Science.gov

Sample records for high-resolution dna melting

  1. High-Resolution DNA Melting Analysis in Plant Research.

    PubMed

    Simko, Ivan

    2016-06-01

    Genetic and genomic studies provide valuable insight into the inheritance, structure, organization, and function of genes. The knowledge gained from the analysis of plant genes is beneficial to all aspects of plant research, including crop improvement. New methods and tools are continually being developed to facilitate rapid and accurate mapping, sequencing, and analyzing of genes. Here, I review the recent progress in the application of high-resolution melting (HRM) analysis of DNA, a method that allows detecting polymorphism in double-stranded DNA by comparing profiles of melting curves. Use of HRM has expanded considerably in the past few years as the method was successfully applied for high-throughput genotyping, mapping genes, testing food products and seeds, and other areas of plant research. PMID:26827247

  2. Automated DNA extraction, quantification, dilution, and PCR preparation for genotyping by high-resolution melting.

    PubMed

    Seipp, Michael T; Herrmann, Mark; Wittwer, Carl T

    2010-12-01

    Genotyping by high-resolution amplicon melting uses only two PCR primers per locus and a generic, saturating DNA dye that detects heteroduplexes as well as homoduplexes. Heterozygous genotypes have a characteristic melting curve shape and a broader width than homozygous genotypes, which are usually differentiated by their melting temperature (T(m)). The H63D mutation, associated with hemochromatosis, is a single nucleotide polymorphism, which is impossible to genotype based on T(m), as the homozygous WT and mutant amplicons melt at the same temperature. To distinguish such homozygous variants, WT DNA can be added to controls and unknown samples to create artificial heterozygotes with all genotypes distinguished by quantitative heteroduplex analysis. By automating DNA extraction, quantification, and PCR preparation, a hands-off integrated solution for genotyping is possible. A custom Biomek® NX robot with an onboard spectrophotometer and custom programming was used to extract DNA from whole blood, dilute the DNA to appropriate concentrations, and add the sample DNA to preprepared PCR plates. Agencourt® Genfind™ v.2 chemistry was used for DNA extraction. PCR was performed on a plate thermocycler, high-resolution melting data collected on a LightScanner-96, followed by analysis and automatic genotyping using custom software. In a blinded study of 42 H63D samples, 41 of the 42 sample genotypes were concordant with dual hybridization probe genotyping. The incorrectly assigned genotype was a heterozygote that appeared to be a homozygous mutant as a result of a low sample DNA concentration. Automated DNA extraction from whole blood with quantification, dilution, and PCR preparation was demonstrated using quantitative heteroduplex analysis. Accuracy is critically dependent on DNA quantification.

  3. High resolution melting (HRM) analysis of DNA--its role and potential in food analysis.

    PubMed

    Druml, Barbara; Cichna-Markl, Margit

    2014-09-01

    DNA based methods play an increasing role in food safety control and food adulteration detection. Recent papers show that high resolution melting (HRM) analysis is an interesting approach. It involves amplification of the target of interest in the presence of a saturation dye by the polymerase chain reaction (PCR) and subsequent melting of the amplicons by gradually increasing the temperature. Since the melting profile depends on the GC content, length, sequence and strand complementarity of the product, HRM analysis is highly suitable for the detection of single-base variants and small insertions or deletions. The review gives an introduction into HRM analysis, covers important aspects in the development of an HRM analysis method and describes how HRM data are analysed and interpreted. Then we discuss the potential of HRM analysis based methods in food analysis, i.e. for the identification of closely related species and cultivars and the identification of pathogenic microorganisms.

  4. Differential effect of three base modifications on DNA thermostability revealed by high resolution melting.

    PubMed

    López, Carlos M Rodríguez; Lloyd, Amanda J; Leonard, Kate; Wilkinson, Mike J

    2012-09-01

    High resolution melting (HRM) can detect and quantify the presence of 5-methylcytosine (5mC) in DNA samples, but the ability of HRM to diagnose other DNA modifications remains unexplored. The DNA bases N6-methyladenine and 5-hydroxymethylcytosine occur across almost all phyla. While their function remains controversial, their presence perturbs DNA structure. Such modifications could affect gene regulation, chromatin condensation and DNA packaging. Here, we reveal that DNA containing N6-methyladenine or 5-hydroxymethylcytosine exhibits reduced thermal stability compared to cytosine-methylated DNA. These thermostability changes are sufficiently divergent to allow detection and quantification by HRM analysis. Thus, we report that HRM distinguishes between sequence-identical DNA differing only in the modification type of one base. This approach is also able to distinguish between two DNA fragments carrying both N6-methyladenine and 5-methylcytosine but differing only in the distance separating the modified bases. This finding provides scope for the development of new methods to characterize DNA chemically and to allow for low cost screening of mutant populations of genes involved in base modification. More fundamentally, contrast between the thermostabilizing effects of 5mC on dsDNA compared with the destabilizing effects of N6-methyladenine (m6A) and 5-hydroxymethylcytosine (5hmC) raises the intriguing possibility of an antagonistic relationship between modification types with functional significance.

  5. Refining DNA Barcoding Coupled High Resolution Melting for Discrimination of 12 Closely Related Croton Species.

    PubMed

    Osathanunkul, Maslin; Suwannapoom, Chatmongkon; Ounjai, Sarawut; Rora, Jantarika A; Madesis, Panagiotis; de Boer, Hugo

    2015-01-01

    DNA barcoding coupled high resolution melting (Bar-HRM) is an emerging method for species discrimination based on DNA dissociation kinetics. The aim of this work was to evaluate the suitability of different primer sets, derived from selected DNA regions, for Bar-HRM analysis of species in Croton (Euphorbiaceae), one of the largest genera of plants with over 1,200 species. Seven primer pairs were evaluated (matK, rbcL1, rbcL2, rbcL3, rpoC, trnL and ITS1) from four plastid regions, matK, rbcL, rpoC, and trnL, and the nuclear ribosomal marker ITS1. The primer pair derived from the ITS1 region was the single most effective region for the identification of the tested species, whereas the rbcL1 primer pair gave the lowest resolution. It was observed that the ITS1 barcode was the most useful DNA barcoding region overall for species discrimination out of all of the regions and primers assessed. Our Bar-HRM results here also provide further support for the hypothesis that both sequence and base composition affect DNA duplex stability.

  6. High-resolution melt analysis of DNA methylation to discriminate semen in biological stains.

    PubMed

    Antunes, Joana; Silva, Deborah S B S; Balamurugan, Kuppareddi; Duncan, George; Alho, Clarice S; McCord, Bruce

    2016-02-01

    The goal of this study was to develop a method for the detection of semen in biological stains using high-resolution melt (HRM) analysis and DNA methylation. To perform this task, we used an epigenetic locus that targets a tissue-specific differentially methylated region for semen. This specific locus, ZC3H12D, contains methylated CpG sites that are hypomethylated in semen and hypermethylated in blood and saliva. Using this procedure, DNA from forensic stains can be isolated, processed using bisulfite-modified polymerase chain reaction (PCR), and detected by real-time PCR with HRM capability. The method described in this article is robust; we were able to obtain results from samples with as little as 1 ng of genomic DNA. Samples inhibited by humic acid still produced reliable results. Furthermore, the procedure is specific and will not amplify non-bisulfite-modified DNA. Because this process can be performed using real-time PCR and is quantitative, it fits nicely within the workflow of current forensic DNA laboratories. As a result, it should prove to be a useful technique for processing trace evidence samples for serological analysis.

  7. Refining DNA Barcoding Coupled High Resolution Melting for Discrimination of 12 Closely Related Croton Species

    PubMed Central

    Osathanunkul, Maslin; Suwannapoom, Chatmongkon; Ounjai, Sarawut; Rora, Jantarika A.; Madesis, Panagiotis; de Boer, Hugo

    2015-01-01

    DNA barcoding coupled high resolution melting (Bar-HRM) is an emerging method for species discrimination based on DNA dissociation kinetics. The aim of this work was to evaluate the suitability of different primer sets, derived from selected DNA regions, for Bar-HRM analysis of species in Croton (Euphorbiaceae), one of the largest genera of plants with over 1,200 species. Seven primer pairs were evaluated (matK, rbcL1, rbcL2, rbcL3, rpoC, trnL and ITS1) from four plastid regions, matK, rbcL, rpoC, and trnL, and the nuclear ribosomal marker ITS1. The primer pair derived from the ITS1 region was the single most effective region for the identification of the tested species, whereas the rbcL1 primer pair gave the lowest resolution. It was observed that the ITS1 barcode was the most useful DNA barcoding region overall for species discrimination out of all of the regions and primers assessed. Our Bar-HRM results here also provide further support for the hypothesis that both sequence and base composition affect DNA duplex stability. PMID:26406615

  8. Genotyping of beta thalassemia trait by high-resolution DNA melting analysis.

    PubMed

    Saetung, Rattika; Ongchai, Siriwan; Charoenkwan, Pimlak; Sanguansermsri, Torpong

    2013-11-01

    Beta thalassemia is a common hereditary hemalogogical disease in Thailand, with a prevalence of 5-8%. In this study, we evaluated the high resolution DNA melting (HRM) assay to identify beta thalassemia mutation in samples from 143 carriers of the beta thalassemia traits in at risk couples. The DNA was isolated from venous blood samples and tested for mutation under a series of 5 PCR-HRM (A, B, C, D and E primers) protocols. The A primers were for detection of beta thalassemia mutations in the HBB promoter region, the B primers for mutations in exon I, the C primers for exon II, the D primers for exon III and the E primers for the 3.4 kb deletion mutation. The mutations were diagnosed by comparing the complete melting curve profiles of a wild type control with those for each mutant sample. With the PCR-HRM technique, fourteen types of beta thalassemia mutations were detected. Each mutation had a unique and specific melting profile. The mutations included 36.4% (52 cases) codon 41/42-CTTT, 26.6% (38 cases) codon 17 A-T, 11.2% (16 cases) IVS1-1 G-T, 8.4% (12 cases) codon 71/72 +A, 8.4% (12 cases) of the 3.4 kb deletion and 3.5% (5 cases) -28 A-G. The remainder included one instance each of -87 C-A, -31 A-C, codon 27/28 +C, codon 30 G-A, IVS1-5 G-C, codon 35 C-A, codon 41-C and IVSII -654 C-T. Of the total cases, 85.8% of the mutations could be detected by primers B and C. The PCR-HRM method provides a rapid, simple and highly feasible strategy for mutation screening of beta thalassemia traits. PMID:24450243

  9. Sex Determination in Highly Fragmented Human DNA by High-Resolution Melting (HRM) Analysis

    PubMed Central

    Álvarez-Sandoval, Brenda A.; Manzanilla, Linda R.; Montiel, Rafael

    2014-01-01

    Sex identification in ancient human remains is a common problem especially if the skeletons are sub-adult, incomplete or damaged. In this paper we propose a new method to identify sex, based on real-time PCR amplification of small fragments (61 and 64 bp) of the third exon within the amelogenin gene covering a 3-bp deletion on the AMELX-allele, followed by a High Resolution Melting analysis (HRM). HRM is based on the melting curves of amplified fragments. The amelogenin gene is located on both chromosomes X and Y, showing dimorphism in length. This molecular tool is rapid, sensitive and reduces the risk of contamination from exogenous genetic material when used for ancient DNA studies. The accuracy of the new method described here has been corroborated by using control samples of known sex and by contrasting our results with those obtained with other methods. Our method has proven to be useful even in heavily degraded samples, where other previously published methods failed. Stochastic problems such as the random allele drop-out phenomenon are expected to occur in a less severe form, due to the smaller fragment size to be amplified. Thus, their negative effect could be easier to overcome by a proper experimental design. PMID:25098828

  10. Sex determination in highly fragmented human DNA by high-resolution melting (HRM) analysis.

    PubMed

    Álvarez-Sandoval, Brenda A; Manzanilla, Linda R; Montiel, Rafael

    2014-01-01

    Sex identification in ancient human remains is a common problem especially if the skeletons are sub-adult, incomplete or damaged. In this paper we propose a new method to identify sex, based on real-time PCR amplification of small fragments (61 and 64 bp) of the third exon within the amelogenin gene covering a 3-bp deletion on the AMELX-allele, followed by a High Resolution Melting analysis (HRM). HRM is based on the melting curves of amplified fragments. The amelogenin gene is located on both chromosomes X and Y, showing dimorphism in length. This molecular tool is rapid, sensitive and reduces the risk of contamination from exogenous genetic material when used for ancient DNA studies. The accuracy of the new method described here has been corroborated by using control samples of known sex and by contrasting our results with those obtained with other methods. Our method has proven to be useful even in heavily degraded samples, where other previously published methods failed. Stochastic problems such as the random allele drop-out phenomenon are expected to occur in a less severe form, due to the smaller fragment size to be amplified. Thus, their negative effect could be easier to overcome by a proper experimental design. PMID:25098828

  11. Sex determination in highly fragmented human DNA by high-resolution melting (HRM) analysis.

    PubMed

    Álvarez-Sandoval, Brenda A; Manzanilla, Linda R; Montiel, Rafael

    2014-01-01

    Sex identification in ancient human remains is a common problem especially if the skeletons are sub-adult, incomplete or damaged. In this paper we propose a new method to identify sex, based on real-time PCR amplification of small fragments (61 and 64 bp) of the third exon within the amelogenin gene covering a 3-bp deletion on the AMELX-allele, followed by a High Resolution Melting analysis (HRM). HRM is based on the melting curves of amplified fragments. The amelogenin gene is located on both chromosomes X and Y, showing dimorphism in length. This molecular tool is rapid, sensitive and reduces the risk of contamination from exogenous genetic material when used for ancient DNA studies. The accuracy of the new method described here has been corroborated by using control samples of known sex and by contrasting our results with those obtained with other methods. Our method has proven to be useful even in heavily degraded samples, where other previously published methods failed. Stochastic problems such as the random allele drop-out phenomenon are expected to occur in a less severe form, due to the smaller fragment size to be amplified. Thus, their negative effect could be easier to overcome by a proper experimental design.

  12. High-resolution DNA melt curve analysis of the clustered, regularly interspaced short-palindromic-repeat locus of Campylobacter jejuni.

    PubMed

    Price, Erin P; Smith, Helen; Huygens, Flavia; Giffard, Philip M

    2007-05-01

    A novel method for genotyping the clustered, regularly interspaced short-palindromic-repeat (CRISPR) locus of Campylobacter jejuni is described. Following real-time PCR, CRISPR products were subjected to high-resolution melt (HRM) analysis, a new technology that allows precise melt profile determination of amplicons. This investigation shows that the CRISPR HRM assay provides a powerful addition to existing C. jejuni genotyping methods and emphasizes the potential of HRM for genotyping short sequence repeats in other species.

  13. Application of High-Resolution DNA Melting for Genotyping in Lepidopteran Non-Model Species: Ostrinia furnacalis (Crambidae)

    PubMed Central

    Li, FengBo; Niu, BaoLong; Huang, YongPing; Meng, ZhiQi

    2012-01-01

    Development of an ideal marker system facilitates a better understanding of the genetic diversity in lepidopteran non-model organisms, which have abundant species, but relatively limited genomic resources. Single nucleotide polymorphisms (SNPs) discovered within single-copy genes have proved to be desired markers, but SNP genotyping by current techniques remain laborious and expensive. High resolution melting (HRM) curve analysis represents a simple, rapid and inexpensive genotyping method that is primarily confined to clinical and diagnostic studies. In this study, we evaluated the potential of HRM analysis for SNP genotyping in the lepidopteran non-model species Ostrinia furnacalis (Crambidae). Small amplicon and unlabeled probe assays were developed for the SNPs, which were identified in 30 females of O. furnacalis from 3 different populations by our direct sequencing. Both assays were then applied to genotype 90 unknown female DNA by prior mixing with known wild-type DNA. The genotyping results were compared with those that were obtained using bi-directional sequencing analysis. Our results demonstrated the efficiency and reliability of the HRM assays. HRM has the potential to provide simple, cost-effective genotyping assays and facilitates genotyping studies in any non-model lepidopteran species of interest. PMID:22253755

  14. Rapid identification of bovine mastitis pathogens by high-resolution melt analysis of 16S rDNA sequences.

    PubMed

    Ajitkumar, Praseeda; Barkema, Herman W; De Buck, Jeroen

    2012-03-23

    Accurate identification of mastitis pathogens is often compromised when using conventional culture-based methods. Here, we report a novel, rapid assay tested for speciation of bacterial mastitis pathogens using high-resolution melt analysis (HRMA) of 16S rDNA sequences. Real-time PCR amplification of 16S rRNA gene fragment, spanning the variable region V5 and V6 was performed with a resulting amplicon of 290bp. First, a library was generated of melt curves of 9 common pathogens that are implicated in bovine mastitis. Six of the isolates, Escherichia coli, Streptococcus agalactiae, Klebsiella pneumoniae, Streptococcus uberis, Staphylococcus aureus and Mycoplasma bovis, were type strains while the other 3, Arcanobacterium pyogenes, Corynebacterium bovis and Streptococcus dysgalactiae, were bovine mastitis field isolates. Four of the type strains, E. coli, S. agalactiae, K. pneumoniae and S. aureus, were found to be of human origin, while the other 3 type strains were isolated from bovine infections. Secondly, the melt curves and corresponding amplicon sequences of A. pyogenes, E. coli, S. agalactiae, S. dysgalactiae, K. pneumoniae, S. uberis and S. aureus were compared with 10 bovine mastitis field isolates of each pathogen. Based on the distinct differences in melt curves and sequences between human and bovine isolates of E. coli and K. pneumoniae, it was deemed necessary to select a set of bovine strains for these pathogens to be used as reference strains in the HRMA. Next, the HRMA was validated by three interpreters analyzing the differential clustering pattern of melt curves of 60 bacterial cultures obtained from mastitis milk samples. The three test interpreters were blinded to the culture and sequencing results of the isolates. Overall accuracy of the validation assay was 95% as there was difficulty in identifying the streptococci due to heterogeneity observed in the PCR amplicons of S. uberis. The present study revealed that broad-range real-time PCR with

  15. Comparative study of IDH1 mutations in gliomas by high resolution melting analysis, immunohistochemistry and direct DNA sequencing.

    PubMed

    Li, Juan; Zhang, Haiyan; Wang, Li; Yang, Chuanhong; Lai, Huangwen; Zhang, Wei; Chen, Xiaodong; Wang, Jie

    2015-09-01

    Patients with glioblastomas with a specific mutation in the isocitrate dehydrogenase 1 (IDH1) gene have a better prognosis than those with gliomas with wild‑type IDH1. IDH1 analysis has become part of the standard diagnostic procedure and a promising tool used for stratification in clinical trials. The present study aimed to compare high resolution melting (HRM) analysis, immunohistochemistry (IHC) and direct DNA sequencing for the detection of IDH mutations in gliomas. Fifty‑one formalin‑fixed paraffin‑embedded tumor samples were selected. For the HRM analysis and direct DNA sequencing, DNA was extracted from the tissues. For IHC, sections were stained with an anti‑IDH1‑R132H specific antibody. The HRM analysis method identified 33 cases of IDH1 gene mutations, and all mutations occurred at the R132H site. There were 33 cases of IDH1 gene mutations found by IHC, which was consistent with that identified using the HRM analysis method. However, only 30 IDH1 samples were confirmed by sequencing, in which mutations occurred at the IDH1 exon 4 R132H site. No mutation was detected in the other three of these 33 cases (two grade II oligodendroglioma and one grade II diffuse astrocytoma) by sequencing, while IHC was positive for IDH1‑R132H. The results showed that the mutation detection rate was not identified to be significantly different (P=0.250) when determined by the HRM analysis method or by direct DNA sequencing, as the concordant rate between the two methods was high (κ=0.866). The HRM analysis method in glioma IDH1 gene mutation detection has advantages of high sensitivity, good repeatability, simple operation and accurate results. It provides a novel method for detecting mutations of the IDH1 gene in paraffin embedded tissue samples of clinical glioma. Related to a small amount of sample, there was no evidence showing that HRM analysis method is superior to IHC. Direct DNA sequencing, HRM analysis and IHC results were consistent; however, HRM and

  16. Detection of plant oil DNA using high resolution melting (HRM) post PCR analysis: a tool for disclosure of olive oil adulteration.

    PubMed

    Vietina, Michelangelo; Agrimonti, Caterina; Marmiroli, Nelson

    2013-12-15

    Extra virgin olive oil is frequently subjected to adulterations with addition of oils obtained from plants other than olive. DNA analysis is a fast and economic tool to identify plant components in oils. Extraction and amplification of DNA by PCR was tested in olives, in milled seeds and in oils, to investigate its use in olive oil traceability. DNA was extracted from different oils made of hazelnut, maize, sunflower, peanut, sesame, soybean, rice and pumpkin. Comparing the DNA melting profiles in reference plant materials and in the oils, it was possible to identify any plant components in oils and mixtures of oils. Real-Time PCR (RT-PCR) platform has been added of the new methodology of high resolution melting (HRM), both were used to analyse olive oils mixed with different percentage of other oils. Results showed HRM a cost effective method for efficient detection of adulterations in olive oils.

  17. High Resolution Melting (HRM) applied to wine authenticity.

    PubMed

    Pereira, Leonor; Gomes, Sónia; Castro, Cláudia; Eiras-Dias, José Eduardo; Brazão, João; Graça, António; Fernandes, José R; Martins-Lopes, Paula

    2017-02-01

    Wine authenticity methods are in increasing demand mainly in Denomination of Origin designations. The DNA-based methodologies are a reliable means of tracking food/wine varietal composition. The main aim of this work was the study of High Resolution Melting (HRM) application as a screening method for must and wine authenticity. Three sample types (leaf, must and wine) were used to validate the three developed HRM assays (Vv1-705bp; Vv2-375bp; and Vv3-119bp). The Vv1 HRM assay was only successful when applied to leaf and must samples. The Vv2 HRM assay successfully amplified all sample types, allowing genotype discrimination based on melting temperature values. The smallest amplicon, Vv3, produced a coincident melting curve shape in all sample types (leaf and wine) with corresponding genotypes. This study presents sensitive, rapid and efficient HRM assays applied for the first time to wine samples suitable for wine authenticity purposes.

  18. High Resolution Melting (HRM) applied to wine authenticity.

    PubMed

    Pereira, Leonor; Gomes, Sónia; Castro, Cláudia; Eiras-Dias, José Eduardo; Brazão, João; Graça, António; Fernandes, José R; Martins-Lopes, Paula

    2017-02-01

    Wine authenticity methods are in increasing demand mainly in Denomination of Origin designations. The DNA-based methodologies are a reliable means of tracking food/wine varietal composition. The main aim of this work was the study of High Resolution Melting (HRM) application as a screening method for must and wine authenticity. Three sample types (leaf, must and wine) were used to validate the three developed HRM assays (Vv1-705bp; Vv2-375bp; and Vv3-119bp). The Vv1 HRM assay was only successful when applied to leaf and must samples. The Vv2 HRM assay successfully amplified all sample types, allowing genotype discrimination based on melting temperature values. The smallest amplicon, Vv3, produced a coincident melting curve shape in all sample types (leaf and wine) with corresponding genotypes. This study presents sensitive, rapid and efficient HRM assays applied for the first time to wine samples suitable for wine authenticity purposes. PMID:27596395

  19. High resolution optical DNA mapping

    NASA Astrophysics Data System (ADS)

    Baday, Murat

    Many types of diseases including cancer and autism are associated with copy-number variations in the genome. Most of these variations could not be identified with existing sequencing and optical DNA mapping methods. We have developed Multi-color Super-resolution technique, with potential for high throughput and low cost, which can allow us to recognize more of these variations. Our technique has made 10--fold improvement in the resolution of optical DNA mapping. Using a 180 kb BAC clone as a model system, we resolved dense patterns from 108 fluorescent labels of two different colors representing two different sequence-motifs. Overall, a detailed DNA map with 100 bp resolution was achieved, which has the potential to reveal detailed information about genetic variance and to facilitate medical diagnosis of genetic disease.

  20. Differentiation of Staphylococcus spp. by high-resolution melting analysis.

    PubMed

    Slany, Michal; Vanerkova, Martina; Nemcova, Eva; Zaloudikova, Barbora; Ruzicka, Filip; Freiberger, Tomas

    2010-12-01

    High-resolution melting analysis (HRMA) is a fast (post-PCR) high-throughput method to scan for sequence variations in a target gene. The aim of this study was to test the potential of HRMA to distinguish particular bacterial species of the Staphylococcus genus even when using a broad-range PCR within the 16S rRNA gene where sequence differences are minimal. Genomic DNA samples isolated from 12 reference staphylococcal strains (Staphylococcus aureus, Staphylococcus capitis, Staphylococcus caprae, Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus hominis, Staphylococcus intermedius, Staphylococcus saprophyticus, Staphylococcus sciuri, Staphylococcus simulans, Staphylococcus warneri, and Staphylococcus xylosus) were subjected to a real-time PCR amplification of the 16S rRNA gene in the presence of fluorescent dye EvaGreen™, followed by HRMA. Melting profiles were used as molecular fingerprints for bacterial species differentiation. HRMA of S. saprophyticus and S. xylosus resulted in undistinguishable profiles because of their identical sequences in the analyzed 16S rRNA region. The remaining reference strains were fully differentiated either directly or via high-resolution plots obtained by heteroduplex formation between coamplified PCR products of the tested staphylococcal strain and phylogenetically unrelated strain.

