walking will help rapid characterization of the 3' structure of a gene, even when it encodes a very large transcript.