  1. Hypervariable pili and flagella genes provide suitable new targets for DNA high-resolution melt-based genotyping of dairy Geobacillus spp.

    PubMed

    Chauhan, Kanika; Seale, R Brent; Deeth, Hilton C; Turner, Mark S

    2014-10-01

    Although nonpathogenic in nature, spores of Geobacillus are able to attach to surfaces, germinate, and form biofilms, allowing rapid multiplication and persistence within milk powder processing plants, causing final product contamination, and eventually leading to a loss of revenue in terms of downgraded product quality. As a result, Geobacillus spp. have been found to be common contaminants of milk powder worldwide. Genotyping methods can help in gaining insight into the ecology and transmission of these thermophilic bacteria within and between dairy processing plants. The objective of this study was to use the assembled draft genomes of two Geobacillus spp. to identify and test new hypervariable genotyping targets for differentiating closely related dairy Geobacillus isolates. The two Geobacillus spp. strains obtained from high spore count powders were obtained in 2010 (isolate 7E) and in 1995 (isolate 126) and were previously shown to be of same genotype based on a variable number tandem repeat genotyping method. Significant nucleotide sequence variation was found in genes encoding pili and flagella, which were further investigated as suitable loci for a new high-resolution melt analysis (HRMA)-based genotyping method. Three genes encoding pulG (containing prepilin-type N-terminal cleavage domain), pilT (pili retraction protein), and fliW (flagellar assembly protein) were selected as targets for the new pili/flagella gene (PilFla) HRMA genotyping method. The three-gene-based PilFla-HRMA genotyping method differentiated 35 milk powder Geobacillus spp. isolates into 19 different genotype groups (D = 0.93), which compared favorably to the previous method (which used four variable number tandem repeat loci) that generated 16 different genotype groups (D = 0.90). In conclusion, through comparative genomics of two closely related dairy Geobacillus strains, we have identified new hypervariable regions that prove to be useful targets for highly discriminatory genotyping.

  2. Hypervariable pili and flagella genes provide suitable new targets for DNA high-resolution melt-based genotyping of dairy Geobacillus spp.

    PubMed

    Chauhan, Kanika; Seale, R Brent; Deeth, Hilton C; Turner, Mark S

    2014-10-01

    Although nonpathogenic in nature, spores of Geobacillus are able to attach to surfaces, germinate, and form biofilms, allowing rapid multiplication and persistence within milk powder processing plants, causing final product contamination, and eventually leading to a loss of revenue in terms of downgraded product quality. As a result, Geobacillus spp. have been found to be common contaminants of milk powder worldwide. Genotyping methods can help in gaining insight into the ecology and transmission of these thermophilic bacteria within and between dairy processing plants. The objective of this study was to use the assembled draft genomes of two Geobacillus spp. to identify and test new hypervariable genotyping targets for differentiating closely related dairy Geobacillus isolates. The two Geobacillus spp. strains obtained from high spore count powders were obtained in 2010 (isolate 7E) and in 1995 (isolate 126) and were previously shown to be of same genotype based on a variable number tandem repeat genotyping method. Significant nucleotide sequence variation was found in genes encoding pili and flagella, which were further investigated as suitable loci for a new high-resolution melt analysis (HRMA)-based genotyping method. Three genes encoding pulG (containing prepilin-type N-terminal cleavage domain), pilT (pili retraction protein), and fliW (flagellar assembly protein) were selected as targets for the new pili/flagella gene (PilFla) HRMA genotyping method. The three-gene-based PilFla-HRMA genotyping method differentiated 35 milk powder Geobacillus spp. isolates into 19 different genotype groups (D = 0.93), which compared favorably to the previous method (which used four variable number tandem repeat loci) that generated 16 different genotype groups (D = 0.90). In conclusion, through comparative genomics of two closely related dairy Geobacillus strains, we have identified new hypervariable regions that prove to be useful targets for highly discriminatory genotyping

  3. Automated Microfluidic Platform for Serial Polymerase Chain Reaction and High-Resolution Melting Analysis.

    PubMed

    Cao, Weidong; Bean, Brian; Corey, Scott; Coursey, Johnathan S; Hasson, Kenton C; Inoue, Hiroshi; Isano, Taisuke; Kanderian, Sami; Lane, Ben; Liang, Hongye; Murphy, Brian; Owen, Greg; Shinoda, Nobuhiko; Zeng, Shulin; Knight, Ivor T

    2016-06-01

    We report the development of an automated genetic analyzer for human sample testing based on microfluidic rapid polymerase chain reaction (PCR) with high-resolution melting analysis (HRMA). The integrated DNA microfluidic cartridge was used on a platform designed with a robotic pipettor system that works by sequentially picking up different test solutions from a 384-well plate, mixing them in the tips, and delivering mixed fluids to the DNA cartridge. A novel image feedback flow control system based on a Canon 5D Mark II digital camera was developed for controlling fluid movement through a complex microfluidic branching network without the use of valves. The same camera was used for measuring the high-resolution melt curve of DNA amplicons that were generated in the microfluidic chip. Owing to fast heating and cooling as well as sensitive temperature measurement in the microfluidic channels, the time frame for PCR and HRMA was dramatically reduced from hours to minutes. Preliminary testing results demonstrated that rapid serial PCR and HRMA are possible while still achieving high data quality that is suitable for human sample testing. PMID:25827436

  4. Automated Microfluidic Platform for Serial Polymerase Chain Reaction and High-Resolution Melting Analysis.

    PubMed

    Cao, Weidong; Bean, Brian; Corey, Scott; Coursey, Johnathan S; Hasson, Kenton C; Inoue, Hiroshi; Isano, Taisuke; Kanderian, Sami; Lane, Ben; Liang, Hongye; Murphy, Brian; Owen, Greg; Shinoda, Nobuhiko; Zeng, Shulin; Knight, Ivor T

    2016-06-01

    We report the development of an automated genetic analyzer for human sample testing based on microfluidic rapid polymerase chain reaction (PCR) with high-resolution melting analysis (HRMA). The integrated DNA microfluidic cartridge was used on a platform designed with a robotic pipettor system that works by sequentially picking up different test solutions from a 384-well plate, mixing them in the tips, and delivering mixed fluids to the DNA cartridge. A novel image feedback flow control system based on a Canon 5D Mark II digital camera was developed for controlling fluid movement through a complex microfluidic branching network without the use of valves. The same camera was used for measuring the high-resolution melt curve of DNA amplicons that were generated in the microfluidic chip. Owing to fast heating and cooling as well as sensitive temperature measurement in the microfluidic channels, the time frame for PCR and HRMA was dramatically reduced from hours to minutes. Preliminary testing results demonstrated that rapid serial PCR and HRMA are possible while still achieving high data quality that is suitable for human sample testing.

  5. Rapid Genetic Analysis of X-Linked Chronic Granulomatous Disease by High-Resolution Melting

    PubMed Central

    Hill, Harry R.; Augustine, Nancy H.; Pryor, Robert J.; Reed, Gudrun H.; Bagnato, Joshua D.; Tebo, Anne E.; Bender, Jeffrey M.; Pasi, Brian M.; Chinen, Javier; Hanson, I. Celine; de Boer, Martin; Roos, Dirk; Wittwer, Carl T.

    2010-01-01

    High-resolution melting analysis was applied to X-linked chronic granulomatous disease, a rare disorder resulting from mutations in CYBB. Melting curves of the 13 PCR products bracketing CYBB exons were predicted by Poland's algorithm and compared with observed curves from 96 normal individuals. Primer plates were prepared robotically in batches and dried, greatly simplifying the 3- to 6-hour workflow that included DNA isolation, PCR, melting, and cycle sequencing of any positive products. Small point mutations or insertions/deletions were detected by mixing the hemizygous male DNA with normal male DNA to produce artificial heterozygotes, whereas detection of gross deletions was performed on unmixed samples. Eighteen validation samples and 22 clinical kindreds were analyzed for CYBB mutations. All blinded validation samples were correctly identified. The clinical probands were identified after screening for neutrophil oxidase activity. Nineteen different mutations were found, including seven near intron-exon boundaries predicting splicing defects, five substitutions within exons, three small deletions predicting premature termination, and four gross deletions of multiple exons. Ten novel mutations were found, including (c.) two missense (730T>A, 134T>G), one nonsense (90C>A), four splice site defects (45 + 1G>T, 674 + 4A>G, 1461 + 2delT, and 1462-2A>C), two small deletions (636delT, 1661_1662delCT), and one gross deletion of exons 6 to 8. High-resolution melting can provide timely diagnosis at low cost for effective clinical management of rare, genetic primary immunodeficiency disorders. PMID:20228266

  6. Rapid Diagnosis of Old World Leishmaniasis by High-Resolution Melting Analysis of the 7SL RNA Gene▿ †

    PubMed Central

    Nasereddin, Abedelmajeed; Jaffe, Charles L.

    2010-01-01

    High-resolution melt analysis PCR (HRM PCR) for diagnosis of Old World Leishmania was developed using the 7SL RNA gene. Cutaneous leishmaniasis samples were analyzed. Sensitivity and specificity of HRM PCR were significantly better (P < 0.001) than those of internal transcribed spacer 1 PCR and similar to those of kinetoplast DNA PCR. PMID:20392923

  7. Rapid fingerprinting of methanogenic communities by high-resolution melting analysis.

    PubMed

    Kim, Jaai; Lee, Changsoo

    2014-12-01

    Characterizing microbial community structure using molecular techniques is becoming a popular approach in studies of waste/wastewater treatment processes. A rapid and robust tool to analyze microbial communities is required for efficient process monitoring and control. In this study, a new community fingerprinting method based on high-resolution melting (HRM) analysis was developed and applied to compare methanogenic community structures of five different anaerobic digesters. The new method produced robust community clustering and ordination results comparable to the results from the commonly used denaturing gradient gel electrophoresis (DGGE) performed in parallel. This method transforms melting peak plots (MPs) of community DNA samples generated by HRM analysis to molecular fingerprints and estimates the relationships between the communities based on the fingerprints. The MP-based fingerprinting would provide a good alternative to monitor variations in microbial community structure especially when handling large sample numbers due to its high-throughput capacity and short analysis time.

  8. Automated Classification and Cluster Visualization of Genotypes Derived from High Resolution Melt Curves

    PubMed Central

    Kanderian, Sami; Jiang, Lingxia; Knight, Ivor

    2015-01-01

    Introduction High Resolution Melting (HRM) following PCR has been used to identify DNA genotypes. Fluorescent dyes bounded to double strand DNA lose their fluorescence with increasing temperature, yielding different signatures for different genotypes. Recent software tools have been made available to aid in the distinction of different genotypes, but they are not fully automated, used only for research purposes, or require some level of interaction or confirmation from an analyst. Materials and Methods We describe a fully automated machine learning software algorithm that classifies unknown genotypes. Dynamic melt curves are transformed to multidimensional clusters of points whereby a training set is used to establish the distribution of genotype clusters. Subsequently, probabilistic and statistical methods were used to classify the genotypes of unknown DNA samples on 4 different assays (40 VKORC1, CYP2C9*2, CYP2C9*3 samples in triplicate, and 49 MTHFR c.665C>T samples in triplicate) run on the Roche LC480. Melt curves of each of the triplicates were genotyped separately. Results Automated genotyping called 100% of VKORC1, CYP2C9*3 and MTHFR c.665C>T samples correctly. 97.5% of CYP2C9*2 melt curves were genotyped correctly with the remaining 2.5% given a no call due to the inability to decipher 3 melt curves in close proximity as either homozygous mutant or wild-type with greater than 99.5% posterior probability. Conclusions We demonstrate the ability to fully automate DNA genotyping from HRM curves systematically and accurately without requiring any user interpretation or interaction with the data. Visualization of genotype clusters and quantification of the expected misclassification rate is also available to provide feedback to assay scientists and engineers as changes are made to the assay or instrument. PMID:26605797

  9. Improved protocol for rapid identification of certain spa types using high resolution melting curve analysis.

    PubMed

    Mayerhofer, Benjamin; Stöger, Anna; Pietzka, Ariane T; Fernandez, Haizpea Lasa; Prewein, Bernhard; Sorschag, Sieglinde; Kunert, Renate; Allerberger, Franz; Ruppitsch, Werner

    2015-01-01

    Methicillin-resistant Staphylococcus aureus is one of the most significant pathogens associated with health care. For efficient surveillance, control and outbreak investigation, S. aureus typing is essential. A high resolution melting curve analysis was developed and evaluated for rapid identification of the most frequent spa types found in an Austrian hospital consortium covering 2,435 beds. Among 557 methicillin-resistant Staphylococcus aureus isolates 38 different spa types were identified by sequence analysis of the hypervariable region X of the protein A gene (spa). Identification of spa types through their characteristic high resolution melting curve profiles was considerably improved by double spiking with genomic DNA from spa type t030 and spa type t003 and allowed unambiguous and fast identification of the ten most frequent spa types t001 (58%), t003 (12%), t190 (9%), t041 (5%), t022 (2%), t032 (2%), t008 (2%), t002 (1%), t5712 (1%) and t2203 (1%), representing 93% of all isolates within this hospital consortium. The performance of the assay was evaluated by testing samples with unknown spa types from the daily routine and by testing three different high resolution melting curve analysis real-time PCR instruments. The ten most frequent spa types were identified from all samples and on all instruments with 100% specificity and 100% sensitivity. Compared to classical spa typing by sequence analysis, this gene scanning assay is faster, cheaper and can be performed in a single closed tube assay format. Therefore it is an optimal screening tool to detect the most frequent endemic spa types and to exclude non-endemic spa types within a hospital. PMID:25768007

  10. Improved protocol for rapid identification of certain spa types using high resolution melting curve analysis.

    PubMed

    Mayerhofer, Benjamin; Stöger, Anna; Pietzka, Ariane T; Fernandez, Haizpea Lasa; Prewein, Bernhard; Sorschag, Sieglinde; Kunert, Renate; Allerberger, Franz; Ruppitsch, Werner

    2015-01-01

    Methicillin-resistant Staphylococcus aureus is one of the most significant pathogens associated with health care. For efficient surveillance, control and outbreak investigation, S. aureus typing is essential. A high resolution melting curve analysis was developed and evaluated for rapid identification of the most frequent spa types found in an Austrian hospital consortium covering 2,435 beds. Among 557 methicillin-resistant Staphylococcus aureus isolates 38 different spa types were identified by sequence analysis of the hypervariable region X of the protein A gene (spa). Identification of spa types through their characteristic high resolution melting curve profiles was considerably improved by double spiking with genomic DNA from spa type t030 and spa type t003 and allowed unambiguous and fast identification of the ten most frequent spa types t001 (58%), t003 (12%), t190 (9%), t041 (5%), t022 (2%), t032 (2%), t008 (2%), t002 (1%), t5712 (1%) and t2203 (1%), representing 93% of all isolates within this hospital consortium. The performance of the assay was evaluated by testing samples with unknown spa types from the daily routine and by testing three different high resolution melting curve analysis real-time PCR instruments. The ten most frequent spa types were identified from all samples and on all instruments with 100% specificity and 100% sensitivity. Compared to classical spa typing by sequence analysis, this gene scanning assay is faster, cheaper and can be performed in a single closed tube assay format. Therefore it is an optimal screening tool to detect the most frequent endemic spa types and to exclude non-endemic spa types within a hospital.

  11. High resolution melting analysis as a new approach to discriminate gluten-containing cereals.

    PubMed

    Martín-Fernández, Begoña; Costa, Joana; de-Los-Santos-Álvarez, Noemí; López-Ruiz, Beatriz; Oliveira, M Beatriz P P; Mafra, Isabel

    2016-11-15

    With this work, it is intended to propose a novel approach based on high resolution melting (HRM) analysis to detect wheat and discriminate it from other gluten-containing cereals. The method consisted of a real-time PCR assay targeting the gene encoding for the germ agglutinin isolectin A protein (Tri a 18 allergen), using the fluorescent Evagreen dye combined with HRM analysis. The results enabled wheat differentiation from other phylogenetically related cereals, namely barley, rye and oat with high level of confidence. Additionally, a quantitative real-time PCR approach was proposed, allowing detecting and quantifying wheat down to 20mg/kg in rice flour and 20pg of wheat DNA (∼1.1 DNA copies). Its application was successfully achieved in the analysis of processed foods to verify labelling compliance, being considered as a cost-effective tool for the specific detection of cereals in gluten-free foods. PMID:27283646

  12. High resolution melting analysis as a new approach to discriminate gluten-containing cereals.

    PubMed

    Martín-Fernández, Begoña; Costa, Joana; de-Los-Santos-Álvarez, Noemí; López-Ruiz, Beatriz; Oliveira, M Beatriz P P; Mafra, Isabel

    2016-11-15

    With this work, it is intended to propose a novel approach based on high resolution melting (HRM) analysis to detect wheat and discriminate it from other gluten-containing cereals. The method consisted of a real-time PCR assay targeting the gene encoding for the germ agglutinin isolectin A protein (Tri a 18 allergen), using the fluorescent Evagreen dye combined with HRM analysis. The results enabled wheat differentiation from other phylogenetically related cereals, namely barley, rye and oat with high level of confidence. Additionally, a quantitative real-time PCR approach was proposed, allowing detecting and quantifying wheat down to 20mg/kg in rice flour and 20pg of wheat DNA (∼1.1 DNA copies). Its application was successfully achieved in the analysis of processed foods to verify labelling compliance, being considered as a cost-effective tool for the specific detection of cereals in gluten-free foods.

  13. Protein-DNA binding in high-resolution

    PubMed Central

    Mahony, Shaun; Pugh, B. Franklin

    2015-01-01

    Recent advances in experimental and computational methodologies are enabling ultra-high resolution genome-wide profiles of protein-DNA binding events. For example, the ChIP-exo protocol precisely characterizes protein-DNA crosslinking patterns by combining chromatin immunoprecipitation (ChIP) with 5′ → 3′ exonuclease digestion. Similarly, deeply sequenced chromatin accessibility assays (e.g. DNase-seq and ATACseq) enable the detection of protected footprints at protein-DNA binding sites. With these techniques and others, we have the potential to characterize the individual nucleotides that interact with transcription factors, nucleosomes, RNA polymerases, and other regulatory proteins in a particular cellular context. In this review, we explain the experimental assays and computational analysis methods that enable high-resolution profiling of protein-DNA binding events. We discuss the challenges and opportunities associated with such approaches. PMID:26038153

  14. Identification of forensically important blowfly species (Diptera: Calliphoridae) by high-resolution melting PCR analysis.

    PubMed

    Malewski, Tadeusz; Draber-Mońko, Agnieszka; Pomorski, Jan; Łoś, Marta; Bogdanowicz, Wiesław

    2010-07-01

    We describe here the successful coupling of real-time polymerase chain reaction (PCR) and high-resolution melting (HRM) analysis to rapidly identify 15 forensically important species of blowfly from the family Calliphoridae (Diptera), which occur in Poland. Two short regions (119 and 70 base pairs, respectively) of cytochrome oxidase gene subunit I with sufficient sequence diversity were selected. In the case of lacking taxa (e.g., reference species) these amplicons can be synthesized using sequences deposited in gene banks. The technique utilizes low template DNA concentration and is highly reproducible. The melting profile was not altered up to a 10,000-fold difference in DNA template concentration (ranging from 5 pg to 50 ng). The several HRM runs performed on different specimens from Poland belonging to the same species and on different days resulted in only minor variations in the amplification curves and in melting temperatures of the peaks. Intraspecific variation in a larger scale was tested using synthesized oligonucleotides from cosmopolitan Lucilia illustris originating from Poland, France, Great Britain, India, and USA. As HRM PCR analysis is sensitive to even single base changes, all geographic variants of this species were identified. This technique is also cost-effective and simple, and it may even be used by non-geneticists. A working protocol was ultimately constructed for the purpose of rapid and accurate species identification in most countries in Europe regardless of which stage or which part of a blowfly was collected.

  15. First screening for Brachyspira hampsonii in Swiss pigs applying a new high resolution melting assay.

    PubMed

    Scherrer, Simone; Borgström, Anna; Frei, Daniel; Wittenbrink, Max M

    2016-09-25

    A new High Resolution Melting (HRM) assay was developed for the rapid detection of Brachyspira (B.) hampsonii. B. hampsonii occurs in different European countries, however, until today it has not been encountered in Switzerland. Four B. hampsonii reference strains were used to develop the HRM assay: B. hampsonii clade I ATCC BAA2463 and clade II ATCC BAA2464 strain, as well as two isolated strains P280/1 from the UK and the German isolate 5369-1x/12. A conserved region of the nox gene was used to design B. hampsonii-specific primers. The HRM melting curves for the four reference strains showed reproducible difference graphs with distinct differences between the four strains based on a slight variation between the four amplicon sequences. In addition, DNA from 22 B. hampsonii strains representing four genetic B. hampsonii groups was used to validate the method. Melting temperatures in the interval between 73.1 and 74°C were obtained for all B. hampsonii strains and allow differentiating B. hampsonii from other Brachyspira species. In total 897 Swiss porcine fecal Brachyspira isolates, cultured between 2009 and 2015, were analysed by the HRM protocol. B. hampsonii was not detected among these Swiss Brachyspira isolates. In conclusion, the rapid and low-cost HRM approach allows a sensitive and specific identification of B. hampsonii. PMID:27599925

  16. Limitation of high-resolution melting curve analysis for genotyping simple sequence repeats in sheep.

    PubMed

    Yang, M; Yue, Y J; Guo, T T; Han, J L; Liu, J B; Guo, J; Sun, X P; Feng, R L; Wu, Y Y; Wang, C F; Wang, L P; Yang, B H

    2014-04-08

    Variation in microsatellite or simple sequence repeat (SSR) loci has, until recently, relied heavily on the use of gel-based methods that can be both time consuming and difficult to genotype. Non gel-based systems are therefore important to increase simplicity and improve turn-around time without compromising assay sensitivity and accuracy. In this report, we assessed the latest of the non-gel-based methods, high-resolution melting (HRM) curve analysis. HRM is a technique that monitors exactly the decreasing fluorescence of intercalating dye in the process of dissociation of double-stranded DNA. The measurement immediately follows polymerase chain reaction in a one-step, closed-tube method. Four SSR loci of different complexity in sheep, namely MAF209, MCM140, CB226, and SRCRSP5, were assessed using the LightScanners System with LC Greens PLUS DNA binding dye. In order to improve the accuracy of genotyping, we applied internal oligo nucleotide calibrators while performing HRM. DNA polymorphisms were previously identified using capillary electrophoresis analysis (CE). The result showed that CE detected more genotypes than HRM in the same loci regardless of the level of polymorphism at the SSR loci. We demonstrate current limitations of the HRM method for the analysis of SSR loci.

  17. High-Resolution Melting Curve Analysis for Identification of Pasteurellaceae Species in Experimental Animal Facilities.

    PubMed

    Miller, Manuel; Zorn, Julia; Brielmeier, Markus

    2015-01-01

    Pasteurellaceae are among the most prevalent bacterial pathogens isolated from mice housed in experimental animal facilities. Reliable detection and differentiation of Pasteurellaceae are essential for high-quality health monitoring. In this study, we combined a real-time PCR assay amplifying a variable region in the 16S rRNA sequence with high-resolution melting curve analysis (HRM) to identify and differentiate among the commonly isolated species Pasteurella pneumotropica biotypes "Jawetz" and "Heyl", Actinobacillus muris, and Haemophilus influenzaemurium. We used a set of six reference strains for assay development, with the melting profiles of these strains clearly distinguishable due to DNA sequence variations in the amplicon. For evaluation, we used real-time PCR/HRM to test 25 unknown Pasteurellaceae isolates obtained from an external diagnostic laboratory and found the results to be consistent with those of partial 16S rRNA sequencing. The real-time PCR/HRM method provides a sensitive, rapid, and closed-tube approach for Pasteurellaceae species identification for health monitoring of laboratory mice. PMID:26556281

  18. [Molecular identification of hairy antler by analysis of high resolution melting].

    PubMed

    Chen, Kang; Jiang, Chao; Yuan, Yuan; Huang, Lu-qi; Jin, Yan

    2015-02-01

    High resolution melting (HRM) , an important technology for genotyping and mutation scanning, has broad prospects in the authenticity of traditional Chinese medicine. This paper selected universal CO I primers and used HRM to establish a new method for authenticity of Hairy Antler. PCR was conducted at the annealing temperature of 60 °C and 45 cycles. The range of the DNA template concentration, the primer concentration and the Mg2+ ion concentration were further optimized. The results showed that the Tm values of Cervus nippon were (81.96 ± 0.07), (84.51 ± 0.03) °C and Cervus elaphus was(82.58 ± 0.13), (85.95 ± 0.05) °C with 10-100 mg · L(-1) DNA template, 0.2 µLmol · L(-1) primer, 2.0 mmol · L(-1) Mg2+. This method can authenticate of hairy antler and is simple, fast, high-throughput, visualization. PMID:26137679

  19. Cancer mutation screening: Comparison of high-resolution melt analysis between two platforms.

    PubMed

    Ebili, Henry O; Ilyas, Mohammad

    2015-01-01

    High-resolution melt analysis (HRMA) is a cheap and reliable post-polymerase chain reaction (PCR) cancer mutation screening technique, which is fast gaining clinical relevance. The HRMA capabilities of the LightScanner (Idaho Technology) have been severally studied. However, the ABI 7500 HRM has not been tested against the purpose-built HRM instrument such as the LightScanner. DNA from formalin-fixed, paraffin-embedded gastric cancer, colorectal cancer, and normal tissue as well as from colorectal cancer cell lines were amplified at exons 2, 3, and 4 of KRAS, and at exons 11 and 15 of BRAF in the ABI 7500 fast real-time PCR machine and subjected to melting both on the ABI and on the LightScanner. HRMA data were analysed with the ABI HRM software v2.0.1 and the LightScanner Call-IT 2.5. We tested the ABI 7500 HRM for internal precision, accuracy, sensitivity, and specificity at mutation screening relative to the LightScanner, using crude percentage concordance, kappa statistics, and the area under the receiver operator characteristics (AUROC) curve on SPSS version 19. The results show that the ABI 7500 HRMA has a high internal precision, and excellent concordance, sensitivity, and specificity at mutation screening compared with the LightScanner. However, in contrast to the LightScanner HRM software analysis, the ABI HRM software v.2.0.1, cannot distinguish real from certain pseudovariations in PCR amplicons that are sometimes brought about by the artefacts of the melting process. In conclusion, the ABI HRM has a comparable performance level with the LightScanner, although in certain respects mentioned previously, the LightScanner has an edge over the ABI. PMID:25932046

  20. Cancer mutation screening: Comparison of high-resolution melt analysis between two platforms.

    PubMed

    Ebili, Henry O; Ilyas, Mohammad

    2015-01-01

    High-resolution melt analysis (HRMA) is a cheap and reliable post-polymerase chain reaction (PCR) cancer mutation screening technique, which is fast gaining clinical relevance. The HRMA capabilities of the LightScanner (Idaho Technology) have been severally studied. However, the ABI 7500 HRM has not been tested against the purpose-built HRM instrument such as the LightScanner. DNA from formalin-fixed, paraffin-embedded gastric cancer, colorectal cancer, and normal tissue as well as from colorectal cancer cell lines were amplified at exons 2, 3, and 4 of KRAS, and at exons 11 and 15 of BRAF in the ABI 7500 fast real-time PCR machine and subjected to melting both on the ABI and on the LightScanner. HRMA data were analysed with the ABI HRM software v2.0.1 and the LightScanner Call-IT 2.5. We tested the ABI 7500 HRM for internal precision, accuracy, sensitivity, and specificity at mutation screening relative to the LightScanner, using crude percentage concordance, kappa statistics, and the area under the receiver operator characteristics (AUROC) curve on SPSS version 19. The results show that the ABI 7500 HRMA has a high internal precision, and excellent concordance, sensitivity, and specificity at mutation screening compared with the LightScanner. However, in contrast to the LightScanner HRM software analysis, the ABI HRM software v.2.0.1, cannot distinguish real from certain pseudovariations in PCR amplicons that are sometimes brought about by the artefacts of the melting process. In conclusion, the ABI HRM has a comparable performance level with the LightScanner, although in certain respects mentioned previously, the LightScanner has an edge over the ABI.

  1. High-resolution melt analysis of the minisatellite D1S80: a potential forensic screening tool.

    PubMed

    Pomeroy, Robert S; Balamurugan, Kuppareddi; Wong, Helena; Duncan, George

    2014-11-01

    High-resolution melt (HRM) analysis of the VNTR region of the human D1S80 locus, a 16-bp repeat minisatellite from approximately 400 to over 700 bp in length, was investigated. A Qiagen Rotor-Gene Q using the Type-it PCR HRM kit was used to acquire HRM curves for 14 single, and 16 biallelic, dsDNA samples. The HRM analysis was applicable over a range of DNA concentrations; however the characteristics of the melt curve did depend on the forward and reverse primer ratio. Despite the large amplicon size and the similarities of the repeat sequences, it was possible to discriminate different genotypes. Heterozygotes were clearly different from the homozygous variants and even small differences in the repeat sequence could be differentiated. However, the melt analysis requires a high-resolution system with temperature resolution of 0.02°C or better in order to sort out differences in these large amplicons of near identical GC content (in this case 56%). HRM analysis of amplicons with large repeat sequences can be used as a means of comparing DNA fragments. Examination of multiple sequences can be used to differentiate DNA samples and demonstrate the potential of HRM analysis as a rapid and inexpensive prescreening technique in forensic applications.

  2. High resolution melt analysis to track infections due to ribotype 027 Clostridium difficile.

    PubMed

    Grando, Danilla; Said, Mohamed M; Mayall, Barrie C; Gurtler, Volker

    2012-05-01

    The increased prevalence of hypervirulent ribotype 027 Clostridium difficile requires rapid identification of isolates in order to implement timely infection control strategies. High resolution melt (HRM) analysis of PCR products can identify strain variation amongst genera of bacteria. The intergenic (16S-23S rDNA) spacer region contains sequence regions conserved within genera and other sequence region variables between species within genera. We wished to investigate whether HRM analysis of PCR ribotyping products could identify ribotype 027 C. difficile. Ribotyping was performed on 93 clinical isolates and five control strains and band patterns were analysed using GelCompar II (Applied Maths, USA). Real-time PCR using ribotyping primers was performed and normalised melt curves were generated. The HRM data was then imported into ScreenClust software (QIAGEN) to generate principal component analysis graphs depicting clustered relationships of strains. Ribotyping produced clear PCR bands for 88/98 isolates tested. Dendrograms generated by GelCompar showed a diversity of ribotype patterns amongst these 88 isolates with 18 groups identified with 70% homology. One clinical isolate showed 100% homology with the control 027 strains. ScreenClust analysis of the same 88 HRM results showed clustering of isolates, with 027 strains identifiable as a unique cluster. HRM analysis correctly identified the control 027 stains and the clinical isolate shown to be 027. HRM combined with ScreenClust analysis of real-time PCR products of the 16S-23S rDNA spacer region successfully identified ribotype 027 strains. For infection control purposes this was achieved within 2-3 h of colony isolation.

  3. Nested Machine Learning Facilitates Increased Sequence Content for Large-Scale Automated High Resolution Melt Genotyping

    PubMed Central

    Fraley, Stephanie I.; Athamanolap, Pornpat; Masek, Billie J.; Hardick, Justin; Carroll, Karen C.; Hsieh, Yu-Hsiang; Rothman, Richard E.; Gaydos, Charlotte A.; Wang, Tza-Huei; Yang, Samuel

    2016-01-01

    High Resolution Melt (HRM) is a versatile and rapid post-PCR DNA analysis technique primarily used to differentiate sequence variants among only a few short amplicons. We recently developed a one-vs-one support vector machine algorithm (OVO SVM) that enables the use of HRM for identifying numerous short amplicon sequences automatically and reliably. Herein, we set out to maximize the discriminating power of HRM + SVM for a single genetic locus by testing longer amplicons harboring significantly more sequence information. Using universal primers that amplify the hypervariable bacterial 16 S rRNA gene as a model system, we found that long amplicons yield more complex HRM curve shapes. We developed a novel nested OVO SVM approach to take advantage of this feature and achieved 100% accuracy in the identification of 37 clinically relevant bacteria in Leave-One-Out-Cross-Validation. A subset of organisms were independently tested. Those from pure culture were identified with high accuracy, while those tested directly from clinical blood bottles displayed more technical variability and reduced accuracy. Our findings demonstrate that long sequences can be accurately and automatically profiled by HRM with a novel nested SVM approach and suggest that clinical sample testing is feasible with further optimization. PMID:26778280

  4. Nested Machine Learning Facilitates Increased Sequence Content for Large-Scale Automated High Resolution Melt Genotyping.

    PubMed

    Fraley, Stephanie I; Athamanolap, Pornpat; Masek, Billie J; Hardick, Justin; Carroll, Karen C; Hsieh, Yu-Hsiang; Rothman, Richard E; Gaydos, Charlotte A; Wang, Tza-Huei; Yang, Samuel

    2016-01-01

    High Resolution Melt (HRM) is a versatile and rapid post-PCR DNA analysis technique primarily used to differentiate sequence variants among only a few short amplicons. We recently developed a one-vs-one support vector machine algorithm (OVO SVM) that enables the use of HRM for identifying numerous short amplicon sequences automatically and reliably. Herein, we set out to maximize the discriminating power of HRM + SVM for a single genetic locus by testing longer amplicons harboring significantly more sequence information. Using universal primers that amplify the hypervariable bacterial 16 S rRNA gene as a model system, we found that long amplicons yield more complex HRM curve shapes. We developed a novel nested OVO SVM approach to take advantage of this feature and achieved 100% accuracy in the identification of 37 clinically relevant bacteria in Leave-One-Out-Cross-Validation. A subset of organisms were independently tested. Those from pure culture were identified with high accuracy, while those tested directly from clinical blood bottles displayed more technical variability and reduced accuracy. Our findings demonstrate that long sequences can be accurately and automatically profiled by HRM with a novel nested SVM approach and suggest that clinical sample testing is feasible with further optimization. PMID:26778280

  5. Application of high-resolution melting analysis for differentiation of spoilage yeasts.

    PubMed

    Erdem, Mine; Kesmen, Zülal; Özbekar, Esra; Çetin, Bülent; Yetim, Hasan

    2016-09-01

    A new method based on high resolution melting (HRM) analysis was developed for the differentiation and classification of the yeast species that cause food spoilage. A total 134 strains belonging to 21 different yeast species were examined to evaluate the discriminative power of HRM analysis. Two different highly variable DNA regions on the 26 rRNA gene were targeted to produce the HRM profiles of each strain. HRM-based grouping was compared and confirmed by (GTG)5 rep-PCR fingerprinting analysis. All of the yeast species belonging to the genera Pichia, Candida, Kazachstania, Kluyveromyces, Debaryomyces, Dekkera, Saccharomyces, Torulaspora, Ustilago, and Yarrowia, which were produced as species-specific HRM profiles, allowed discrimination at species and/or strain level. The HRM analysis of both target regions provided successful discrimination that correlated with rep-PCR fingerprinting analysis. Consequently, the HRM analysis has the potential for use in the rapid and accurate classification and typing of yeast species isolated from different foods to determine their sources and routes as well as to prevent contamination. PMID:27572511

  6. Identification of gene mutation in patients with osteogenesis imperfect using high resolution melting analysis

    PubMed Central

    Wang, Jianhai; Ren, Xiuzhi; Bai, Xue; Zhang, Tianke; Wang, Yi; Li, Keqiu; Li, Guang

    2015-01-01

    Osteogenesis imperfecta (OI), a congenital bone disorder, is caused by mutations in COL1A1 and COL1A2 genes, leading to deficiency of type I collagen. The high resolution melting (HRM) analysis has been used for detecting mutations, polymorphisms and epigenetic alteration in double-stranded DNAs. This study was to evaluate the potential application of HRM analysis for identifying gene mutations in patients with OI. This study included four children with OI and their parents and fifty normal people as controls. Blood samples were collected for HRM analysis of PCR-amplified exons and flanking DNA sequences of COL1A1 and COL1A2 genes. Direct gene sequencing was performed to validate HRM-identified gene mutations. As compared to controls, HRM analysis of samples form children with OI showed abnormal melting curves in exons 11 and 33–34 of the COL1A1 gene and exons 19 and 48 of the COL1A2 gene, which indicates the presence of heterozygous mutations in COL1A1 and COL1A2 genes. In addition to two known mutations in the COL1A2 gene, c.982G > A and c.3197G > T, sequencing analysis identified two novel mutations in the COL1A1 gene, c.2321delC and c.768dupC mutations, which function as premature stop codons. These results support future studies of applying HRM analysis as a diagnostic approach for OI. PMID:26307460

  7. Identification of gene mutation in patients with osteogenesis imperfect using high resolution melting analysis.

    PubMed

    Wang, Jianhai; Ren, Xiuzhi; Bai, Xue; Zhang, Tianke; Wang, Yi; Li, Keqiu; Li, Guang

    2015-01-01

    Osteogenesis imperfecta (OI), a congenital bone disorder, is caused by mutations in COL1A1 and COL1A2 genes, leading to deficiency of type I collagen. The high resolution melting (HRM) analysis has been used for detecting mutations, polymorphisms and epigenetic alteration in double-stranded DNAs. This study was to evaluate the potential application of HRM analysis for identifying gene mutations in patients with OI. This study included four children with OI and their parents and fifty normal people as controls. Blood samples were collected for HRM analysis of PCR-amplified exons and flanking DNA sequences of COL1A1 and COL1A2 genes. Direct gene sequencing was performed to validate HRM-identified gene mutations. As compared to controls, HRM analysis of samples form children with OI showed abnormal melting curves in exons 11 and 33-34 of the COL1A1 gene and exons 19 and 48 of the COL1A2 gene, which indicates the presence of heterozygous mutations in COL1A1 and COL1A2 genes. In addition to two known mutations in the COL1A2 gene, c.982G > A and c.3197G > T, sequencing analysis identified two novel mutations in the COL1A1 gene, c.2321delC and c.768dupC mutations, which function as premature stop codons. These results support future studies of applying HRM analysis as a diagnostic approach for OI. PMID:26307460

  8. Rapid real-time PCR and high resolution melt analysis in a self-filling thermoplastic chip.

    PubMed

    Sposito, A; Hoang, V; DeVoe, D L

    2016-09-21

    A microfluidic platform designed for point-of-care PCR-based nucleic acid diagnostics is described. Compared to established microfluidic PCR technologies, the system is unique in its ability to achieve exceptionally rapid PCR amplification in a low cost thermoplastic format, together with high temperature accuracy enabling effective validation of reaction product by high resolution melt analysis performed in the same chamber as PCR. In addition, the system employs capillary pumping for automated loading of sample into the reaction chamber, combined with an integrated hydrophilic valve for precise self-metering of sample volumes into the device. Using the microfluidic system to target a mutation in the G6PC gene, efficient PCR from human genomic DNA template is achieved with cycle times as low as 14 s, full amplification in 8.5 min, and final melt analysis accurately identifying the desired amplicon. PMID:27460504

  9. Detection of Indel Mutations in Drosophila by High-Resolution Melt Analysis (HRMA).

    PubMed

    Housden, Benjamin E; Perrimon, Norbert

    2016-01-01

    Although CRISPR technology allows specific genome alterations to be created with relative ease, detection of these events can be problematic. For example, CRISPR-induced double-strand breaks are often repaired imprecisely to generate unpredictable short indel mutations. Detection of these events requires the use of molecular screening techniques such as endonuclease assays, restriction profiling, or high-resolution melt analysis (HRMA). Here, we provide detailed protocols for HRMA-based mutation screening in Drosophila and analysis of the resulting data using the online tool HRMAnalyzer. PMID:27587781

  10. High resolution melting method to detect single nucleotide polymorphism of VKORC1 and CYP2C9.

    PubMed

    Chen, Chunxia; Li, Siyue; Lu, Xiaojun; Tan, Bin; Huang, Chunyan; Qin, Li

    2014-01-01

    Single nucleotide polymorphisms (SNPs) of VKORC (1173T/C, rs9934438) and CYP2C9 (1075A/C, rs1057910) are major contributory factors on the sensitivity of warfarin in Chinese. Analysis of the two genomic loci could help warfarin treatment individual from bleeding or thrombosis events. An assay with the advantages of simplicity, speed, high sensitivity and low cost for genotyping is calling for clinical laboratories. High resolution method (HRM) meets these callings, but no study with large sample tested its performance in genotyping of rs9934438 and rs1057910. In this study, we identified polymorphisms of rs9934438 and rs1057910 in 255 unrelated Chinese heart valve replacement patients of Han ethnic group from West China Hospital. The two genomic loci were genotyped by HRM using LightCycler® 480 High Resolution Melting Master on LightCycler® 480 Real-Time PCR instruments (Roche Diagnostics), and all amplified PCR products were sent for direct DNA sequencing. The genotyping of rs1057910 between HRM and sequencing showed perfect 100% concordance. While the concordance of rs9934438 between HRM and sequencing was 99.2%. Unexpected mutation interfered genotyping results of HRM when genotyping rs9934438. HRM is a valuable technique for genotype detection of rs9934438 and rs1057910 to assess individual sensitivity to warfarin, where DNA sequencing is added inevitably sometimes.

  11. Primate genotyping via high resolution melt analysis: rapid and reliable identification of color vision status in wild lemurs.

    PubMed

    Jacobs, Rachel L; Spriggs, Amanda N; MacFie, Tammie S; Baden, Andrea L; Irwin, Mitchell T; Wright, Patricia C; Louis, Edward E; Lawler, Richard R; Mundy, Nicholas I; Bradley, Brenda J

    2016-10-01

    Analyses of genetic polymorphisms can aid our understanding of intra- and interspecific variation in primate sociality, ecology, and behavior. Studies of primate opsin genes are prime examples of this, as single nucleotide variants (SNVs) in the X-linked opsin gene underlie variation in color vision. For primate species with polymorphic trichromacy, genotyping opsin SNVs can generally indicate whether individual primates are red-green color-blind (denoted homozygous M or homozygous L) or have full trichromatic color vision (heterozygous ML). Given the potential influence of color vision on behavior and fitness, characterizing the color vision status of study subjects is becoming commonplace for many primate field projects. Such studies traditionally involve a multi-step sequencing-based method that can be costly and time-consuming. Here we present a new reliable, rapid, and relatively inexpensive method for characterizing color vision in primate populations using high resolution melt analysis (HRMA). Using lemurs as a case study, we characterized variation at exons 3 and/or 5 of the X-linked opsin gene for 87 individuals representing nine species. We scored opsin genotypes and color vision status using both traditional sequencing-based methods as well as our novel melting-curve based HRMA protocol. For each species, the melting curves of varying genotypes (homozygous M, homozygous L, heterozygous ML) differed in melting temperature and/or shape. Melting curves for each sample were consistent across replicates, and genotype-specific melting curves were consistent across DNA sources (blood vs. feces). We show that opsin genotypes can be quickly and reliably scored using HRMA once lab-specific reference curves have been developed based on known genotypes. Although the protocol presented here focuses on genotyping lemur opsin loci, we also consider the larger potential for applying this approach to various types of genetic studies of primate populations. PMID:27271303

  12. Primate genotyping via high resolution melt analysis: rapid and reliable identification of color vision status in wild lemurs.

    PubMed

    Jacobs, Rachel L; Spriggs, Amanda N; MacFie, Tammie S; Baden, Andrea L; Irwin, Mitchell T; Wright, Patricia C; Louis, Edward E; Lawler, Richard R; Mundy, Nicholas I; Bradley, Brenda J

    2016-10-01

    Analyses of genetic polymorphisms can aid our understanding of intra- and interspecific variation in primate sociality, ecology, and behavior. Studies of primate opsin genes are prime examples of this, as single nucleotide variants (SNVs) in the X-linked opsin gene underlie variation in color vision. For primate species with polymorphic trichromacy, genotyping opsin SNVs can generally indicate whether individual primates are red-green color-blind (denoted homozygous M or homozygous L) or have full trichromatic color vision (heterozygous ML). Given the potential influence of color vision on behavior and fitness, characterizing the color vision status of study subjects is becoming commonplace for many primate field projects. Such studies traditionally involve a multi-step sequencing-based method that can be costly and time-consuming. Here we present a new reliable, rapid, and relatively inexpensive method for characterizing color vision in primate populations using high resolution melt analysis (HRMA). Using lemurs as a case study, we characterized variation at exons 3 and/or 5 of the X-linked opsin gene for 87 individuals representing nine species. We scored opsin genotypes and color vision status using both traditional sequencing-based methods as well as our novel melting-curve based HRMA protocol. For each species, the melting curves of varying genotypes (homozygous M, homozygous L, heterozygous ML) differed in melting temperature and/or shape. Melting curves for each sample were consistent across replicates, and genotype-specific melting curves were consistent across DNA sources (blood vs. feces). We show that opsin genotypes can be quickly and reliably scored using HRMA once lab-specific reference curves have been developed based on known genotypes. Although the protocol presented here focuses on genotyping lemur opsin loci, we also consider the larger potential for applying this approach to various types of genetic studies of primate populations.

  13. Barcode High Resolution Melting (Bar-HRM) analysis for detection and quantification of PDO "Fava Santorinis" (Lathyrus clymenum) adulterants.

    PubMed

    Ganopoulos, Ioannis; Madesis, Panagiotis; Darzentas, Nikos; Argiriou, Anagnostis; Tsaftaris, Athanasios

    2012-07-15

    Legumes considered as one of the most important crops worldwide. Due to high price as a PDO product, commercial products of "Fava Santorinis" are often subjected to adulterations from other legume products coming from other Lathyrus or Vicia and Pisum species. Using plant DNA barcoding regions (trnL and rpoC) coupled with High Resolution Melting (Bar-HRM) we have developed a method allowing us to detect and authenticate PDO "Fava Santorinis". Bar-HRM proved to be a very sensitive tool able to genotype Lathyrus and its closed relative species and to detect admixtures, being sensitive enough to as low as 1:100 of non-"Fava Santorinis" in "Fava Santorinis" commercial products. In conclusion, Bar-HRM analysis can be a faster, with higher resolution and cost effectiveness alternative method to authenticate PDO "Fava Santorinis" and to quantitatively detect adulterations in "Fava Santorinis" with other relative commercial "Fava" food products. PMID:25683426

  14. Genotyping of classical swine fever virus using high-resolution melt analysis.

    PubMed

    Titov, Ilya; Tsybanov, Sodnom; Malogolovkin, Alexander

    2015-11-01

    Discrimination between different field and vaccine strains of classical swine fever virus (CSFV) is crucial for meaningful disease diagnosis and epidemiological investigation. In this study, a rapid method for differentiating vaccine strains and outbreak CSFV isolates by combined RT-PCR and high-resolution melt (HRM) analysis has been developed. The assay is based on PCR amplification of short fragments from the most variable region of CSFVgene E2, followed by HRM analysis of amplicons. Real-Time PCR/HRM for CSFV detection and differentiation analysis has sensitivity comparable to RT-qPCR and genotyping resolution comparable to E2 nucleotide sequencing. This assay in one step enables rapid and sensitive identification and genotype discrimination of CSFV in field samples, and thus will be valuable for CSF outbreak response and disease control. PMID:26300371

  15. Methods to Increase the Sensitivity of High Resolution Melting Single Nucleotide Polymorphism Genotyping in Malaria.

    PubMed

    Daniels, Rachel; Hamilton, Elizabeth J; Durfee, Katelyn; Ndiaye, Daouda; Wirth, Dyann F; Hartl, Daniel L; Volkman, Sarah K

    2015-01-01

    Despite decades of eradication efforts, malaria remains a global burden. Recent renewed interest in regional elimination and global eradication has been accompanied by increased genomic information about Plasmodium parasite species responsible for malaria, including characteristics of geographical populations as well as variations associated with reduced susceptibility to anti-malarial drugs. One common genetic variation, single-nucleotide polymorphisms (SNPs), offers attractive targets for parasite genotyping. These markers are useful not only for tracking drug resistance markers but also for tracking parasite populations using markers not under drug or other selective pressures. SNP genotyping methods offer the ability to track drug resistance as well as to fingerprint individual parasites for population surveillance, particularly in response to malaria control efforts in regions nearing elimination status. While informative SNPs have been identified that are agnostic to specific genotyping technologies, high-resolution melting (HRM) analysis is particularly suited to field-based studies. Compared to standard fluorescent-probe based methods that require individual SNPs in a single labeled probe and offer at best 10% sensitivity to detect SNPs in samples that contain multiple genomes (polygenomic), HRM offers 2-5% sensitivity. Modifications to HRM, such as blocked probes and asymmetric primer concentrations as well as optimization of amplification annealing temperatures to bias PCR towards amplification of the minor allele, further increase the sensitivity of HRM. While the sensitivity improvement depends on the specific assay, we have increased detection sensitivities to less than 1% of the minor allele. In regions approaching malaria eradication, early detection of emerging or imported drug resistance is essential for prompt response. Similarly, the ability to detect polygenomic infections and differentiate imported parasite types from cryptic local reservoirs

  16. Methods to Increase the Sensitivity of High Resolution Melting Single Nucleotide Polymorphism Genotyping in Malaria

    PubMed Central

    Daniels, Rachel; Hamilton, Elizabeth J.; Durfee, Katelyn; Ndiaye, Daouda; Wirth, Dyann F.; Hartl, Daniel L.; Volkman, Sarah K.

    2015-01-01

    Despite decades of eradication efforts, malaria remains a global burden. Recent renewed interest in regional elimination and global eradication has been accompanied by increased genomic information about Plasmodium parasite species responsible for malaria, including characteristics of geographical populations as well as variations associated with reduced susceptibility to anti-malarial drugs. One common genetic variation, single-nucleotide polymorphisms (SNPs), offers attractive targets for parasite genotyping. These markers are useful not only for tracking drug resistance markers but also for tracking parasite populations using markers not under drug or other selective pressures. SNP genotyping methods offer the ability to track drug resistance as well as to fingerprint individual parasites for population surveillance, particularly in response to malaria control efforts in regions nearing elimination status. While informative SNPs have been identified that are agnostic to specific genotyping technologies, high-resolution melting (HRM) analysis is particularly suited to field-based studies. Compared to standard fluorescent-probe based methods that require individual SNPs in a single labeled probe and offer at best 10% sensitivity to detect SNPs in samples that contain multiple genomes (polygenomic), HRM offers 2-5% sensitivity. Modifications to HRM, such as blocked probes and asymmetric primer concentrations as well as optimization of amplification annealing temperatures to bias PCR towards amplification of the minor allele, further increase the sensitivity of HRM. While the sensitivity improvement depends on the specific assay, we have increased detection sensitivities to less than 1% of the minor allele. In regions approaching malaria eradication, early detection of emerging or imported drug resistance is essential for prompt response. Similarly, the ability to detect polygenomic infections and differentiate imported parasite types from cryptic local reservoirs

  17. Applying high-resolution melting (HRM) technology to identify five commonly used Artemisia species

    PubMed Central

    Song, Ming; Li, Jingjian; Xiong, Chao; Liu, Hexia; Liang, Junsong

    2016-01-01

    Many members of the genus Artemisia are important for medicinal purposes with multiple pharmacological properties. Often, these herbal plants sold on the markets are in processed forms so it is difficult to authenticate. Routine testing and identification of these herbal materials should be performed to ensure that the raw materials used in pharmaceutical products are suitable for their intended use. In this study, five commonly used Artemisia species included Artemisia argyi, Artemisia annua, Artemisia lavandulaefolia, Artemisia indica, and Artemisia atrovirens were analyzed using high resolution melting (HRM) analysis based on the internal transcribed spacer 2 (ITS2) sequences. The melting profiles of the ITS2 amplicons of the five closely related herbal species are clearly separated so that they can be differentiated by HRM method. The method was further applied to authenticate commercial products in powdered. HRM curves of all the commercial samples tested are similar to the botanical species as labeled. These congeneric medicinal products were also clearly separated using the neighbor-joining (NJ) tree. Therefore, HRM method could provide an efficient and reliable authentication system to distinguish these commonly used Artemisia herbal products on the markets and offer a technical reference for medicines quality control in the drug supply chain. PMID:27698485

  18. Exploring crystallization kinetics in natural rhyolitic melts using high resolution CT imagery of spherulites

    NASA Astrophysics Data System (ADS)

    Clow, T. W.; Befus, K. S.; Gardner, J. E.

    2014-12-01

    Little of our understanding of crystallization kinetics has been directly derived from studies of natural samples. We examine crystallization of rhyolitic melts by quantifying spherulite sizes and number densities in obsidian collected from Yellowstone caldera using high-resolution x-ray computed tomography (CT) imagery. Spherulites are spherical to ellipsoidal masses of intergrown alkali feldspar and quartz in a radiating, fibrous structure. They are thought to form in response to relatively rapid crystallization of melt in response to large amounts of undercooling. Recent research using compositional gradients that form outside of spherulites has suggested that they nucleate at 700 to 500 ˚C and their growth slows exponentially until it eventually ceases at ~400 ˚C. By quantifying spherulite textures, and using those temperature constraints, we derive new kinetic information regarding crystallization in natural rhyolitic systems. We find that spherulites range from 0.2 to 12.3 mm in diameter, and are 0.004 to 49.5 mm3 in volume. Such values generate number densities of 70 to 185 spherulites cm-3. Histograms of size display positively skewed distributions indicating small spherulites are far more abundant than larger ones. Those distributions imply nucleation rates change as a function of temperature. At higher temperatures where the melt is undercooled by 400-500 ˚C, nucleation is rare and growth is favored. With decreasing temperature, nucleation rates increase rapidly until cold enough temperatures are reached that diffusion limits crystallization and causes it to cease (undercoolings of ~650 ˚C). Assuming a cooling rate for the host obsidian of 10-5 ˚C s-1, then overall spherulite nucleation rates are 0.01 to 0.03 spherulites cm-3 hour-1.

  19. High Resolution Melting Analysis Targeting hsp70 as a Fast and Efficient Method for the Discrimination of Leishmania Species

    PubMed Central

    Zampieri, Ricardo Andrade; Laranjeira-Silva, Maria Fernanda; Muxel, Sandra Marcia; Stocco de Lima, Ana Carolina; Shaw, Jeffrey Jon; Floeter-Winter, Lucile Maria

    2016-01-01

    Background Protozoan parasites of the genus Leishmania cause a large spectrum of clinical manifestations known as Leishmaniases. These diseases are increasingly important public health problems in many countries both within and outside endemic regions. Thus, an accurate differential diagnosis is extremely relevant for understanding epidemiological profiles and for the administration of the best therapeutic protocol. Methods/Principal Findings Exploring the High Resolution Melting (HRM) dissociation profiles of two amplicons using real time polymerase chain reaction (real-time PCR) targeting heat-shock protein 70 coding gene (hsp70) revealed differences that allowed the discrimination of genomic DNA samples of eight Leishmania species found in the Americas, including Leishmania (Leishmania) infantum chagasi, L. (L.) amazonensis, L. (L.) mexicana, L. (Viannia) lainsoni, L. (V.) braziliensis, L. (V.) guyanensis, L. (V.) naiffi and L. (V.) shawi, and three species found in Eurasia and Africa, including L. (L.) tropica, L. (L.) donovani and L. (L.) major. In addition, we tested DNA samples obtained from standard promastigote culture, naturally infected phlebotomines, experimentally infected mice and clinical human samples to validate the proposed protocol. Conclusions/Significance HRM analysis of hsp70 amplicons is a fast and robust strategy that allowed for the detection and discrimination of all Leishmania species responsible for the Leishmaniases in Brazil and Eurasia/Africa with high sensitivity and accuracy. This method could detect less than one parasite per reaction, even in the presence of host DNA. PMID:26928050

  20. Applications of the method of high resolution melting analysis for diagnosis of Leber's disease and the three primary mutation spectrum of LHON in the Han Chinese population.

    PubMed

    Cui, Guanglin; Ding, Hu; Xu, Yujun; Li, Bin; Wang, Dao Wen

    2013-01-01

    Current screening methods, such as single strand conformational polymorphism (SSCP), denaturing high performance liquid chromatography (dHPLC) and direct DNA sequencing that are used for detecting mutation in Leber's hereditary optic neuropathy (LHON) subjects are time consuming and costly. Here we tested high-resolution melt (HRM) analysis for mtDNA primary mutations in LHON patients. In this study, we applied the high resolution melting (HRM) technology to screen mtDNA primary mutations in 50 LHON patients from their peripheral blood. In order to evaluate the reliability of this technique, we compared the results obtained by HRM and direct mtDNA sequencing. We also investigated the spectrum of three most common mtDNA mutations implicated in LHON in the Han Chinese population. The results showed HRM analysis differentiated all of the mtDNA primary mutations and identified 4 additional mtDNA mutations from 50 patients in the blind study. The prevalence of three primary mutations were 11778G>A (87.9%), 14484T>C (6.5%) and 3460G>A (1.7%) in the Han Chinese population. In conclusion, HRM analysis is a rapid, reliable, and low-cost tool for detecting mtDNA primary mutations and has practical applications in molecular genetics.

  1. Molecular Differentiation of Opisthorchis viverrini and Clonorchis sinensis Eggs by Multiplex Real-Time PCR with High Resolution Melting Analysis

    PubMed Central

    Kaewkong, Worasak; Intapan, Pewpan M.; Sanpool, Oranuch; Janwan, Penchom; Thanchomnang, Tongjit; Laummaunwai, Porntip; Lulitanond, Viraphong; Doanh, Pham Ngoc

    2013-01-01

    Opisthorchis viverrini and Clonorchis sinensis are parasites known to be carcinogenic and causative agents of cholangiocarcinoma in Asia. The standard method for diagnosis for those parasite infections is stool examination to detect parasite eggs. However, the method has low sensitivity, and eggs of O. viverrini and C. sinensis are difficult to distinguish from each other and from those of some other trematodes. Here, we report a multiplex real-time PCR coupled with high resolution melting (HRM) analysis for the differentiation of O. viverrini and C. sinensis eggs in fecal samples. Using 2 pairs of species-specific primers, DNA sequences from a portion of the mitochondrial NADH dehydrogenase subunit 2 (nad 2) gene, were amplified to generate 209 and 165 bp products for O. viverrini and C. sinensis, respectively. The distinct characteristics of HRM patterns were analyzed, and the melting temperatures peaked at 82.4±0.09℃ and 85.9±0.08℃ for O. viverrini and C. sinensis, respectively. This technique was able to detect as few as 1 egg of O. viverrini and 2 eggs of C. sinensis in a 150 mg fecal sample, which is equivalent to 7 and 14 eggs per gram of feces, respectively. The method is species-specific, rapid, simple, and does not require fluorescent probes or post-PCR processing for discrimination of eggs of the 2 species. It offers a new tool for differentiation and detection of Asian liver fluke infections in stool specimens. PMID:24516275

  2. Rapid detection of fungal pathogens in bronchoalveolar lavage samples using panfungal PCR combined with high resolution melting analysis.

    PubMed

    Bezdicek, Matej; Lengerova, Martina; Ricna, Dita; Weinbergerova, Barbora; Kocmanova, Iva; Volfova, Pavlina; Drgona, Lubos; Poczova, Miroslava; Mayer, Jiri; Racil, Zdenek

    2016-10-01

    Despite advances in the treatment of invasive fungal diseases (IFD), mortality rates remain high. Moreover, due to the expanding spectrum of causative agents, fast and accurate pathogen identification is necessary. We designed a panfungal polymerase chain reaction (PCR), which targets the highly variable ITS2 region of rDNA genes and uses high resolution melting analysis (HRM) for subsequent species identification. The sensitivity and specificity of this method was tested on a broad spectrum of the most clinically important fungal pathogens including Aspergillus spp., Candida spp. and mucormycetes. Despite the fact that fluid from bronchoalveolar lavage (BAL) is one of the most frequently tested materials there is a lack of literature sources aimed at panfungal PCR as an IFD diagnostic tool from BAL samples. The applicability of this method in routine practice was evaluated on 104 BAL samples from immunocompromised patients. Due to high ITS region variability, we obtained divergent melting peaks for different fungal species. Thirteen out of 18 patients with proven or probable IFD were positive. Therefore, the sensitivity, specificity, positive predictive value and negative predictive value of our method were 67%, 100%, 100%, and 94%, respectively. In our assay, fungal pathogens identification is based on HRM, therefore omitting the expensive and time consuming sequencing step. With the high specificity, positive and negative predictive values, short time needed to obtain a result, and low price, the presented assay is intended to be used as a quick screening method for patients at risk of IFD. PMID:27161789

  3. Differentiation of Campylobacter jejuni and Campylobacter coli Using Multiplex-PCR and High Resolution Melt Curve Analysis.

    PubMed

    Banowary, Banya; Dang, Van Tuan; Sarker, Subir; Connolly, Joanne H; Chenu, Jeremy; Groves, Peter; Ayton, Michelle; Raidal, Shane; Devi, Aruna; Vanniasinkam, Thiru; Ghorashi, Seyed A

    2015-01-01

    Campylobacter spp. are important causes of bacterial gastroenteritis in humans in developed countries. Among Campylobacter spp. Campylobacter jejuni (C. jejuni) and C. coli are the most common causes of human infection. In this study, a multiplex PCR (mPCR) and high resolution melt (HRM) curve analysis were optimized for simultaneous detection and differentiation of C. jejuni and C. coli isolates. A segment of the hippuricase gene (hipO) of C. jejuni and putative aspartokinase (asp) gene of C. coli were amplified from 26 Campylobacter isolates and amplicons were subjected to HRM curve analysis. The mPCR-HRM was able to differentiate between C. jejuni and C. coli species. All DNA amplicons generated by mPCR were sequenced. Analysis of the nucleotide sequences from each isolate revealed that the HRM curves were correlated with the nucleotide sequences of the amplicons. Minor variation in melting point temperatures of C. coli or C. jejuni isolates was also observed and enabled some intraspecies differentiation between C. coli and/or C. jejuni isolates. The potential of PCR-HRM curve analysis for the detection and speciation of Campylobacter in additional human clinical specimens and chicken swab samples was also confirmed. The sensitivity and specificity of the test were found to be 100% and 92%, respectively. The results indicated that mPCR followed by HRM curve analysis provides a rapid (8 hours) technique for differentiation between C. jejuni and C. coli isolates.

  4. High resolution DNA content measurements of mammalian sperm

    SciTech Connect

    Pinkel, D.; Lake, S.; Gledhill, B.L.; Van Dilla, M.A.; Stephenson, D.; Watchmaker, G.

    1982-01-01

    The high condensation and flat shape of the mammalian sperm nucleus present unique difficulties to flow cytometric measurement of DNA content. Chromatin compactness makes quantitative fluorescent staining for DNA difficult and causes a high index of refraction. The refractive index makes optical measurements sensitive to sperm head orientation. We demonstrate that the optical problems can be overcome using the commercial ICP22 epiillumination flow cytometer (Ortho Instruments, Westwood, MA) or a specially built cell orientating flow cytometer (OFCM). The design and operation of the OFCM are described. Measurements of the angular dependence of fluorescence from acriflavine stained rabbit sperm show that it is capable of orienting flat sperm with a tolerance of +-7/sup 0/. Differences in the angular dependence for the similarly shaped bull and rabbit sperm allow discrimination of these cells. We show that DNA staining with 4-6 diamidino-2-phenylindole (DAPI) or an ethidium bromide mithramycin combination allows resolution of the X and Y populations in mouse sperm. They have also been successful with sperm from the bull, ram, rabbit, and boar. Reliable results with human sperm are not obtained. The accuracy of the staining and measurement techniques are verified by the correct determination of the relative content of these two populations in sperm from normal mice and those with the Cattanach (7 to X) translocation. Among the potential uses of these techniques are measurement of DNA content errors induced in sperm due to mutagen exposure, and assessment of the fractions of X and Y sperm in semen that may have one population artifically enriched.

  5. Development of High Resolution Melting Analysis for the Diagnosis of Human Malaria

    PubMed Central

    Chua, Kek Heng; Lim, Siew Chee; Ng, Ching Ching; Lee, Ping Chin; Lim, Yvonne Ai Lian; Lau, Tze Pheng; Chai, Hwa Chia

    2015-01-01

    Molecular detection has overcome limitations of microscopic examination by providing greater sensitivity and specificity in Plasmodium species detection. The objective of the present study was to develop a quantitative real-time polymerase chain reaction coupled with high-resolution melting (qRT-PCR-HRM) assay for rapid, accurate and simultaneous detection of all five human Plasmodium spp. A pair of primers targeted the 18S SSU rRNA gene of the Plasmodium spp. was designed for qRT-PCR-HRM assay development. Analytical sensitivity and specificity of the assay were evaluated. Samples collected from 229 malaria suspected patients recruited from Sabah, Malaysia were screened using the assay and results were compared with data obtained using PlasmoNexTM, a hexaplex PCR system. The qRT-PCR-HRM assay was able to detect and discriminate the five Plasmodium spp. with lowest detection limits of 1–100 copy numbers without nonspecific amplifications. The detection of Plasmodium spp. in clinical samples using this assay also achieved 100% concordance with that obtained using PlasmoNexTM. This indicated that the diagnostic sensitivity and specificity of this assay in Plasmodium spp. detection is comparable with those of PlasmoNexTM. The qRT-PCR-HRM assay is simple, produces results in two hours and enables high-throughput screening. Thus, it is an alternative method for rapid and accurate malaria diagnosis. PMID:26507008

  6. High Resolution Melting Analysis: A Rapid and Accurate Method to Detect CALR Mutations

    PubMed Central

    Moreno, Melania; Torres, Laura; Santana-Lopez, Gonzalo; Rodriguez-Medina, Carlos; Perera, María; Bellosillo, Beatriz; de la Iglesia, Silvia; Molero, Teresa; Gomez-Casares, Maria Teresa

    2014-01-01

    Background The recent discovery of CALR mutations in essential thrombocythemia (ET) and primary myelofibrosis (PMF) patients without JAK2/MPL mutations has emerged as a relevant finding for the molecular diagnosis of these myeloproliferative neoplasms (MPN). We tested the feasibility of high-resolution melting (HRM) as a screening method for rapid detection of CALR mutations. Methods CALR was studied in wild-type JAK2/MPL patients including 34 ET, 21 persistent thrombocytosis suggestive of MPN and 98 suspected secondary thrombocytosis. CALR mutation analysis was performed through HRM and Sanger sequencing. We compared clinical features of CALR-mutated versus 45 JAK2/MPL-mutated subjects in ET. Results Nineteen samples showed distinct HRM patterns from wild-type. Of them, 18 were mutations and one a polymorphism as confirmed by direct sequencing. CALR mutations were present in 44% of ET (15/34), 14% of persistent thrombocytosis suggestive of MPN (3/21) and none of the secondary thrombocytosis (0/98). Of the 18 mutants, 9 were 52 bp deletions, 8 were 5 bp insertions and other was a complex mutation with insertion/deletion. No mutations were found after sequencing analysis of 45 samples displaying wild-type HRM curves. HRM technique was reproducible, no false positive or negative were detected and the limit of detection was of 3%. Conclusions This study establishes a sensitive, reliable and rapid HRM method to screen for the presence of CALR mutations. PMID:25068507

  7. High Resolution Melting Analysis for JAK2 Exon 14 and Exon 12 Mutations

    PubMed Central

    Rapado, Inmaculada; Grande, Silvia; Albizua, Enriqueta; Ayala, Rosa; Hernández, José-Angel; Gallardo, Miguel; Gilsanz, Florinda; Martinez-Lopez, Joaquin

    2009-01-01

    JAK2 mutations are important criteria for the diagnosis of Philadelphia chromosome-negative myeloproliferative neoplasms. We aimed to assess JAK2 exon 14 and exon 12 mutations by high-resolution melting (HRM) analysis, which allows variation screening. The exon 14 analysis included 163 patients with polycythemia vera, secondary erythrocytoses, essential thrombocythemia, or secondary thrombocytoses, and 126 healthy subjects. The study of exon 12 included 40 JAK2 V617F-negative patients (nine of which had polycythemia vera, and 31 with splanchnic vein thrombosis) and 30 healthy subjects. HRM analyses of JAK2 exons 14 and 12 gave analytical sensitivities near 1% and both intra- and interday coefficients of variation of less than 1%. For HRM analysis of JAK2 exon 14 in polycythemia vera and essential thrombocythemia, clinical sensitivities were 93.5% and 67.9%, clinical specificities were 98.8% and 97.0%, positive predictive values were 93.5% and 79.2%, and negative predictive values were 98.8% and 94.6, respectively. Correlations were observed between the results from HRM and three commonly used analytical methods. The JAK2 exon 12 HRM results agreed completely with those from sequencing analysis, and the three mutations in exon 12 were detected by both methods. Hence, HRM analysis of exons 14 and 12 in JAK2 shows better diagnostic values than three other routinely used methods against which it was compared. In addition, HRM analysis has the advantage of detecting unknown mutations. PMID:19225136

  8. Rapid Detection and Differentiation of Clonorchis sinensis and Opisthorchis viverrini Using Real-Time PCR and High Resolution Melting Analysis

    PubMed Central

    Cai, Xian-Quan; Yu, Hai-Qiong; Li, Rong; Yue, Qiao-Yun; Liu, Guo-Hua; Bai, Jian-Shan; Deng, Yan; Qiu, De-Yi; Zhu, Xing-Quan

    2014-01-01

    Clonorchis sinensis and Opisthorchis viverrini are both important fish-borne pathogens, causing serious public health problem in Asia. The present study developed an assay integrating real-time PCR and high resolution melting (HRM) analysis for the specific detection and rapid identification of C. sinensis and O. viverrini. Primers targeting COX1 gene were highly specific for these liver flukes, as evidenced by the negative amplification of closely related trematodes. Assays using genomic DNA extracted from the two flukes yielded specific amplification and their identity was confirmed by sequencing, having the accuracy of 100% in reference to conventional methods. The assay was proved to be highly sensitive with a detection limit below 1 pg of purified genomic DNA, 5 EPG, or 1 metacercaria of C. sinensis. Moreover, C. sinensis and O. viverrini were able to be differentiated by their HRM profiles. The method can reduce labor of microscopic examination and the contamination of agarose electrophoresis. Moreover, it can differentiate these two flukes which are difficult to be distinguished using other methods. The established method provides an alternative tool for rapid, simple, and duplex detection of C. sinensis and O. viverrini. PMID:25386621

  9. Diagnostic accuracy of high resolution melting analysis for detection of KRAS mutations: a systematic review and meta-analysis.

    PubMed

    Liu, Yue-Ping; Wu, Hai-Yan; Yang, Xiang; Xu, Han-Qing; Chen, Dong; Huang, Qing; Fu, Wei-Ling

    2014-01-01

    Increasing evidence points to a negative correlation between KRAS mutations and patients' responses to anti-EGFR monoclonal antibody treatment. Therefore, patients must undergo KRAS mutation detection to be eligible for treatment. High resolution melting analysis (HRM) is gaining increasing attention in KRAS mutation detection. However, its accuracy has not been systematically evaluated. We conducted a meta-analysis of published articles, involving 13 articles with 1,520 samples, to assess its diagnostic accuracy compared with DNA sequencing. The quality of included articles was assessed using the revised Quality Assessment for Studies of Diagnostic Accuracy (QUADAS-2) tools. Random effects models were applied to analyze the performance of pooled characteristics. The overall sensitivity and specificity of HRM were 0.99 (95% confidence interval [CI]: 0.98-1.00) and 0.96 (95%CI: 0.94-0.97), respectively. The area under the summary receiver operating characteristic curve was 0.996. High sensitivity and specificity, less labor, rapid turn-around and the closed-tube format of HRM make it an attractive choice for rapid detection of KRAS mutations in clinical practice. The burden of DNA sequencing can be reduced dramatically by the implementation of HRM, but positive results still need to be sequenced for diagnostic confirmation. PMID:25515911

  10. High resolution melting curve analysis as a new tool for rapid identification of canine parvovirus type 2 strains.

    PubMed

    Bingga, Gali; Liu, Zhicheng; Zhang, Jianfeng; Zhu, Yujun; Lin, Lifeng; Ding, Shuangyang; Guo, Pengju

    2014-01-01

    A high resolution melting (HRM) curve method was developed to identify canine parvovirus type 2 (CPV-2) strains by nested PCR. Two sets of primers, CPV-426F/426R and CPV-87R/87F, were designed that amplified a 52 bp and 53 bp product from the viral VP2 capsid gene. The region amplified by CPV-426F/426R included the A4062G and T4064A mutations in CPV-2a, CPV-2b and CPV-2c. The region amplified by CPV-87F/87R included the A3045T mutation in the vaccine strains of CPV-2 and CPV-2a, CPV-2b and CPV-2c. Faecal samples were obtained from 30 dogs that were CPV antigen-positive. The DNA was isolated from the faecal samples and PCR-amplified using the two sets of primers, and genotyped by HRM curve analysis. The PCR-HRM assay was able to distinguish single nucleotide polymorphisms between CPV-2a, CPV-2b and CPV-2c using CPV-426F/426R. CPV-2a was distinguished from CPV-2b and CPV-2c by differences in the melting temperature. CPV-2b and CPV-2c could be distinguished based on the shape of the melting curve after generating heteroduplexes using a CPV-2b reference sample. The vaccine strains of CPV-2 were identified using CPV-87F/87R. Conventional methods for genotyping CPV strains are labor intensive, expensive or time consuming; the present PCR-based HRM assay might be an attractive alternative.

  11. High-resolution melt analysis for species identification of coagulase-negative staphylococci derived from bovine milk.

    PubMed

    Ajitkumar, Praseeda; Barkema, Herman W; Zadoks, Ruth N; Morck, Douglas W; van der Meer, Frank J U M; De Buck, Jeroen

    2013-03-01

    Coagulase-negative staphylococci (CNS) are the most frequently isolated pathogens isolated from bovine milk. In this study, we report a rapid assay for species identification of CNS using high-resolution melt analysis (HRMA) of 16S rDNA sequences. Real-time polymerase chain reaction amplification of 16S rRNA gene fragment, spanning the variable region V1 and V2, was performed with a resulting amplicon of 215 bp. A library of distinct melt curves of reference strains of 13 common CNS species was created using HRMA. Sequencing of 16S rRNA and rpoB genes, and, when needed, tuf gene, of 100 CNS isolates obtained from Canadian Bovine Mastitis Research Network was done to determine their species identity, allowing for subsequent evaluation of the performance of HRMA for field isolates of bovine CNS. A combination of HRMA and sequencing revealed that Staphylococcus chromogenes, S. xylosus, S. simulans, and S. sciuri had multiple genotypes, complicating their resolution by HRMA. As the 3 genotypes of S. chromogenes had distinct melt curves, the 3 distinct genotypes were employed as reference strains in a blinded trial of 156 CNS isolates to identify S. chromogenes. HRMA correctly identified all S. chromogenes isolates which were later confirmed by sequencing. Staphylococcus chromogenes (68%) was most frequently found among the CNS isolates, followed by S. haemolyticus (10%) and S. xylosus (6%). The present study revealed that HRMA of 16S rRNA gene (V1-V2) could be used as a rapid, efficient, low-cost, and minimally cumbersome technique for S. chromogenes identification, the most common CNS derived from bovine milk. PMID:23273337

  12. Analysis of bla(SHV) codon 238 and 240 allele mixtures using Sybr green high-resolution melting analysis.

    PubMed

    Andersson, Patiyan; Harris, Tegan; Tong, Steven Y C; Giffard, Philip M

    2009-06-01

    Klebsiella pneumoniae isolates frequently contain complex mixtures of bla(SHV) alleles. A high-resolution melting-based method for interrogating the extended-spectrum activity conferring codon 238 and 240 polymorphisms was developed. This detects minority extended-spectrum beta-lactamase-encoding alleles, allows estimation of allele ratios, and discriminates between single and double mutants.

  13. Validation of a blood group genotyping method based on high-resolution melting curve analysis.

    PubMed

    Gong, Tianxiang; Hong, Ying; Wang, Naihong; Fu, Xuemei; Zhou, Changhua

    2014-01-01

    The detection of polymorphism is the basis of blood group genotyping and phenotype prediction. Genotyping may be useful to determine blood groups when serologic results are unclear. The development and application of different methods for blood group genotyping may be needed as a substitute for blood group typing. The purpose of this study is to establish an approach for blood group genotyping based on a melting curve analysis of real-time polymerase chain reaction (PCR). Using DNA extracted from whole blood, we developed and validated a DNA typing method for detecting DO*01/DO*02, DO*01/DI*02, LU*01/LU*02, and GYPB*03/GYBP*04 alleles using a melting curve analysis. All assays were confirmed with a commercial reagent containing sequence-specific primers (PCR-SSP), and a cohort of the samples was confirmed with sequencing. Results for all blood groups were within the range of specificity and assay variability. Genotypes of 300 blood donors were fully consistent with PCR-SSP data. The obtained genotype distribution is in complete concordance with existing data for the Chinese population. There are several advantages for this approach of blood group genotyping: lower contamination rates with PCR products in this laboratory, ease of performance, automation potential, and rapid cycling time.

  14. Quantitative high-resolution melting PCR analysis for monitoring of fermentation microbiota in sourdough.

    PubMed

    Lin, Xiaoxi B; Gänzle, Michael G

    2014-09-01

    Current methods of monitoring the microbial ecology of food fermentation system are generally labor intensive and/or time consuming. This study developed two methods based on high-resolution melting curves (HRM) to monitor sourdough microbiota during fermentation and to investigate the effect of cereal substrate on microbial ecology. A strain cocktail of Lactobacillus fermentum FUA3165, Lactobacillus plantarum FUA3309, Lactobacillus paracasei FUA3166 and Lactobacillus reuteri FUA3168 was used to ferment red (Town and PAN8609) and white (commercial and Segaolane) sorghum sourdough, and wheat sourdough. The microbial composition of sourdoughs was determined by plate count and HRM-qPCR to differentiate at the species level. The resistance of each species to sorghum phenolic extract was measured. There was no difference in microbial composition among the four sorghum sourdoughs, with L. fermentum FUA3165 in all sourdoughs. The competiveness of the strains in sorghum sourdoughs corresponded to their resistance to sorghum phenolic extract. In a second experiment, five L. reuteri strains, the human-lineage strains FUA3400 and 3401 isolated from wheat sourdough, the rodent-lineage strain FUA5448 isolated from rye sourdough and the sorghum isolates FUA3168 and 3324, were used to ferment wheat, rye and sorghum sourdoughs. The microbial composition of sourdoughs was determined by plate counts and HRM-qPCR to different L. reuteri strains representing different host-adapted lineages. No difference among different substrates was observed; indicating cereal type had no selective effect on sourdough microbial ecology. In conclusion, HRM-qPCR assays were established as rapid and highly specific tool for monitoring of sourdough microbiota. The ability to distinguish highly similar microbes in samples containing only few genotypes makes HRM-qPCR suitable for quality control in other food fermentation systems. The presence of phenolic compounds in sorghum sourdough favored organisms

  15. Evaluation of High Resolution Melting for MTHFR C677T Genotyping in Congenital Heart Disease

    PubMed Central

    Yue, Shuying; Zhang, Kun; Wang, Hui; Dong, Rui; Yang, Xiaomeng; Liu, Yi; Ma, Yanhui

    2016-01-01

    Background High resolution melting (HRM) is a simple, flexible and low-cost mutation screening technique. The methylenetetrahydrofolate reductase (MTHFR) gene encoding a critical enzyme, potentially affects susceptibility to some congenital defects like congenital heart disease (CHD). We evaluate the performance of HRM for genotyping of the MTHFR gene C677T locus in CHD cases and healthy controls of Chinese Han population. Methods A total of 315 blood samples from 147 CHD patients (male72, female 75) and 168 healthy controls (male 92, female 76) were enrolled in the study. HRM was utilized to genotype MTHFR C677T locus of all the samples. The results were compared to that of PCR-RFLP and Sanger sequencing. The association of the MTHFR C677T genotypes and the risk of CHD was analyzed using odds ratio with their 95% confidence interval (CIs) from unconditional logistic regression. Results All the samples were successfully genotyped by HRM within 1 hour and 30 minutes while at least 6 hours were needed for PCR-RFLP and sequencing. The genotypes of MTHFR C677T CC, CT, and TT were 9.52%, 49.66%, and 40.82% in CHD group but 29.17%, 50% and 20.83% in control group, which were identical using both methods of HRM and PCR-RFLP, demonstrating the sensitivity and specificity of HRM were all 100%. Conclusion MTHFR C677T is a potential risk factor for CHD in our local residents of Shandong province in China. HRM is a fast, sensitive, specific and reliable method for clinical application of genotyping. PMID:26990189

  16. Rapid and high throughput molecular identification of diverse mosquito species by high resolution melting analysis

    PubMed Central

    Ajamma, Yvonne Ukamaka; Mararo, Enock; Omondi, David; Onchuru, Thomas; Muigai, Anne W. T.; Masiga, Daniel; Villinger, Jandouwe

    2016-01-01

    Mosquitoes are a diverse group of invertebrates, with members that are among the most important vectors of diseases. The correct identification of mosquitoes is paramount to the control of the diseases that they transmit. However, morphological techniques depend on the quality of the specimen and often unavailable taxonomic expertise, which may still not be able to distinguish mosquitoes among species complexes (sibling and cryptic species). High resolution melting (HRM) analyses, a closed-tube, post-polymerase chain reaction (PCR) method used to identify variations in nucleic acid sequences, has been used to differentiate species within the Anopheles gambiae and Culex pipiens complexes. We validated the use of PCR-HRM analyses to differentiate species within Anopheles and within each of six genera of culicine mosquitoes, comparing primers targeting cytochrome b ( cyt b), NADH dehydrogenase subunit 1 (ND1), intergenic spacer region (IGS) and cytochrome c oxidase subunit 1 ( COI) gene regions. HRM analyses of amplicons from all the six primer pairs successfully differentiated two or more mosquito species within one or more genera ( Aedes ( Ae. vittatus from Ae. metallicus), Culex ( Cx. tenagius from Cx. antennatus, Cx. neavei from Cx. duttoni, cryptic Cx. pipiens species), Anopheles ( An. gambiae s.s. from An. arabiensis) and Mansonia ( Ma. africana from Ma. uniformis)) based on their HRM profiles. However, PCR-HRM could not distinguish between species within Aedeomyia ( Ad. africana and Ad. furfurea), Mimomyia ( Mi. hispida and Mi. splendens) and Coquillettidia ( Cq. aurites, Cq. chrysosoma, Cq. fuscopennata, Cq. metallica, Cq. microannulatus, Cq. pseudoconopas and Cq. versicolor) genera using any of the primers. The IGS and COI barcode region primers gave the best and most definitive separation of mosquito species among anopheline and culicine mosquito genera, respectively, while the other markers may serve to confirm identifications of closely related sub

  17. Quantitative high-resolution melting PCR analysis for monitoring of fermentation microbiota in sourdough.

    PubMed

    Lin, Xiaoxi B; Gänzle, Michael G

    2014-09-01

    Current methods of monitoring the microbial ecology of food fermentation system are generally labor intensive and/or time consuming. This study developed two methods based on high-resolution melting curves (HRM) to monitor sourdough microbiota during fermentation and to investigate the effect of cereal substrate on microbial ecology. A strain cocktail of Lactobacillus fermentum FUA3165, Lactobacillus plantarum FUA3309, Lactobacillus paracasei FUA3166 and Lactobacillus reuteri FUA3168 was used to ferment red (Town and PAN8609) and white (commercial and Segaolane) sorghum sourdough, and wheat sourdough. The microbial composition of sourdoughs was determined by plate count and HRM-qPCR to differentiate at the species level. The resistance of each species to sorghum phenolic extract was measured. There was no difference in microbial composition among the four sorghum sourdoughs, with L. fermentum FUA3165 in all sourdoughs. The competiveness of the strains in sorghum sourdoughs corresponded to their resistance to sorghum phenolic extract. In a second experiment, five L. reuteri strains, the human-lineage strains FUA3400 and 3401 isolated from wheat sourdough, the rodent-lineage strain FUA5448 isolated from rye sourdough and the sorghum isolates FUA3168 and 3324, were used to ferment wheat, rye and sorghum sourdoughs. The microbial composition of sourdoughs was determined by plate counts and HRM-qPCR to different L. reuteri strains representing different host-adapted lineages. No difference among different substrates was observed; indicating cereal type had no selective effect on sourdough microbial ecology. In conclusion, HRM-qPCR assays were established as rapid and highly specific tool for monitoring of sourdough microbiota. The ability to distinguish highly similar microbes in samples containing only few genotypes makes HRM-qPCR suitable for quality control in other food fermentation systems. The presence of phenolic compounds in sorghum sourdough favored organisms

  18. Rapid and high throughput molecular identification of diverse mosquito species by high resolution melting analysis

    PubMed Central

    Ajamma, Yvonne Ukamaka; Mararo, Enock; Omondi, David; Onchuru, Thomas; Muigai, Anne W. T.; Masiga, Daniel; Villinger, Jandouwe

    2016-01-01

    Mosquitoes are a diverse group of invertebrates, with members that are among the most important vectors of diseases. The correct identification of mosquitoes is paramount to the control of the diseases that they transmit. However, morphological techniques depend on the quality of the specimen and often unavailable taxonomic expertise, which may still not be able to distinguish mosquitoes among species complexes (sibling and cryptic species). High resolution melting (HRM) analyses, a closed-tube, post-polymerase chain reaction (PCR) method used to identify variations in nucleic acid sequences, has been used to differentiate species within the Anopheles gambiae and Culex pipiens complexes. We validated the use of PCR-HRM analyses to differentiate species within Anopheles and within each of six genera of culicine mosquitoes, comparing primers targeting cytochrome b ( cyt b), NADH dehydrogenase subunit 1 (ND1), intergenic spacer region (IGS) and cytochrome c oxidase subunit 1 ( COI) gene regions. HRM analyses of amplicons from all the six primer pairs successfully differentiated two or more mosquito species within one or more genera ( Aedes ( Ae. vittatus from Ae. metallicus), Culex ( Cx. tenagius from Cx. antennatus, Cx. neavei from Cx. duttoni, cryptic Cx. pipiens species), Anopheles ( An. gambiae s.s. from An. arabiensis) and Mansonia ( Ma. africana from Ma. uniformis)) based on their HRM profiles. However, PCR-HRM could not distinguish between species within Aedeomyia ( Ad. africana and Ad. furfurea), Mimomyia ( Mi. hispida and Mi. splendens) and Coquillettidia ( Cq. aurites, Cq. chrysosoma, Cq. fuscopennata, Cq. metallica, Cq. microannulatus, Cq. pseudoconopas and Cq. versicolor) genera using any of the primers. The IGS and COI barcode region primers gave the best and most definitive separation of mosquito species among anopheline and culicine mosquito genera, respectively, while the other markers may serve to confirm identifications of closely related sub

  19. Putative hybrids between two Anisakis cryptic species: molecular genotyping using High Resolution Melting.

    PubMed

    Cavallero, S; Costa, A; Caracappa, S; Gambetta, B; D'Amelio, S

    2014-11-01

    The genus Anisakis includes nine recognized species and the complex of cryptic species Anisakis simplex s. l. is often associated with the human disease known as anisakiasis. During the last decades the use of nuclear ribosomal ITS allowed the identification and description of numerous anisakid nematodes and the discovery of recombinant genotypes or putative hybrids even in other parasitic helminths, such as those between A. simplex sensu stricto and A. pegreffii. The existence of pure hybrids of the two sibling species has been long debated due to the large recovery of larval forms from sympatric areas and the rare observation of adult hybrids. The aims of the present report were to identify anisakid nematodes collected from Stenella coeruleoalba using PCR-RFLP of ITS and to focus the interest on hybrid forms using a High Resolution Melting (HRM) and direct sequencing analyses, since the new record of putative hybrid at adult stage. The PCR-RFLP analysis enabled to identify A. simplex s.s., A. pegreffii, the heterozygous genotype of the two species and A. physeteris. The use of the genotyping approach based on HRM confirmed the profiles of the two species A. simplex s.s. and A. pegreffii, and of the hybrid individuals. The new record of adult hybrids in definitive hosts rekindles the long debate about their existence and their evolutionary meaning. Since the reproductive isolation between A. simplex s.s. and A. pegreffii is the assumption for their existence as separated species, the use of alternative molecular markers and population genetic studies on adult anisakids are recommended. PMID:25241034

  20. Rapid molecular typing of Prototheca zopfii by high resolution melting real-time PCR (PCR-HRM).

    PubMed

    Sobukawa, Hideto; Ibaraki, Masato; Kano, Rui; Ito, Takaaki; Suzuki, Kazuyuki; Kamata, Hiroshi; Hasegawa, Atsuhiko

    2013-01-01

    Prototheca zopfii is an achlorophyllic alga that is ubiquitous around cow sheds. The alga is associated with bovine mastitis, which causes a reduction in milk production and secretion of thin watery milk containing white flakes. Isolates of P. zopfii from bovine mastitis were almost all identified as P. zopfii genotype 2, suggesting that it is the main causative agent of bovine protothecal mastitis. The ability to differentiate between genotype 1 and genotype 2 is therefore very important for preventing bovine mastitis. In this study, high resolution melting real-time PCR (PCR-HRM) analysis of the protothecal 18S rDNA domain successfully differentiated between genotypes of P. zopfii in less than 3 hours, while conventional sequence analysis requires more than 48 hours to differentiate between genotypes. PCR-HRM analysis clustered P. zopfii genotype 1 isolates separately from P. zopfii genotype 2 isolates, indicating that this molecular typing method is an effective tool for rapidly diagnosing bovine protothecal mastitis. PMID:24292136

  1. Rapid detection of HLA-B*51 by real-time polymerase chain reaction and high-resolution melting analysis.

    PubMed

    Imperiali, C; Alía-Ramos, P; Padró-Miquel, A

    2015-08-01

    HLA-B*51, a class I human leukocyte antigen (HLA) molecule, is the strongest known genetic risk factor for Behçet disease. However, there are only few articles reporting methods to determine the presence or absence of HLA-B51. For this reason, we designed and developed an easy, fast, and inexpensive real-time high-resolution melting (HRM) assay to detect HLA-B*51. We genotyped 61 samples by our HRM assay and by conventional polymerase chain reaction, and no discrepancies were found between results. Besides, a subgroup of 25 samples was also genotyped in a different laboratory, and another subgroup of 16 samples was obtained from the International Histocompatibility Working Group DNA Bank, and a full concordance of results was observed with those obtained by HRM. Regarding the identifying system evaluated, we obtained 100% of specificity, sensibility, and repeatability, and 0% of false positive and false negative rates. Therefore, this HRM analysis is easily applicable to the rapid detection of HLA-B*51, exhibits a high speed, and requires a very low budget. PMID:26176813

  2. Rapid detection of HLA-B*51 by real-time polymerase chain reaction and high-resolution melting analysis.

    PubMed

    Imperiali, C; Alía-Ramos, P; Padró-Miquel, A

    2015-08-01

    HLA-B*51, a class I human leukocyte antigen (HLA) molecule, is the strongest known genetic risk factor for Behçet disease. However, there are only few articles reporting methods to determine the presence or absence of HLA-B51. For this reason, we designed and developed an easy, fast, and inexpensive real-time high-resolution melting (HRM) assay to detect HLA-B*51. We genotyped 61 samples by our HRM assay and by conventional polymerase chain reaction, and no discrepancies were found between results. Besides, a subgroup of 25 samples was also genotyped in a different laboratory, and another subgroup of 16 samples was obtained from the International Histocompatibility Working Group DNA Bank, and a full concordance of results was observed with those obtained by HRM. Regarding the identifying system evaluated, we obtained 100% of specificity, sensibility, and repeatability, and 0% of false positive and false negative rates. Therefore, this HRM analysis is easily applicable to the rapid detection of HLA-B*51, exhibits a high speed, and requires a very low budget.

  3. Forensic age prediction for dead or living samples by use of methylation-sensitive high resolution melting.

    PubMed

    Hamano, Yuya; Manabe, Sho; Morimoto, Chie; Fujimoto, Shuntaro; Ozeki, Munetaka; Tamaki, Keiji

    2016-07-01

    Age prediction with epigenetic information is now edging closer to practical use in forensic community. Many age-related CpG (AR-CpG) sites have proven useful in predicting age in pyrosequencing or DNA chip analyses. In this study, a wide range methylation status in the ELOVL2 and FHL2 promoter regions were detected with methylation-sensitive high resolution melting (MS-HRM) in a labor-, time-, and cost-effective manner. Non-linear-distributions of methylation status and chronological age were newly fitted to the logistic curve. Notably, these distributions were revealed to be similar in 22 living blood samples and 52 dead blood samples. Therefore, the difference of methylation status between living and dead samples suggested to be ignorable by MS-HRM. Additionally, the information from ELOVL2 and FHL2 were integrated into a logistic curve fitting model to develop a final predictive model through the multivariate linear regression of logit-linked methylation rates and chronological age with adjusted R(2)=0.83. Mean absolute deviation (MAD) was 7.44 for 74 training set and 7.71 for 30 additional independent test set, indicating that the final predicting model is accurate. This suggests that our MS-HRM-based method has great potential in predicting actual forensic age.

  4. Combined Use of Molecular Markers and High-Resolution Melting (HRM) to Assess Chromosome Dosage in Potato Hybrids.

    PubMed

    Villano, Clizia; Miraglia, Valeria; Iorizzo, Massimo; Aversano, Riccardo; Carputo, Domenico

    2016-03-01

    In plants, the most widely used cytological techniques to assess parental genome contributions are based on in situ hybridization (FISH and GISH), but they are time-consuming and need specific expertise and equipment. Recent advances in genomics and molecular biology have made PCR-based markers a straightforward, affordable technique for chromosome typing. Here, we describe the development of a molecular assay that uses single-copy conserved ortholog set II (COSII)-based single nucleotide polymorphisms (SNPs) and the high-resolution melting (HRM) technique to assess the chromosome dosage of interspecific hybrids between a Solanum phureja-S. tuberosum diploid (2n = 2x = 24) hybrid and its wild relative S. commersonii. Screening and analysis of 45 COSII marker sequences allowed S. commersonii-specific SNPs to be identified for all 12 chromosomes. Combining the HRM technique with the establishment of synthetic DNA hybrids, SNP markers were successfully used to predict the expected parental chromosome ratio of 5 interspecific triploid hybrids. These results demonstrate the ability of this strategy to distinguish diverged genomes from each other, and to estimate chromosome dosage. The method could potentially be applied to any species as a tool to assess paternal to maternal ratios in the framework of a breeding program or following transformation techniques. PMID:26663623

  5. Forensic age prediction for dead or living samples by use of methylation-sensitive high resolution melting.

    PubMed

    Hamano, Yuya; Manabe, Sho; Morimoto, Chie; Fujimoto, Shuntaro; Ozeki, Munetaka; Tamaki, Keiji

    2016-07-01

    Age prediction with epigenetic information is now edging closer to practical use in forensic community. Many age-related CpG (AR-CpG) sites have proven useful in predicting age in pyrosequencing or DNA chip analyses. In this study, a wide range methylation status in the ELOVL2 and FHL2 promoter regions were detected with methylation-sensitive high resolution melting (MS-HRM) in a labor-, time-, and cost-effective manner. Non-linear-distributions of methylation status and chronological age were newly fitted to the logistic curve. Notably, these distributions were revealed to be similar in 22 living blood samples and 52 dead blood samples. Therefore, the difference of methylation status between living and dead samples suggested to be ignorable by MS-HRM. Additionally, the information from ELOVL2 and FHL2 were integrated into a logistic curve fitting model to develop a final predictive model through the multivariate linear regression of logit-linked methylation rates and chronological age with adjusted R(2)=0.83. Mean absolute deviation (MAD) was 7.44 for 74 training set and 7.71 for 30 additional independent test set, indicating that the final predicting model is accurate. This suggests that our MS-HRM-based method has great potential in predicting actual forensic age. PMID:27497326

  6. Detection of Schistosoma mansoni and Schistosoma haematobium by Real-Time PCR with High Resolution Melting Analysis

    PubMed Central

    Sady, Hany; Al-Mekhlafi, Hesham M.; Ngui, Romano; Atroosh, Wahib M.; Al-Delaimy, Ahmed K.; Nasr, Nabil A.; Dawaki, Salwa; Abdulsalam, Awatif M.; Ithoi, Init; Lim, Yvonne A. L.; Chua, Kek Heng; Surin, Johari

    2015-01-01

    The present study describes a real-time PCR approach with high resolution melting-curve (HRM) assay developed for the detection and differentiation of Schistosoma mansoni and S. haematobium in fecal and urine samples collected from rural Yemen. The samples were screened by microscopy and PCR for the Schistosoma species infection. A pair of degenerate primers were designed targeting partial regions in the cytochrome oxidase subunit I (cox1) gene of S. mansoni and S. haematobium using real-time PCR-HRM assay. The overall prevalence of schistosomiasis was 31.8%; 23.8% of the participants were infected with S. haematobium and 9.3% were infected with S. mansoni. With regards to the intensity of infections, 22.1% and 77.9% of S. haematobium infections were of heavy and light intensities, respectively. Likewise, 8.1%, 40.5% and 51.4% of S. mansoni infections were of heavy, moderate and light intensities, respectively. The melting points were distinctive for S. mansoni and S. haematobium, categorized by peaks of 76.49 ± 0.25 °C and 75.43 ± 0.26 °C, respectively. HRM analysis showed high detection capability through the amplification of Schistosoma DNA with as low as 0.0001 ng/µL. Significant negative correlations were reported between the real-time PCR-HRM cycle threshold (Ct) values and microscopic egg counts for both S. mansoni in stool and S. haematobium in urine (p < 0.01). In conclusion, this closed-tube HRM protocol provides a potentially powerful screening molecular tool for the detection of S. mansoni and S. haematobium. It is a simple, rapid, accurate, and cost-effective method. Hence, this method is a good alternative approach to probe-based PCR assays. PMID:26193254

  7. Genotyping single-nucleotide polymorphisms of human genes involved in organophosphate detoxification by high-resolution melting.

    PubMed

    Kurdyukov, Ivan; Rodionov, Gennady; Radilov, Andrey; Babakov, Vladimir

    2014-08-01

    Paraoxonase-1 (PON1) and butyrylcholinesterase (BCHE) are natural bioscavengers of organophosphate acetylcholinesterase inhibitors in the human body, which can determine individual sensitivity to organophosphate toxicity. Interindividual differences in activity of PON1 (catalytic bioscavenger) and substrate specificity are strongly associated with the substitution of two amino acids: Leu/Met (L/M) at position 55 (rs854560) and Gln/Arg (Q/R) at position 192 (rs662). In the case of BCHE (stoichiometric bioscavenger) substitution, Ala/Thr (A/T) at position 539 produces the so-called "K-variant" of the enzyme (rs1803274). Threonine allele is often co-inherited with an atypical BCHE allele (rs1799807). The atypical variant of BCHE displays a lower affinity for cholinesterase inhibitors. Genotyping rs662 and rs1803274 single-nucleotide polymorphisms (SNP) by high-resolution melting (HRM) is facilitated by the nucleotide substitution A>G (G>A), which resulted in a changed number of hydrogen bonds in the PCR product and, consequently, shifted T m. In the case of rs854560, genotyping is complicated by the nucleotide substitution T>A, which has no significant effect on the T m of the PCR product. An addition of a small quantity of LL homozygote DNA into the reaction mixture before PCR discriminates the three genotypes by the melt curves due to different amounts of heteroduplexes formed in the LM and MM samples. HRM analysis can be applied for genotyping human rs854560, rs662, and rs1803274 SNPs. PMID:24705954

  8. Multiplex Real-Time PCR Assay with High-Resolution Melting Analysis for Characterization of Antimicrobial Resistance in Neisseria gonorrhoeae.

    PubMed

    Donà, Valentina; Kasraian, Sara; Lupo, Agnese; Guilarte, Yuvia N; Hauser, Christoph; Furrer, Hansjakob; Unemo, Magnus; Low, Nicola; Endimiani, Andrea

    2016-08-01

    Resistance to antibiotics used against Neisseria gonorrhoeae infections is a major public health concern. Antimicrobial resistance (AMR) testing relies on time-consuming culture-based methods. Development of rapid molecular tests for detection of AMR determinants could provide valuable tools for surveillance and epidemiological studies and for informing individual case management. We developed a fast (<1.5-h) SYBR green-based real-time PCR method with high-resolution melting (HRM) analysis. One triplex and three duplex reactions included two sequences for N. gonorrhoeae identification and seven determinants of resistance to extended-spectrum cephalosporins (ESCs), azithromycin, ciprofloxacin, and spectinomycin. The method was validated by testing 39 previously fully characterized N. gonorrhoeae strains, 19 commensal Neisseria species strains, and an additional panel of 193 gonococcal isolates. Results were compared with results of culture-based AMR determination. The assay correctly identified N. gonorrhoeae and the presence or absence of the seven AMR determinants. There was some cross-reactivity with nongonococcal Neisseria species, and the detection limit was 10(3) to 10(4) genomic DNA (gDNA) copies/reaction. Overall, the platform accurately detected resistance to ciprofloxacin (sensitivity and specificity, 100%), ceftriaxone (sensitivity, 100%; specificity, 90%), cefixime (sensitivity, 92%; specificity, 94%), azithromycin (sensitivity and specificity, 100%), and spectinomycin (sensitivity and specificity, 100%). In conclusion, our methodology accurately detects mutations that generate resistance to antibiotics used to treat gonorrhea. Low assay sensitivity prevents direct diagnostic testing of clinical specimens, but this method can be used to screen collections of gonococcal isolates for AMR more quickly than current culture-based AMR testing. PMID:27225407

  9. Multiplex Real-Time PCR Assay with High-Resolution Melting Analysis for Characterization of Antimicrobial Resistance in Neisseria gonorrhoeae.

    PubMed

    Donà, Valentina; Kasraian, Sara; Lupo, Agnese; Guilarte, Yuvia N; Hauser, Christoph; Furrer, Hansjakob; Unemo, Magnus; Low, Nicola; Endimiani, Andrea

    2016-08-01

    Resistance to antibiotics used against Neisseria gonorrhoeae infections is a major public health concern. Antimicrobial resistance (AMR) testing relies on time-consuming culture-based methods. Development of rapid molecular tests for detection of AMR determinants could provide valuable tools for surveillance and epidemiological studies and for informing individual case management. We developed a fast (<1.5-h) SYBR green-based real-time PCR method with high-resolution melting (HRM) analysis. One triplex and three duplex reactions included two sequences for N. gonorrhoeae identification and seven determinants of resistance to extended-spectrum cephalosporins (ESCs), azithromycin, ciprofloxacin, and spectinomycin. The method was validated by testing 39 previously fully characterized N. gonorrhoeae strains, 19 commensal Neisseria species strains, and an additional panel of 193 gonococcal isolates. Results were compared with results of culture-based AMR determination. The assay correctly identified N. gonorrhoeae and the presence or absence of the seven AMR determinants. There was some cross-reactivity with nongonococcal Neisseria species, and the detection limit was 10(3) to 10(4) genomic DNA (gDNA) copies/reaction. Overall, the platform accurately detected resistance to ciprofloxacin (sensitivity and specificity, 100%), ceftriaxone (sensitivity, 100%; specificity, 90%), cefixime (sensitivity, 92%; specificity, 94%), azithromycin (sensitivity and specificity, 100%), and spectinomycin (sensitivity and specificity, 100%). In conclusion, our methodology accurately detects mutations that generate resistance to antibiotics used to treat gonorrhea. Low assay sensitivity prevents direct diagnostic testing of clinical specimens, but this method can be used to screen collections of gonococcal isolates for AMR more quickly than current culture-based AMR testing.

  10. DNA bound to polypyrrole films: high-resolution imaging, DNA binding kinetics and internal migration.

    PubMed

    Pande, R; Ruben, G C; Lim, J O; Tripathy, S; Marx, K A

    1998-09-01

    We have studied the time-dependent uptake of 35S radiolabeled DNA with electrochemically prepared polypyrrole films. The two distinct polypyrrole film surfaces, a rough (solution polymeric growth face, R) and a smooth surface (electrode face, S) were characterized by low-resolution AFM and high-resolution transmission electron microscopy (TEM). These studies showed the presence of steep contours and defects in the form of large and small surface holes and valleys on the rough surface of polypyrrole. The void dimensions ranged from the nanoscale to micron size. By contrast, the smooth surface was flatter and largely devoid of significant structural defects and exhibited closer packing of the polypyrrole chains over large areas. Both surfaces were comprised largely of chains whose average diameters were 1.0-1.2 +/- 0.3 nm. The surface characterization studies were complemented by time-dependent DNA uptake studies which showed a t1/2-dependent total uptake of 35S DNA at higher levels on the rough surface compared to the smooth surface. This is consistent with the apparent higher effective surface area of the rough surface compared to the smooth. Using a proportional counter the time-dependent ratio (R/S) of the 35S DNA detected from the rough surface of the polypyrrole disk to that detected from the smooth surface suggested that DNA was migrating into the disk interior from its uptake surface. The rough side defect dimensions measured by TEM were more than sufficient to allow for the penetration and migration of DNA into the disk interior. Both R/S ratios were extrapolated and found to intersect at an R/S value close to 1.0, suggesting a kinetic process leading ultimately towards a nearly uniform radiolabeled DNA distribution in the disk. These kinetic results were in agreement with the surface characterization studies and suggest a model in which sizeable internal pores exist throughout the electrochemically prepared polypyrrole, that could account for the DNA

  11. Rigidity of melting DNA.

    PubMed

    Pal, Tanmoy; Bhattacharjee, Somendra M

    2016-05-01

    The temperature dependence of DNA flexibility is studied in the presence of stretching and unzipping forces. Two classes of models are considered. In one case the origin of elasticity is entropic due to the polymeric correlations, and in the other the double-stranded DNA is taken to have an intrinsic rigidity for bending. In both cases single strands are completely flexible. The change in the elastic constant for the flexible case due to thermally generated bubbles is obtained exactly. For the case of intrinsic rigidity, the elastic constant is found to be proportional to the square root of the bubble number fluctuation. PMID:27300825

  12. Rigidity of melting DNA

    NASA Astrophysics Data System (ADS)

    Pal, Tanmoy; Bhattacharjee, Somendra M.

    2016-05-01

    The temperature dependence of DNA flexibility is studied in the presence of stretching and unzipping forces. Two classes of models are considered. In one case the origin of elasticity is entropic due to the polymeric correlations, and in the other the double-stranded DNA is taken to have an intrinsic rigidity for bending. In both cases single strands are completely flexible. The change in the elastic constant for the flexible case due to thermally generated bubbles is obtained exactly. For the case of intrinsic rigidity, the elastic constant is found to be proportional to the square root of the bubble number fluctuation.

  13. High resolution melting analysis of the NR1I3 genetic variants: Is there an association with neonatal hyperbilirubinemia?

    PubMed

    Cheung, Tian Pei; Van Rostenberghe, Hans; Ismail, Rosliza; Nawawi, Noor Namirah; Abdullah, Nurul Amierah; Ramli, Noraida; Ibrahim, Nor Rosidah; Hj Abd Majid, Noorizan; Mohd Yusoff, Narazah; Nishio, Hisahide; Yusoff, Surini

    2015-12-01

    Constitutive androstane receptor (CAR) encoded by the nuclear receptor subfamily 1, group I, member 3 (NR1I3) gene regulates the elimination of bilirubin through activating the components of the bilirubin clearance pathway. Hence, NR1I3 genetic variants may affect bilirubin metabolism and result in neonatal hyperbilirubinemia. Thus far, research which investigates the association between NR1I3 variants and neonatal hyperbilirubinemia has not been undertaken in any population. The present study aimed to evaluate the influence of MPJ6_1I3008 (rs10157822), IVS8+116T>G (rs4073054) and 540A>G (rs2307424) on neonatal hyperbilirubinemia development in the Malay population. Buccal swabs were collected from 232 hyperbilirubinemia and 277 control term newborns with gestational age ≥37weeks and birth weight ≥2500g. The NR1I3 variants were genotyped by using high resolution melting (HRM) assays and verified by DNA sequencing. Gender, mode of delivery and birth weight did not differ between hyperbilirubinemia and control groups. The genotypic and allelic frequencies of MPJ6_1I3008, IVS8+116T>G and 540A>G were not significantly different between the groups. However, stratification by gender revealed a significant inverse association between homozygous variant genotype of MPJ6_1I3008 and risk of neonatal hyperbilirubinemia in the females (OR, 0.44; 95% CI, 0.20-0.95; p=0.034). This study demonstrates that the homozygous variant genotype of MPJ6_1I3008 was associated with a significant reduced risk of neonatal hyperbilirubinemia in the females. PMID:26188155

  14. Comparison of a high-resolution melting assay to next-generation sequencing for analysis of HIV diversity.

    PubMed

    Cousins, Matthew M; Ou, San-San; Wawer, Maria J; Munshaw, Supriya; Swan, David; Magaret, Craig A; Mullis, Caroline E; Serwadda, David; Porcella, Stephen F; Gray, Ronald H; Quinn, Thomas C; Donnell, Deborah; Eshleman, Susan H; Redd, Andrew D

    2012-09-01

    Next-generation sequencing (NGS) has recently been used for analysis of HIV diversity, but this method is labor-intensive, costly, and requires complex protocols for data analysis. We compared diversity measures obtained using NGS data to those obtained using a diversity assay based on high-resolution melting (HRM) of DNA duplexes. The HRM diversity assay provides a single numeric score that reflects the level of diversity in the region analyzed. HIV gag and env from individuals in Rakai, Uganda, were analyzed in a previous study using NGS (n = 220 samples from 110 individuals). Three sequence-based diversity measures were calculated from the NGS sequence data (percent diversity, percent complexity, and Shannon entropy). The amplicon pools used for NGS were analyzed with the HRM diversity assay. HRM scores were significantly associated with sequence-based measures of HIV diversity for both gag and env (P < 0.001 for all measures). The level of diversity measured by the HRM diversity assay and NGS increased over time in both regions analyzed (P < 0.001 for all measures except for percent complexity in gag), and similar amounts of diversification were observed with both methods (P < 0.001 for all measures except for percent complexity in gag). Diversity measures obtained using the HRM diversity assay were significantly associated with those from NGS, and similar increases in diversity over time were detected by both methods. The HRM diversity assay is faster and less expensive than NGS, facilitating rapid analysis of large studies of HIV diversity and evolution.

  15. Hybrid analysis (barcode-high resolution melting) for authentication of Thai herbal products, Andrographis paniculata (Burm.f.) Wall.ex Nees

    PubMed Central

    Osathanunkul, Maslin; Suwannapoom, Chatmongkon; Khamyong, Nuttaluck; Pintakum, Danupol; Lamphun, Santisuk Na; Triwitayakorn, Kanokporn; Osathanunkul, Kitisak; Madesis, Panagiotis

    2016-01-01

    Background: Andrographis paniculata Nees is a medicinal plant with multiple pharmacological properties. It has been used over many centuries as a household remedy. A. paniculata products sold on the markets are in processed forms so it is difficult to authenticate. Therefore buying the herbal products poses a high-risk of acquiring counterfeited, substituted and/or adulterated products. Due to these issues, a reliable method to authenticate products is needed. Materials and Methods: High resolution melting analysis coupled with DNA barcoding (Bar-HRM) was applied to detect adulteration in commercial herbal products. The rbcL barcode was selected to use in primers design for HRM analysis to produce standard melting profile of A. paniculata species. DNA of the tested commercial products was isolated and their melting profiles were then generated and compared with the standard A. paniculata. Results: The melting profiles of the rbcL amplicons of the three closely related herbal species (A. paniculata, Acanthus ebracteatus and Rhinacanthus nasutus) are clearly separated so that they can be distinguished by the developed method. The method was then used to authenticate commercial herbal products. HRM curves of all 10 samples tested are similar to A. paniculata which indicated that all tested products were contained the correct species as labeled. Conclusion: The method described in this study has been proved to be useful in aiding identification and/or authenticating A. paniculata. This Bar-HRM analysis has allowed us easily to determine the A. paniculata species in herbal products on the markets even they are in processed forms. SUMMARY We propose the use of DNA barcoding combined with High Resolution Melting analysis for authenticating of Andrographis paniculata products.The developed method can be used regardless of the type of the DNA template (fresh or dried tissue, leaf, and stem).rbcL region was chosen for the analysis and work well with our samplesWe can easily

  16. Molecular Differentiation of Schistosoma japonicum and Schistosoma mekongi by Real-Time PCR with High Resolution Melting Analysis

    PubMed Central

    Kongklieng, Amornmas; Kaewkong, Worasak; Intapan, Pewpan M.; Sanpool, Oranuch; Janwan, Penchom; Thanchomnang, Tongjit; Lulitanond, Viraphong; Sri-Aroon, Pusadee; Limpanont, Yanin

    2013-01-01

    Human schistosomiasis caused by Schistosoma japonicum and Schistosoma mekongi is a chronic and debilitating helminthic disease still prevalent in several countries of Asia. Due to morphological similarities of cercariae and eggs of these 2 species, microscopic differentiation is difficult. High resolution melting (HRM) real-time PCR is developed as an alternative tool for the detection and differentiation of these 2 species. A primer pair was designed for targeting the 18S ribosomal RNA gene to generate PCR products of 156 base pairs for both species. The melting points of S. japonicum and S. mekongi PCR products were 84.5±0.07℃ and 85.7±0.07℃, respectively. The method permits amplification from a single cercaria or an egg. The HRM real-time PCR is a rapid and simple tool for differentiation of S. japonicum and S. mekongi in the intermediate and final hosts. PMID:24516269

  17. Rapid and efficient differentiation of Yersinia species using high-resolution melting analysis.

    PubMed

    Souza, Roberto A; Frazão, Miliane R; Almeida, Alzira M P; Falcão, Juliana P

    2015-08-01

    The primary goal of clinical microbiology is the accurate identification of the causative agent of the disease. Here, we describe a method for differentiation between Yersinia species using PCR-HRMA. The results revealed species-specific melting profiles. The herein developed assay can be used as an effective method to differentiate Yersinia species.

  18. High-resolution melting analysis (HRM) for differentiation of four major Taeniidae species in dogs Taenia hydatigena, Taenia multiceps, Taenia ovis, and Echinococcus granulosus sensu stricto.

    PubMed

    Dehghani, Mansoureh; Mohammadi, Mohammad Ali; Rostami, Sima; Shamsaddini, Saeedeh; Mirbadie, Seyed Reza; Harandi, Majid Fasihi

    2016-07-01

    Tapeworms of the genus Taenia include several species of important parasites with considerable medical and veterinary significance. Accurate identification of these species in dogs is the prerequisite of any prevention and control program. Here, we have applied an efficient method for differentiating four major Taeniid species in dogs, i.e., Taenia hydatigena, T. multiceps, T. ovis, and Echinococcus granulosus sensu stricto. High-resolution melting (HRM) analysis is simpler, less expensive, and faster technique than conventional DNA-based assays and enables us to detect PCR amplicons in a closed system. Metacestode samples were collected from local abattoirs from sheep. All the isolates had already been identified by PCR-sequencing, and their sequence data were deposited in the GenBank. Real-time PCR coupled with HRM analysis targeting mitochondrial cox1 and ITS1 genes was used to differentiate taeniid species. Distinct melting curves were obtained from ITS1 region enabling accurate differentiation of three Taenia species and E. granulosus in dogs. The HRM curves of Taenia species and E .granulosus were clearly separated at Tm of 85 to 87 °C. In addition, double-pick melting curves were produced in mixed infections. Cox1 melting curves were not decisive enough to distinguish four taeniids. In this work, the efficiency of HRM analysis to differentiate four major taeniid species in dogs has been demonstrated using ITS1 gene. PMID:27008188

  19. High-resolution melting analysis for the detection of two erythromycin-resistant Bordetella pertussis strains carried by healthy schoolchildren in China.

    PubMed

    Zhang, Q; Li, M; Wang, L; Xin, T; He, Q

    2013-06-01

    Two erythromycin-resistant strains of Bordetella pertussis were isolated from nasopharyngeal specimens of two asymptomatic schoolchildren in China. High-resolution melting and sequencing analyses confirmed the homogeneous A2047G mutation in 23S rRNA genes of the two isolates. High-resolution melting (HRM) analysis is a useful assay for the rapid detection of erythromycin-resistant B. pertussis. The appearance of erythromycin-resistant B. pertussis strains in China is alarming.

  20. Identification case of evidence in timber tracing of Pinus radiate, using high-resolution melting (HRM) analysis.

    PubMed

    Solano, Jaime; Anabalón, Leonardo; Encina, Francisco

    2016-03-01

    Fast, accurate detection of plant species and their hybrids using molecular tools will facilitate assessment and monitoring of timber tracing evidence. In this study the origin of unknown pine samples is determined for a case of timber theft in the region of Araucania southern Chile. We evaluate the utility of the trnL marker region for species identification applied to pine wood based on High Resolution Melting. This efficient tracing methods can be incorporated into forestry applications such as certification of origin. The object of this work was genotype identification using high-resolution melting (HRM) and trnL approaches for Pinus radiata (Don) in timber tracing evidence. Our results indicate that trnL is a very sensitive marker for delimiting species and HRM analysis was used successfully for genotyping Pinus samples for timber tracing purposes. Genotyping samples by HRM analysis with the trnL1 approach allowed us to differentiate two wood samples from the Pinaceae family: Pinus radiata (Don) and Pseudotsuga menziesii (Mirb.) Franco. The same approach with Pinus trnL wood was not able to discriminate between samples of Pinus radiata, indicating that the samples were genetically indistinguishable, possibly because they have the same genotype at this locus. Timber tracing with HRM analysis is expected to contribute to future forest certification schemes, control of illegal trading, and molecular traceability of Pinus spp.

  1. High-resolution melting analysis using unlabeled probe and amplicon scanning simultaneously detects several lactase persistence variants.

    PubMed

    Janukonyté, Jurgita; Vestergaard, Else M; Ladefoged, Søren A; Nissen, Peter H

    2010-12-01

    Lactase persistence and thereby tolerance to lactose is a common trait in people of Northern European descent. It is linked to the LCT -13910C>T variant located in intron 13 of the MCM6 gene 13.9 kb upstream of the lactase (LCT) gene. In people of African and Middle Eastern descent, lactase persistence can be associated with other variants nearby the -13910C>T variant, limiting the use of the -13910C>T-based SNP analysis, e.g. TaqMan assays for the diagnosis of lactose intolerance. Using high-resolution melting analysis, we identified five samples that were heterozygous for the -13915T>G variant among 78 patients genotyped as -13910C/C by a TaqMan assay. All samples originated from patients of probable Middle Eastern descent. In order to detect the -13910 and -13915 variants simultaneously, we developed a new high-resolution melting (HRM) analysis assay based on unlabeled probe genotyping and simultaneous amplicon scanning analysis. By using this assay we were able to distinguish the -13910 and -13915 genotypes clearly. Furthermore, we identified two rare variants, the -13907C>G and -13913T>C. With this method, based on an inexpensive unlabeled probe, it is possible to simultaneously detect the -13910C>T and -13915T>G variants in addition to rarer variants surrounding the -13910 site. This new method may contribute to improve the diagnostic performance of the genetic analysis for lactose intolerance.

  2. Co-evolution of tidewater glacier calving front morphology and submarine melt rates in a high resolution ocean model

    NASA Astrophysics Data System (ADS)

    Slater, D. A.; Nienow, P. W.; Goldberg, D. N.; Cowton, T. R.; Sole, A. J.

    2015-12-01

    Rapid dynamic changes at the margins of the Greenland Ice Sheet, synchronous with ocean warming, have raised concern that tidewater glaciers can respond rapidly and sensitively to ocean forcing. One way in which ocean forcing would manifest is through the melting of the submerged parts of tidewater glacier calving fronts, with the spatial distribution of submarine melt a control on their morphology. Calving front morphology has thus far received little attention and yet has the potential to significantly impact calving rates and therefore tidewater glacier dynamics. Here we present a model which allows us to study the evolution of calving front morphology in two dimensions. We outline a new routine for calculating submarine melt rates from ocean models at calving fronts of arbitrary geometry, and for adjusting this geometry according to the calculated melt rates. This routine is applied to a high resolution (~1m) non-hydrostatic ocean model (MITgcm) with a glacier boundary (calving front) which evolves in time according to the simulated submarine melt rates. The model shows, consistent with recent observations, that submarine melting leads to undercutting of tidewater glacier calving fronts. We examine how undercut magnitude, undercut depth and potential steady states respond to variation in subglacial discharge, ice velocity, and fjord depth, temperature and stratification. In addition to this analysis we use a diagnostic full-Stokes flow-line ice model to examine how these geometries affect ice internal stress and potential for calving. In undertaking this work we aim to elucidate a process which - supposing tidewater glaciers are sensitive to ocean forcing - must provide a fundamental link between the ocean and the ice.

  3. The utility of high-resolution melting analysis of SNP nucleated PCR amplicons--an MLST based Staphylococcus aureus typing scheme.

    PubMed

    Lilliebridge, Rachael A; Tong, Steven Y C; Giffard, Philip M; Holt, Deborah C

    2011-01-01

    High resolution melting (HRM) analysis is gaining prominence as a method for discriminating DNA sequence variants. Its advantage is that it is performed in a real-time PCR device, and the PCR amplification and HRM analysis are closed tube, and effectively single step. We have developed an HRM-based method for Staphylococcus aureus genotyping. Eight single nucleotide polymorphisms (SNPs) were derived from the S. aureus multi-locus sequence typing (MLST) database on the basis of maximized Simpson's Index of Diversity. Only G↔A, G↔T, C↔A, C↔T SNPs were considered for inclusion, to facilitate allele discrimination by HRM. In silico experiments revealed that DNA fragments incorporating the SNPs give much higher resolving power than randomly selected fragments. It was shown that the predicted optimum fragment size for HRM analysis was 200 bp, and that other SNPs within the fragments contribute to the resolving power. Six DNA fragments ranging from 83 bp to 219 bp, incorporating the resolution optimized SNPs were designed. HRM analysis of these fragments using 94 diverse S. aureus isolates of known sequence type or clonal complex (CC) revealed that sequence variants are resolved largely in accordance with G+C content. A combination of experimental results and in silico prediction indicates that HRM analysis resolves S. aureus into 268 "melt types" (MelTs), and provides a Simpson's Index of Diversity of 0.978 with respect to MLST. There is a high concordance between HRM analysis and the MLST defined CCs. We have generated a Microsoft Excel key which facilitates data interpretation and translation between MelT and MLST data. The potential of this approach for genotyping other bacterial pathogens was investigated using a computerized approach to estimate the densities of SNPs with unlinked allelic states. The MLST databases for all species tested contained abundant unlinked SNPs, thus suggesting that high resolving power is not dependent upon large numbers of SNPs.

  4. Probing the atomic structure of basaltic melts generated by partial melting of upper mantle peridotite (KLB-1): Insights from high-resolution solid-state NMR study

    NASA Astrophysics Data System (ADS)

    Park, S. Y.; Lee, S. K.

    2015-12-01

    Probing the structural disorder in multi-component silicate glasses and melts with varying composition is essential to reveal the change of macroscopic properties in natural silicate melts. While a number of NMR studies for the structure of multi-component silicate glasses and melts including basaltic and andesitic glasses have been reported (e.g., Park and Lee, Geochim. Cosmochim. Acta, 2012, 80, 125; Park and Lee, Geochim. Cosmochim. Acta, 2014, 26, 42), many challenges still remain. The composition of multi-component basaltic melts vary with temperature, pressure, and melt fraction (Kushiro, Annu. Rev. Earth Planet. Sci., 2001, 71, 107). Especially, the eutectic point (the composition of first melt) of nepheline-forsterite-quartz (the simplest model of basaltic melts) moves with pressure from silica-saturated to highly undersaturated and alkaline melts. The composition of basaltic melts generated by partial melting of upper mantle peridotite (KLB-1, the xenolith from Kilbourne Hole) also vary with pressure. In this study we report experimental results for the effects of composition on the atomic structure of Na2O-MgO-Al2O3-SiO2 (NMAS) glasses in nepheline (NaAlSiO4)-forsterite (Mg2SiO4)-quartz (SiO2) eutectic composition and basaltic glasses generated by partial melting of upper mantle peridotite (KLB-1) using high-resolution multi-nuclear solid-state NMR. The Al-27 3QMAS (triple quantum magic angle spinning) NMR spectra of NMAS glasses in nepheline-forsterite-quartz eutectic composition show only [4]Al. The Al-27 3QMAS NMR spectra of KLB-1 basaltic glasses show mostly [4]Al and a non-negligible fraction of [5]Al. The fraction of [5]Al, the degree of configurational disorder, increases from 0 at XMgO [MgO/(MgO+Al2O3)]=0.55 to ~3% at XMgO=0.79 in KLB-1 basaltic glasses while only [4]Al are observed in nepheline-forsterite-quartz eutectic composition. The current experimental results provide that the fraction of [5]Al abruptly increases by the effect of

  5. Detection of Sequence Polymorphism in Rubus Occidentalis L. Monomorphic Microsatellite Markers by High Resolution Melting

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Microsatellite, or simple sequence repeat (SSR) markers, are valuable as co-dominant genetic markers with a variety of applications such as DNA fingerprinting, linkage mapping, and population structure analysis. Development of microsatellite primers through the identification of appropriate repeate...

  6. A refined, rapid and reproducible high resolution melt (HRM)-based method suitable for quantification of global LINE-1 repetitive element methylation

    PubMed Central

    2011-01-01

    Background The methylation of DNA is recognized as a key mechanism in the regulation of genomic stability and evidence for its role in the development of cancer is accumulating. LINE-1 methylation status represents a surrogate measure of genome-wide methylation. Findings Using high resolution melt (HRM) curve analysis technology, we have established an in-tube assay that is linear (r > 0.9986) with a high amplification efficiency (90-105%), capable of discriminating between partcipant samples with small differences in methylation, and suitable for quantifying a wide range of LINE-1 methylation levels (0-100%)--including the biologically relevant range of 50-90% expected in human DNA. We have optimized this procedure to perform using 2 μg of starting DNA and 2 ng of bisulfite-converted DNA for each PCR reaction. Intra- and inter-assay coefficients of variation were 1.44% and 0.49%, respectively, supporting the high reproducibility and precision of this approach. Conclusions In summary, this is a completely linear, quantitative HRM PCR method developed for the measurement of LINE-1 methylation. This cost-efficient, refined and reproducible assay can be performed using minimal amounts of starting DNA. These features make our assay suitable for high throughput analysis of multiple samples from large population-based studies. PMID:22204640

  7. High resolution melting detects sequence polymorphism in rubus occidentalis L. monomorphic microsatellite markers

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Microsatellite, or simple sequence repeat (SSR) markers, are valuable as co-dominant genetic markers with a variety of applications such as DNA fingerprinting, linkage mapping, and population structure analysis. However, primer pairs designed from the regions that flank SSRs often generate fragment...

  8. Rapid Identification of Biothreat and Other Clinically Relevant Bacterial Species by Use of Universal PCR Coupled with High-Resolution Melting Analysis▿

    PubMed Central

    Yang, Samuel; Ramachandran, Padmini; Rothman, Richard; Hsieh, Yu-Hsiang; Hardick, Andrew; Won, Helen; Kecojevic, Aleksandar; Jackman, Joany; Gaydos, Charlotte

    2009-01-01

    A rapid assay for eubacterial species identification is described using high-resolution melt analysis to characterize PCR products. Unique melt profiles generated from multiple hypervariable regions of the 16S rRNA gene for 100 clinically relevant bacterial pathogens, including category A and B biothreat agents and their surrogates, allowed highly specific species identification. PMID:19458181

  9. A method to distinguish morphologically similar Peromyscus species using extracellular RNA and high-resolution melt analysis.

    PubMed

    Seifert, Veronica A; Clarke, Benjamin L; Crossland, Janet P; Bemis, Lynne T

    2016-09-01

    A method applying high-resolution melt (HRM) analysis to PCR products copied and amplified from extracellular RNA (exRNA) has been developed to distinguish two morphologically similar Peromyscus species: Peromyscus leucopus and Peromyscus maniculatus. P. leucopus is considered the primary reservoir host of Borrelia burgdorferi, the causative agent for Lyme disease in North America. In northern Minnesota the habitat ranges of P. leucopus overlaps with that of P. maniculatus. Serum samples from live mice of both species were collected from cheek bleeds, total extracellular RNA (exRNA) was extracted, copied using reverse transcription and amplified by PCR followed by HRM analysis. A circulating ribosomal RNA (rRNA) was identified which differed at seven nucleotides between the two species and a method of HRM analysis was developed allowing rapid species confirmation. In the future, this HRM based method may be adapted for additional species. PMID:27349513

  10. A method to distinguish morphologically similar Peromyscus species using extracellular RNA and high-resolution melt analysis.

    PubMed

    Seifert, Veronica A; Clarke, Benjamin L; Crossland, Janet P; Bemis, Lynne T

    2016-09-01

    A method applying high-resolution melt (HRM) analysis to PCR products copied and amplified from extracellular RNA (exRNA) has been developed to distinguish two morphologically similar Peromyscus species: Peromyscus leucopus and Peromyscus maniculatus. P. leucopus is considered the primary reservoir host of Borrelia burgdorferi, the causative agent for Lyme disease in North America. In northern Minnesota the habitat ranges of P. leucopus overlaps with that of P. maniculatus. Serum samples from live mice of both species were collected from cheek bleeds, total extracellular RNA (exRNA) was extracted, copied using reverse transcription and amplified by PCR followed by HRM analysis. A circulating ribosomal RNA (rRNA) was identified which differed at seven nucleotides between the two species and a method of HRM analysis was developed allowing rapid species confirmation. In the future, this HRM based method may be adapted for additional species.

  11. Duplex High-Resolution Melting Assay for the Simultaneous Genotyping of IL28B rs12979860 and PNPLA3 rs738409 Polymorphisms in Chronic Hepatitis C Patients

    PubMed Central

    Enache, Elena L.; Sin, Anca; Bancu, Ligia; Ramière, Christophe; Diaz, Olivier; André, Patrice; Enache, Liviu S.

    2015-01-01

    Chronic hepatitis C (CHC) is a major burden for public health worldwide. Although newer direct-acting antivirals show good efficacy, their cost precludes their wide adoption in resource-limited regions. Thus, strategies are being developed to help identify patients with high susceptibility to response to classic PEG-interferon + ribavirin therapy. IL28B polymorphism rs12979860 C/T is an important predictor for an efficient response to interferon-based therapy. A genetic variant in adiponutrin (PNPLA3) gene, rs738409 C/G, is associated with steatosis, severity, and progression of liver fibrosis in CHC patients, and predicts treatment outcome in difficult-to-cure HCV-infected patients with advanced fibrosis. We developed a rapid and inexpensive assay based on duplex high-resolution melting (HRM) for the simultaneous genotyping of these two polymorphisms. The assay validation was performed on synthetic DNA templates and 132 clinical samples from CHC patients. When compared with allele-specific PCR and sequencing, our assay showed 100% (95% CI: 0.9724–1) accuracy, with 100% sensitivity and specificity. Our assay was robust against concentration and quality of DNA samples, melting curve normalization intervals, HRM analysis algorithm, and sequence variations near the targeted SNPs (single nucleotide polymorphism). This duplex assay should provide useful information for patient-oriented management and clinical decision-making in CHC. PMID:26389885

  12. Rapid molecular identification of Listeria species by use of real-time PCR and high-resolution melting analysis.

    PubMed

    Jin, Dazhi; Luo, Yun; Zhang, Zheng; Fang, Weijia; Ye, Julian; Wu, Fang; Ding, Gangqiang

    2012-05-01

    Identification of Listeria species via a molecular method is critical for food safety and clinical diagnosis. In this study, an assay integrating real-time quantitative PCR (Q-PCR) with high-resolution melting (HRM) curve analysis was developed and assessed for rapid identification of six Listeria species. The ssrA gene, which encodes a transfer-messenger RNA (tmRNA) is conserved and common to all bacterial phyla, contains a variable domain in Listeria spp. Therefore, Q-PCR and a HRM profile were applied to characterize this gene. Fifty-three Listeria species and 45 non-Listeria species were detected using one primer set, with an accuracy of 100% in reference to conventional methods. There was a 93.3% correction rate to 30 artificially contaminated samples. Thus, Q-PCR with melting profiling analysis proved able to identify Listeria species accurately. Consequently, this study demonstrates that the assay we developed is a functional tool for rapidly identifying six Listeria species, and has the potential for discriminating novel species food safety and epidemiological research.

  13. A high-resolution melting (HRM) assay for the differentiation between Israeli field and Neethling vaccine lumpy skin disease viruses.

    PubMed

    Menasherow, Sophia; Erster, Oran; Rubinstein-Giuni, Marisol; Kovtunenko, Anita; Eyngor, Evgeny; Gelman, Boris; Khinich, Evgeny; Stram, Yehuda

    2016-06-01

    Lumpy skin disease (LSD) is a constant threat to the Middle East including the State of Israel. During vaccination programs it is essential for veterinary services and farmers to be able to distinguish between animals affected by the cattle-borne virulent viruses and vaccinated animals, subsequently affected by the vaccine strain. This study describes an improved high resolution-melting (HRM) test that exploits a 27 base pair (bp) fragment of the LSDV126 extracellular enveloped virion (EEV) gene that is present in field viruses but is absent from the Neethling vaccine strain. This difference leads to ∼0.5 °C melting point change in the HRM assay, when testing the quantitative PCR (qPCR) products generated from the virulent field viruses compared to the attenuated vaccine. By exploiting this difference, it could be shown using the newly developed HRM assay that virus isolated from vaccinated cattle that developed disease symptoms behave similarly to vaccine virus control, indicating that the vaccine virus can induce disease symptoms. This assay is not only in full agreement with the previously published PCR gradient and restriction fragment length polymorphism (RFLP) tests but it is faster with, fewer steps, cheaper and dependable.

  14. A high-resolution melting (HRM) assay for the differentiation between Israeli field and Neethling vaccine lumpy skin disease viruses.

    PubMed

    Menasherow, Sophia; Erster, Oran; Rubinstein-Giuni, Marisol; Kovtunenko, Anita; Eyngor, Evgeny; Gelman, Boris; Khinich, Evgeny; Stram, Yehuda

    2016-06-01

    Lumpy skin disease (LSD) is a constant threat to the Middle East including the State of Israel. During vaccination programs it is essential for veterinary services and farmers to be able to distinguish between animals affected by the cattle-borne virulent viruses and vaccinated animals, subsequently affected by the vaccine strain. This study describes an improved high resolution-melting (HRM) test that exploits a 27 base pair (bp) fragment of the LSDV126 extracellular enveloped virion (EEV) gene that is present in field viruses but is absent from the Neethling vaccine strain. This difference leads to ∼0.5 °C melting point change in the HRM assay, when testing the quantitative PCR (qPCR) products generated from the virulent field viruses compared to the attenuated vaccine. By exploiting this difference, it could be shown using the newly developed HRM assay that virus isolated from vaccinated cattle that developed disease symptoms behave similarly to vaccine virus control, indicating that the vaccine virus can induce disease symptoms. This assay is not only in full agreement with the previously published PCR gradient and restriction fragment length polymorphism (RFLP) tests but it is faster with, fewer steps, cheaper and dependable. PMID:26902159

  15. The WAIS Melt Monitor: An automated ice core melting system for meltwater sample handling and the collection of high resolution microparticle size distribution data

    NASA Astrophysics Data System (ADS)

    Breton, D. J.; Koffman, B. G.; Kreutz, K. J.; Hamilton, G. S.

    2010-12-01

    Paleoclimate data are often extracted from ice cores by careful geochemical analysis of meltwater samples. The analysis of the microparticles found in ice cores can also yield unique clues about atmospheric dust loading and transport, dust provenance and past environmental conditions. Determination of microparticle concentration, size distribution and chemical makeup as a function of depth is especially difficult because the particle size measurement either consumes or contaminates the meltwater, preventing further geochemical analysis. Here we describe a microcontroller-based ice core melting system which allows the collection of separate microparticle and chemistry samples from the same depth intervals in the ice core, while logging and accurately depth-tagging real-time electrical conductivity and particle size distribution data. This system was designed specifically to support microparticle analysis of the WAIS Divide WDC06A deep ice core, but many of the subsystems are applicable to more general ice core melting operations. Major system components include: a rotary encoder to measure ice core melt displacement with 0.1 millimeter accuracy, a meltwater tracking system to assign core depths to conductivity, particle and sample vial data, an optical debubbler level control system to protect the Abakus laser particle counter from damage due to air bubbles, a Rabbit 3700 microcontroller which communicates with a host PC, collects encoder and optical sensor data and autonomously operates Gilson peristaltic pumps and fraction collectors to provide automatic sample handling, melt monitor control software operating on a standard PC allowing the user to control and view the status of the system, data logging software operating on the same PC to collect data from the melting, electrical conductivity and microparticle measurement systems. Because microparticle samples can easily be contaminated, we use optical air bubble sensors and high resolution ice core density

  16. Development of a High-Resolution Melting Approach for Scanning Beta Globin Gene Point Mutations in the Greek and Other Mediterranean Populations.

    PubMed

    Chassanidis, Christos; Boutou, Effrossyni; Voskaridou, Ersi; Balassopoulou, Angeliki

    2016-01-01

    Beta-thalassaemia is one of the most common autosomal recessive disorders worldwide. The disease's high incidence, which is observed in the broader Mediterranean area has led to the establishment of molecular diagnostics' assays to prevent affected births. Therefore, the development of a reliable, cost-effective and rapid scanning method for β globin gene point mutations, easily adapted to a routine laboratory, is absolutely essential. Here, we describe, for the first time, the development of a High-Resolution Melting Analysis (HRMA) approach, suitable for scanning the particularly heterogeneous beta globin gene mutations present in the Greek population, and thus adaptable to the Mediterranean and other areas where these mutations have been identified. Within this context, β globin gene regions containing mutations frequently identified in the Greek population were divided in ten overlapping amplicons. Our reactions' setup allowed for the simultaneous amplification of multiple primer sets and partial multiplexing, thereby resulting in significant reduction of the experimental time. DNA samples from β-thalassaemia patients/carriers with defined genotypes were tested. Distinct genotypes displayed distinguishable melting curves, enabling accurate detection of mutations. The described HRMA can be adapted to a high-throughput level. It represents a rapid, simple, cost-effective, reliable, highly feasible and sensitive method for β-thalassaemia gene scanning. PMID:27351925

  17. Development of a High-Resolution Melting Approach for Scanning Beta Globin Gene Point Mutations in the Greek and Other Mediterranean Populations.

    PubMed

    Chassanidis, Christos; Boutou, Effrossyni; Voskaridou, Ersi; Balassopoulou, Angeliki

    2016-01-01

    Beta-thalassaemia is one of the most common autosomal recessive disorders worldwide. The disease's high incidence, which is observed in the broader Mediterranean area has led to the establishment of molecular diagnostics' assays to prevent affected births. Therefore, the development of a reliable, cost-effective and rapid scanning method for β globin gene point mutations, easily adapted to a routine laboratory, is absolutely essential. Here, we describe, for the first time, the development of a High-Resolution Melting Analysis (HRMA) approach, suitable for scanning the particularly heterogeneous beta globin gene mutations present in the Greek population, and thus adaptable to the Mediterranean and other areas where these mutations have been identified. Within this context, β globin gene regions containing mutations frequently identified in the Greek population were divided in ten overlapping amplicons. Our reactions' setup allowed for the simultaneous amplification of multiple primer sets and partial multiplexing, thereby resulting in significant reduction of the experimental time. DNA samples from β-thalassaemia patients/carriers with defined genotypes were tested. Distinct genotypes displayed distinguishable melting curves, enabling accurate detection of mutations. The described HRMA can be adapted to a high-throughput level. It represents a rapid, simple, cost-effective, reliable, highly feasible and sensitive method for β-thalassaemia gene scanning.

  18. Development of a High-Resolution Melting Approach for Scanning Beta Globin Gene Point Mutations in the Greek and Other Mediterranean Populations

    PubMed Central

    Chassanidis, Christos; Boutou, Effrossyni; Voskaridou, Ersi; Balassopoulou, Angeliki

    2016-01-01

    Beta-thalassaemia is one of the most common autosomal recessive disorders worldwide. The disease’s high incidence, which is observed in the broader Mediterranean area has led to the establishment of molecular diagnostics’ assays to prevent affected births. Therefore, the development of a reliable, cost-effective and rapid scanning method for β globin gene point mutations, easily adapted to a routine laboratory, is absolutely essential. Here, we describe, for the first time, the development of a High-Resolution Melting Analysis (HRMA) approach, suitable for scanning the particularly heterogeneous beta globin gene mutations present in the Greek population, and thus adaptable to the Mediterranean and other areas where these mutations have been identified. Within this context, β globin gene regions containing mutations frequently identified in the Greek population were divided in ten overlapping amplicons. Our reactions’ setup allowed for the simultaneous amplification of multiple primer sets and partial multiplexing, thereby resulting in significant reduction of the experimental time. DNA samples from β-thalassaemia patients/carriers with defined genotypes were tested. Distinct genotypes displayed distinguishable melting curves, enabling accurate detection of mutations. The described HRMA can be adapted to a high-throughput level. It represents a rapid, simple, cost-effective, reliable, highly feasible and sensitive method for β-thalassaemia gene scanning. PMID:27351925

  19. Development of a high-resolution melting-based approach for efficient differentiation among Bacillus cereus group isolates.

    PubMed

    Antolinos, Vera; Fernández, Pablo S; Ros-Chumillas, María; Periago, Paula M; Weiss, Julia

    2012-09-01

    Strains belonging to Bacillus cereus Group include six different species, among which are Bacillus thuringiensis, Bacillus weihenstephanensis, and Bacillus cereus sensu stricto, a causative agent of food poisoning. Sequence of the panC-housekeeping gene is used for B. cereus Group affiliation to seven major phylogenetic groups (I-VII) with different ecological niches and variations in thermal growth range and spore heat resistance of B. cereus Group microorganisms varies among phylogenetic groups. We assigned a selection of B. cereus sensu stricto strains related to food poisoning from the Spanish cultivar Collection (Valencia) to Group IV strains based on panC gene sequence. Thermal inactivation assays revealed variability of spore heat resistance within these Group IV strains. Adequate food sanitizing treatments therefore require fast and reliable identification of particular strains. In the present study, feasibility of genotyping via high-resolution melting (HRM) analysis was examined. HRM analysis of amplified polymorphic 16S-23 intergenic spacer region (ISR) region proved to be discriminatory for B. cereus sensu stricto strain typing, while two other polymorphic regions within the bacterial rRNA operon allowed differentiation between Bacillus species, demonstrating its applicability for discrimination on the species and strain level within B. cereus Group.

  20. High-resolution melting curve analysis for genotyping of common SNP in MTHFR gene using fixed-cell suspension.

    PubMed

    Sinthuwiwat, Thivaratana; Poowasanpetch, Phanasit; Wongngamrungroj, Angsana; Promso, Somying; Auewarakul, Chirayu; Mooney, Sean; Tocharoentanaphol, Chintana

    2008-01-01

    Genetic variation in MTHFR might explain the interindividual differences in both therapeutic and toxic responses to the treatment of cancer and rheumatoid arthritis with methotrexate, and can be involved in the sensitivity of developing diseases like cancer and congenital anomalies. We investigated the common sequence variation, C677T, in the MTHFR gene in fixed-cell specimens archived after chromosomal analysis using a novel gene scanning method based on post PCR analysis of high-resolution melting curves (HRM). These fixed specimens were stored after routine chromosomal analysis for 1 year at -20 degrees C in a 3:1 methanol:acetic acid solution. The method revealed a distinct pattern between homozygous and heterozygous alleles. Sensitivity and specificity of the HRM based method were comparable to that obtained by a hybridization probe. While the success rate for genotyping of a common SNP in MTHFR was similar to the hybridization probe approach, the HRM based method was more cost-effective and had a shorter turnaround time. PMID:18725286

  1. Development of a high-resolution melting marker for selecting Fusarium crown and root rot resistance in tomato.

    PubMed

    Kim, Bichseam; Kim, Nahui; Kim, Jun Young; Kim, Byung Sup; Jung, Hee-Jeong; Hwang, Indoek; Noua, Ill-Sup; Sim, Sung-Chur; Park, Younghoon

    2016-03-01

    Fusarium crown and root rot is a severe fungal disease of tomato caused by Fusarium oxysporum f. sp. radicis-lycopersici (FORL). In this study, the genomic location of the FORL-resistance locus was determined using a set of molecular markers on chromosome 9 and an F2 population derived from FORL-resistant inbred 'AV107-4' (Solanum lycopersicum) × susceptible 'L3708' (Solanum pimpinellifolium). Bioassay performed using Korean FORL strain KACC 40031 showed single dominant inheritance of FORL resistance in the F2 population. In all, 13 polymerase chain reaction-based markers encompassing approximately 3.6-72.0 Mb of chromosome 9 were developed based on the Tomato-EXPEN 2000 map and SolCAP Tomato single nucleotide polymorphism array analysis. These markers were genotyped on 345 F2 plants, and the FORL-resistance locus was found to be present on a pericentromeric region of suppressed chromosomal recombination in chromosome 9. The location of the FORL-resistance locus was further confirmed by testing these markers against diverse commercial tomato and stock cultivars resistant to FORL. A restriction fragment length polymorphism marker, PNU-D4, located at approximately 6.1 Mb of chromosome 9 showed the highest match with the resistance locus and was used for conducting high-resolution melting analysis for marker-assisted selection of FORL resistance.

  2. Identification of molecular markers associated with Verticillium wilt resistance in alfalfa (Medicago sativa L.) using high-resolution melting.

    PubMed

    Zhang, Tiejun; Yu, Long-Xi; McCord, Per; Miller, David; Bhamidimarri, Suresh; Johnson, David; Monteros, Maria J; Ho, Julie; Reisen, Peter; Samac, Deborah A

    2014-01-01

    Verticillium wilt, caused by the soilborne fungus, Verticillium alfalfae, is one of the most serious diseases of alfalfa (Medicago sativa L.) worldwide. To identify loci associated with resistance to Verticillium wilt, a bulk segregant analysis was conducted in susceptible or resistant pools constructed from 13 synthetic alfalfa populations, followed by association mapping in two F1 populations consisted of 352 individuals. Simple sequence repeat (SSR) and single nucleotide polymorphism (SNP) markers were used for genotyping. Phenotyping was done by manual inoculation of the pathogen to replicated cloned plants of each individual and disease severity was scored using a standard scale. Marker-trait association was analyzed by TASSEL. Seventeen SNP markers significantly associated with Verticillium wilt resistance were identified and they were located on chromosomes 1, 2, 4, 7 and 8. SNP markers identified on chromosomes 2, 4 and 7 co-locate with regions of Verticillium wilt resistance loci reported in M. truncatula. Additional markers identified on chromosomes 1 and 8 located the regions where no Verticillium resistance locus has been reported. This study highlights the value of SNP genotyping by high resolution melting to identify the disease resistance loci in tetraploid alfalfa. With further validation, the markers identified in this study could be used for improving resistance to Verticillium wilt in alfalfa breeding programs.

  3. Identification of Molecular Markers Associated with Verticillium Wilt Resistance in Alfalfa (Medicago Sativa L.) Using High-Resolution Melting

    PubMed Central

    Zhang, Tiejun; Yu, Long-Xi; McCord, Per; Miller, David; Bhamidimarri, Suresh; Johnson, David; Monteros, Maria J.; Ho, Julie; Reisen, Peter; Samac, Deborah A.

    2014-01-01

    Verticillium wilt, caused by the soilborne fungus, Verticillium alfalfae, is one of the most serious diseases of alfalfa (Medicago sativa L.) worldwide. To identify loci associated with resistance to Verticillium wilt, a bulk segregant analysis was conducted in susceptible or resistant pools constructed from 13 synthetic alfalfa populations, followed by association mapping in two F1 populations consisted of 352 individuals. Simple sequence repeat (SSR) and single nucleotide polymorphism (SNP) markers were used for genotyping. Phenotyping was done by manual inoculation of the pathogen to replicated cloned plants of each individual and disease severity was scored using a standard scale. Marker-trait association was analyzed by TASSEL. Seventeen SNP markers significantly associated with Verticillium wilt resistance were identified and they were located on chromosomes 1, 2, 4, 7 and 8. SNP markers identified on chromosomes 2, 4 and 7 co-locate with regions of Verticillium wilt resistance loci reported in M. truncatula. Additional markers identified on chromosomes 1 and 8 located the regions where no Verticillium resistance locus has been reported. This study highlights the value of SNP genotyping by high resolution melting to identify the disease resistance loci in tetraploid alfalfa. With further validation, the markers identified in this study could be used for improving resistance to Verticillium wilt in alfalfa breeding programs. PMID:25536106

  4. Application of PCR and High-Resolution Melting for Rapid Identification of Yeasts Routinely Isolated in a Clinical Microbiology Laboratory.

    PubMed

    Ninghui, Guo; Bing, Wang; Wei, Ren; Mengmeng, Liu; Meiling, Chu; Dongya, Meng; Liqiong, Yao; Wencheng, Xue

    2015-01-01

    This study aimed to develop a method for rapid and accurate identification of yeasts obtained in the clinic, especially from immunocompromised patients, in order to provide a timely and appropriate antifungal therapy. A total of 112 Candida isolates were analyzed in this study; 28 of them were used to validate the PCR-HRM method in species identification in a blinded manner. Strains were identified by conventional techniques that use VITEK 2 YST cards and Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS). These methods were compared to the newly developed technique based on real-time polymerase-chain reaction high-resolution melting (PCR-HRM). Discordant results were resolved with internal transcribed spacer (ITS) gene sequencing, the "golden standard" used to evaluate the reliability of all methods in identifying yeasts at the species level. PCR-HRM sensitivity was assessed with the isolated strains. VITEK 2, MALDI-TOF-MS, and PCR-HRM accurately identified 89.2% (74/83), 97.6% (81/83), 100% (83/83) of the isolates, respectively. PCR-HRM detection limit was 1fg/μl of yeasts. In validation assays, a 100% accuracy rate was achieved by the use of PCR-HRM. Therefore, the PCR-HRM method is a rapid, sensitive, and specific diagnostic approach, which provides a cost-effective and more suitable alternative for yeast identification in a clinical laboratory. Future research is needed for automation of data acquisition.

  5. Probe-free and sensitive detection of diarrhea-causing pathogens using RT-PCR combined high resolution melting analysis.

    PubMed

    Wang, Hai-Bo; Mo, Qiu-Hua; Wang, Qi; Wu, Bi-Mei; Feng, Zi-Li; Lin, Ji-Can; Yang, Ze

    2016-09-01

    Rapid and sensitive diagnostic methods are needed to help physicians make faster and better treatment decision for patients suffered from diarrhea. In the present study, a probe-free and sensitive RT-PCR combined high resolution melting analysis (HRMA) assay was established successfully for the detection of four major diarrhea-causing pathogens. The lower limit of detection of the assay were 10(0), 10(2), 10(0) and 10(3) copies/reaction for rotaviruses group A, astroviruses serotype 1, noroviruses genogroup II, and sapoviruses genegroup I, respectively, which were 1000-fold, 10-fold, 1000-fold and 10-fold more sensitive than conventional RT-PCR assay developed in parallel and comparable to or higher than commercially available real-time RT-PCR assay. Blinded sample evaluation showed that the assay was 100% concordant to both conventional RT-PCR and commercial real-time RT-PCR, indicating high reliability of the new assay. Therefore, the assay could provide a valuable platform for the probe-free and sensitive diagnosis of these pathogens. PMID:27461241

  6. Mutation Scanning in a Single and a Stacked Genetically Modified (GM) Event by Real-Time PCR and High Resolution Melting (HRM) Analysis

    PubMed Central

    Ben Ali, Sina-Elisabeth; Madi, Zita Erika; Hochegger, Rupert; Quist, David; Prewein, Bernhard; Haslberger, Alexander G.; Brandes, Christian

    2014-01-01

    Genetic mutations must be avoided during the production and use of seeds. In the European Union (EU), Directive 2001/18/EC requires any DNA construct introduced via transformation to be stable. Establishing genetic stability is critical for the approval of genetically modified organisms (GMOs). In this study, genetic stability of two GMOs was examined using high resolution melting (HRM) analysis and real-time polymerase chain reaction (PCR) employing Scorpion primers for amplification. The genetic variability of the transgenic insert and that of the flanking regions in a single oilseed rape variety (GT73) and a stacked maize (MON88017 × MON810) was studied. The GT73 and the 5' region of MON810 showed no instabilities in the examined regions. However; two out of 100 analyzed samples carried a heterozygous point mutation in the 3' region of MON810 in the stacked variety. These results were verified by direct sequencing of the amplified PCR products as well as by sequencing of cloned PCR fragments. The occurrence of the mutation suggests that the 5' region is more suitable than the 3' region for the quantification of MON810. The identification of the single nucleotide polymorphism (SNP) in a stacked event is in contrast to the results of earlier studies of the same MON810 region in a single event where no DNA polymorphism was found. PMID:25365178

  7. Mutation scanning in a single and a stacked genetically modified (GM) event by real-time PCR and high resolution melting (HRM) analysis.

    PubMed

    Ben Ali, Sina-Elisabeth; Madi, Zita Erika; Hochegger, Rupert; Quist, David; Prewein, Bernhard; Haslberger, Alexander G; Brandes, Christian

    2014-01-01

    Genetic mutations must be avoided during the production and use of seeds. In the European Union (EU), Directive 2001/18/EC requires any DNA construct introduced via transformation to be stable. Establishing genetic stability is critical for the approval of genetically modified organisms (GMOs). In this study, genetic stability of two GMOs was examined using high resolution melting (HRM) analysis and real-time polymerase chain reaction (PCR) employing Scorpion primers for amplification. The genetic variability of the transgenic insert and that of the flanking regions in a single oilseed rape variety (GT73) and a stacked maize (MON88017×MON810) was studied. The GT73 and the 5' region of MON810 showed no instabilities in the examined regions. However; two out of 100 analyzed samples carried a heterozygous point mutation in the 3' region of MON810 in the stacked variety. These results were verified by direct sequencing of the amplified PCR products as well as by sequencing of cloned PCR fragments. The occurrence of the mutation suggests that the 5' region is more suitable than the 3' region for the quantification of MON810. The identification of the single nucleotide polymorphism (SNP) in a stacked event is in contrast to the results of earlier studies of the same MON810 region in a single event where no DNA polymorphism was found. PMID:25365178

  8. A novel closed-tube method based on high resolution melting (HRM) analysis for authenticity testing and quantitative detection in Greek PDO Feta cheese.

    PubMed

    Ganopoulos, Ioannis; Sakaridis, Ioannis; Argiriou, Anagnostis; Madesis, Panagiotis; Tsaftaris, Athanasios

    2013-11-15

    Animal species identification of milk and dairy products has received increasing attention concerning food composition, traceability, allergic pathologies and accurate consumer information. Here we sought to develop an easy to use and robust method for species identification in cheese with emphasis on an authenticity control of PDO Feta cheese products. We used specific mitochondrial DNA regions coupled with high resolution melting (HRM) a closed-tube method allowing us to detect bovine, ovine and caprine species and authenticate Greek PDO Feta cheese. The primers successfully amplified DNA isolated from milk and cheese and showed a high degree of specificity. HRM was proven capable of accurately identifying the presence of bovine milk (not allowed in Feta) down to 0.1% and also of quantifying the ratio of sheep to goat milk mixture in different Feta cheese commercial products. In conclusion, HRM analysis can be a faster, with higher resolution and a more cost effective alternative method to authenticate milk and dairy products including PDO Feta cheese and to quantitatively detect its sheep milk adulterations.

  9. Mutation scanning in a single and a stacked genetically modified (GM) event by real-time PCR and high resolution melting (HRM) analysis.

    PubMed

    Ben Ali, Sina-Elisabeth; Madi, Zita Erika; Hochegger, Rupert; Quist, David; Prewein, Bernhard; Haslberger, Alexander G; Brandes, Christian

    2014-10-31

    Genetic mutations must be avoided during the production and use of seeds. In the European Union (EU), Directive 2001/18/EC requires any DNA construct introduced via transformation to be stable. Establishing genetic stability is critical for the approval of genetically modified organisms (GMOs). In this study, genetic stability of two GMOs was examined using high resolution melting (HRM) analysis and real-time polymerase chain reaction (PCR) employing Scorpion primers for amplification. The genetic variability of the transgenic insert and that of the flanking regions in a single oilseed rape variety (GT73) and a stacked maize (MON88017×MON810) was studied. The GT73 and the 5' region of MON810 showed no instabilities in the examined regions. However; two out of 100 analyzed samples carried a heterozygous point mutation in the 3' region of MON810 in the stacked variety. These results were verified by direct sequencing of the amplified PCR products as well as by sequencing of cloned PCR fragments. The occurrence of the mutation suggests that the 5' region is more suitable than the 3' region for the quantification of MON810. The identification of the single nucleotide polymorphism (SNP) in a stacked event is in contrast to the results of earlier studies of the same MON810 region in a single event where no DNA polymorphism was found.

  10. Plasmid-based controls to detect rpoB mutations in Mycobacterium tuberculosis by quantitative polymerase chain reaction-high-resolution melting

    PubMed Central

    da Silva, Joas Lucas; Leite, Gabriela Guimaraes Sousa; Bastos, Gisele Medeiros; Lucas, Beatriz Cacciacarro; Shinohara, Daniel Keniti; Takinami, Joice Sayuri; Miyata, Marcelo; Fajardo, Cristina Moreno; Luchessi, André Ducati; Leite, Clarice Queico Fujimura; Cardoso, Rosilene Fressatti; Hirata, Rosario Dominguez Crespo; Hirata, Mario Hiroyuki

    2013-01-01

    Quantitative polymerase chain reaction-high-resolution melting (qPCR-HRM) analysis was used to screen for mutations related to drug resistance in Mycobacterium tuberculosis. We detected the C526T and C531T mutations in the rifampicin resistance-determining region (RRDR) of the rpoB gene with qPCR-HRM using plasmid-based controls. A segment of the RRDR region from M. tuberculosis H37Rv and from strains carrying C531T or C526T mutations in the rpoB were cloned into pGEM-T vector and these vectors were used as controls in the qPCR-HRM analysis of 54 M. tuberculosis strains. The results were confirmed by DNA sequencing and showed that recombinant plasmids can replace genomic DNA as controls in the qPCR-HRM assay. Plasmids can be handled outside of biosafety level 3 facilities, reducing the risk of contamination and the cost of the assay. Plasmids have a high stability, are normally maintained in Escherichia coli and can be extracted in large amounts. PMID:23440123

  11. Rapid Identification of Echinococcus granulosus and E. canadensis Using High-Resolution Melting (HRM) Analysis by Focusing on a Single Nucleotide Polymorphism.

    PubMed

    Safa, Ahmad Hosseini; Harandi, Majid Fasihi; Tajaddini, Mohammadhasan; Rostami-Nejad, Mohammad; Mohtashami-Pour, Mehdi; Pestehchian, Nader

    2016-07-22

    High-resolution melting (HRM) is a reliable and sensitive scanning method to detect variation in DNA sequences. We used this method to better understand the epidemiology and transmission of Echinococcus granulosus. We tested the use of HRM to discriminate the genotypes of E. granulosus and E. canadensis. One hundred forty-one hydatid cysts were collected from slaughtered animals in different parts of Isfahan-Iran in 2013. After DNA extraction, the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene was amplified using PCR coupled with the HRM curve. The result of HRM analysis using partial the sequences of cox1 gene revealed that 93, 35, and 2 isolates were identified as G1, G3, and G6 genotypes, respectively. A single nucleotide polymorphism (SNP) was found in locus 9867 of the cox1 gene. This is a critical locus for the differentiation between the G6 and G7 genotypes. In the phylogenic tree, the sample with a SNP was located between the G6 and G7 genotypes, which suggest that this isolate has a G6/G7 genotype. The HRM analysis developed in the present study provides a powerful technique for molecular and epidemiological studies on echinococcosis in humans and animals. PMID:26567833

  12. High Resolution Melting Analysis Is a More Sensitive and Effective Alternative to Gel-Based Platforms in Analysis of SSR – An Example in Citrus

    PubMed Central

    Distefano, Gaetano; Caruso, Marco; La Malfa, Stefano; Gentile, Alessandra; Wu, Shu-Biao

    2012-01-01

    High resolution melting curve analysis (HRM) has been used as an efficient, accurate and cost-effective tool to detect single nucleotide polymorphisms (SNPs) or insertions or deletions (INDELs). However, its efficiency, accuracy and applicability to discriminate microsatellite polymorphism have not been extensively assessed. The traditional protocols used for SSR genotyping include PCR amplification of the DNA fragment and the separation of the fragments on electrophoresis-based platform. However, post-PCR handling processes are laborious and costly. Furthermore, SNPs present in the sequences flanking repeat motif cannot be detected by polyacrylamide-gel-electrophoresis based methods. In the present study, we compared the discriminating power of HRM with the traditional electrophoresis-based methods and provided a panel of primers for HRM genotyping in Citrus. The results showed that sixteen SSR markers produced distinct polymorphic melting curves among the Citrus spp investigated through HRM analysis. Among those, 10 showed more genotypes by HRM analysis than capillary electrophoresis owing to the presence of SNPs in the amplicons. For the SSR markers without SNPs present in the flanking region, HRM also gave distinct melting curves which detected same genotypes as were shown in capillary electrophoresis (CE) analysis. Moreover, HRM analysis allowed the discrimination of most of the 15 citrus genotypes and the resulting genetic distance analysis clustered them into three main branches. In conclusion, it has been approved that HRM is not only an efficient and cost-effective alternative of electrophoresis-based method for SSR markers, but also a method to uncover more polymorphisms contributed by SNPs present in SSRs. It was therefore suggested that the panel of SSR markers could be used in a variety of applications in the citrus biodiversity and breeding programs using HRM analysis. Furthermore, we speculate that the HRM analysis can be employed to analyse SSR

  13. Rapid identification of bacterial pathogens in positive blood culture bottles by use of a broad-based PCR assay coupled with high-resolution melt analysis.

    PubMed

    Won, Helen; Rothman, Richard; Ramachandran, Padmini; Hsieh, Yu-Hsiang; Kecojevic, Aleksandar; Carroll, Karen C; Aird, Deborah; Gaydos, Charlotte; Yang, Samuel

    2010-09-01

    We evaluated a broad-based PCR assay coupled with high-resolution melt analysis for rapid bacterial identification in patients with bacterial sepsis. With a reference library of 60 clinically relevant bacterial species, 52 positive blood culture samples were tested. Our assay identified 46/52 samples at the species level, with 100% concordance to culture findings.

  14. High-resolution melt PCR analysis for rapid identification of Chlamydia abortus live vaccine strain 1B among C. abortus strains and field isolates.

    PubMed

    Vorimore, Fabien; Cavanna, Noémie; Vicari, Nadia; Magnino, Simone; Willems, Hermann; Rodolakis, Annie; Siarkou, Victoria I; Laroucau, Karine

    2012-09-01

    We describe a novel high-resolution melt assay that clearly differentiates Chlamydia abortus live vaccine strain 1B from field C. abortus strains and field wild-type isolates based on previously described single nucleotide polymorphisms. This modern genotyping technique is inexpensive, easy to use, and less time-consuming than PCR-RFLP.

  15. A novel method for simultaneous Enterococcus species identification/typing and van genotyping by high resolution melt analysis.

    PubMed

    Gurtler, Volker; Grando, Danilla; Mayall, Barrie C; Wang, Jenny; Ghaly-Derias, Shahbano

    2012-09-01

    In order to develop a typing and identification method for van gene containing Enterococcus faecium, two multiplex PCR reactions were developed for use in HRM-PCR (High Resolution Melt-PCR): (i) vanA, vanB, vanC, vanC23 to detect van genes from different Enterococcus species; (ii) ISR (intergenic spacer region between the 16S and 23S rRNA genes) to detect all Enterococcus species and obtain species and isolate specific HRM curves. To test and validate the method three groups of isolates were tested: (i) 1672 Enterococcus species isolates from January 2009 to December 2009; (ii) 71 isolates previously identified and typed by PFGE (pulsed-field gel electrophoresis) and MLST (multi-locus sequence typing); and (iii) 18 of the isolates from (i) for which ISR sequencing was done. As well as successfully identifying 2 common genotypes by HRM from the Austin Hospital clinical isolates, this study analysed the sequences of all the vanB genes deposited in GenBank and developed a numerical classification scheme for the standardised naming of these vanB genotypes. The identification of Enterococcus faecalis from E. faecium was reliable and stable using ISR PCR. The typing of E. faecium by ISR PCR: (i) detected two variable peaks corresponding to different copy numbers of insertion sequences I and II corresponding to peak I and II respectively; (ii) produced 7 melt profiles for E. faecium with variable copy numbers of sequences I and II; (iii) demonstrated stability and instability of peak heights with equal frequency within the patient sample (36.4±4.5 days and 38.6±5.8 days respectively for 192 patients); (iv) detected ISR-HRM types with as much discrimination as PFGE and more than MLST; and (v) detected ISR-HRM types that differentiated some isolates that were identical by PFGE and MLST. In conjunction with the rapid and accurate van genotyping method described here, this ISR-HRM typing and identification method can be used as a stable identification and typing method with

  16. Establishment of a simple and rapid identification method for Listeria spp. by using high-resolution melting analysis, and its application in food industry.

    PubMed

    Ohshima, Chihiro; Takahashi, Hajime; Phraephaisarn, Chirapiphat; Vesaratchavest, Mongkol; Keeratipibul, Suwimon; Kuda, Takashi; Kimura, Bon

    2014-01-01

    Listeria monocytogenes is the causative bacteria of listeriosis, which has a higher mortality rate than that of other causes of food poisoning. Listeria spp., of which L. monocytogenes is a member, have been isolated from food and manufacturing environments. Several methods have been published for identifying Listeria spp.; however, many of the methods cannot identify newly categorized Listeria spp. Additionally, they are often not suitable for the food industry, owing to their complexity, cost, or time consumption. Recently, high-resolution melting analysis (HRMA), which exploits DNA-sequence differences, has received attention as a simple and quick genomic typing method. In the present study, a new method for the simple, rapid, and low-cost identification of Listeria spp. has been presented using the genes rarA and ldh as targets for HRMA. DNA sequences of 9 Listeria species were first compared, and polymorphisms were identified for each species for primer design. Species specificity of each HRM curve pattern was estimated using type strains of all the species. Among the 9 species, 7 were identified by HRMA using rarA gene, including 3 new species. The remaining 2 species were identified by HRMA of ldh gene. The newly developed HRMA method was then used to assess Listeria isolates from the food industry, and the method efficiency was compared to that of identification by 16S rDNA sequence analysis. The 2 methods were in coherence for 92.6% of the samples, demonstrating the high accuracy of HRMA. The time required for identifying Listeria spp. was substantially low, and the process was considerably simplified, providing a useful and precise method for processing multiple samples per day. Our newly developed method for identifying Listeria spp. is highly valuable; its use is not limited to the food industry, and it can be used for the isolates from the natural environment.

  17. Screening of the BRCA1 gene in Brazilian patients with breast and/or ovarian cancer via high-resolution melting reaction analysis.

    PubMed

    de Oliveira, Eneida Santos; Soares, Bárbara Luisa; Lemos, Sara; Rosa, Reginaldo Cruz Alves; Rodrigues, Angélica Nogueira; Barbosa, Leandro Augusto; de Oliveira Lopes, Débora; dos Santos, Luciana Lara

    2016-04-01

    The aim of this study was to evaluate the profile of BRCA1 mutations among cancer-affected Brazilian women from the Midwest region of Minas Gerais state with clearly defined risk factors for hereditary breast and ovarian cancer (HBOC) syndrome. In this Brazilian region, the first Center for Hereditary Cancer Control began operation in 2011, and 90% of patients receive assistance from the public health service. Eighteen patients at high risk for HBOC were subjected to molecular analysis. Primers were designed for 22 coding exons of the gene; DNA was extracted; and real-time PCR followed by high-resolution melting reaction was performed. The amplicons were sequenced to confirm the identified profiles. Only exon 11 was directly sequenced due its length. Multiplex ligation-dependent probe amplification (MLPA) was performed for those patients in whom no pathogenic mutations were found. Among the 14 alterations identified in this study, the c.5263_5264insC pathogenic mutation was present in two patients (11.1%). Four alterations showed no clinical relevance; one exhibited inconclusive clinical relevance according to the examined databases; and eight alterations presented a divergent classification between the databases. No deletions or duplications were found using the MLPA technique. The HRM methodology was highly sensitive in identifying variants in the BRCA1 gene and can dramatically reduce the amount of sequencing required to identify germline mutations in BRCA genes, enabling cheaper tests and increasing their availability to Brazilian women assisted by the public health service. PMID:26666763

  18. Intracavity DNA melting analysis with optofluidic lasers.

    PubMed

    Lee, Wonsuk; Fan, Xudong

    2012-11-01

    DNA melting analysis holds great promise for simple and fast DNA sequence discrimination. However, conventional fluorescence-based methods suffer from a small differential signal and demanding melting curve analysis, both of which make it difficult to distinguish the target DNA from the mismatched one. Herein, we propose and demonstrate a highly specific intracavity DNA melting analysis scheme utilizing an optofluidic laser. The laser optically amplifies the small yet intrinsic thermal dynamic difference between the target and the single-base-mismatched DNA, resulting in a differential signal that is orders of magnitude greater than with fluorescence-based methods. In particular, the existence of a phase transition between the stimulated laser emission and fluorescence (i.e., spontaneous emission) enables accurate determination of the DNA transition temperature difference. Furthermore, the high differential signal in the intracavity detection allows for scanning of the laser excitation at a fixed temperature to distinguish two DNA sequences, which provides another means for rapid DNA analysis. In this paper, we first theoretically investigate DNA melting analysis using an optofluidic laser and then experimentally explore this scheme with a high-quality optofluidic ring resonator. Distinction of two DNA sequences of up to 100 bases long is demonstrated. The intracavity detection developed here will lead to novel optofluidic devices that enable rapid and simple analysis of DNAs with very long sequences.

  19. High regularity of Z-DNA revealed by ultra high-resolution crystal structure at 0.55;#8201;Å

    SciTech Connect

    Brzezinski, Krzysztof; Brzuszkiewicz, Anna; Dauter, Miroslawa; Kubicki, Maciej; Jaskolski, Mariusz; Dauter, Zbigniew

    2011-12-02

    The crystal structure of a Z-DNA hexamer duplex d(CGCGCG){sub 2} determined at ultra high resolution of 0.55 {angstrom} and refined without restraints, displays a high degree of regularity and rigidity in its stereochemistry, in contrast to the more flexible B-DNA duplexes. The estimations of standard uncertainties of all individually refined parameters, obtained by full-matrix least-squares optimization, are comparable with values that are typical for small-molecule crystallography. The Z-DNA model generated with ultra high-resolution diffraction data can be used to revise the stereochemical restraints applied in lower resolution refinements. Detailed comparisons of the stereochemical library values with the present accurate Z-DNA parameters, shows in general a good agreement, but also reveals significant discrepancies in the description of guanine-sugar valence angles and in the geometry of the phosphate groups.

  20. High resolution mapping of Twist to DNA in Drosophila embryos: Efficient functional analysis and evolutionary conservation.

    PubMed

    Ozdemir, Anil; Fisher-Aylor, Katherine I; Pepke, Shirley; Samanta, Manoj; Dunipace, Leslie; McCue, Kenneth; Zeng, Lucy; Ogawa, Nobuo; Wold, Barbara J; Stathopoulos, Angelike

    2011-04-01

    Cis-regulatory modules (CRMs) function by binding sequence specific transcription factors, but the relationship between in vivo physical binding and the regulatory capacity of factor-bound DNA elements remains uncertain. We investigate this relationship for the well-studied Twist factor in Drosophila melanogaster embryos by analyzing genome-wide factor occupancy and testing the functional significance of Twist occupied regions and motifs within regions. Twist ChIP-seq data efficiently identified previously studied Twist-dependent CRMs and robustly predicted new CRM activity in transgenesis, with newly identified Twist-occupied regions supporting diverse spatiotemporal patterns (>74% positive, n = 31). Some, but not all, candidate CRMs require Twist for proper expression in the embryo. The Twist motifs most favored in genome ChIP data (in vivo) differed from those most favored by Systematic Evolution of Ligands by EXponential enrichment (SELEX) (in vitro). Furthermore, the majority of ChIP-seq signals could be parsimoniously explained by a CABVTG motif located within 50 bp of the ChIP summit and, of these, CACATG was most prevalent. Mutagenesis experiments demonstrated that different Twist E-box motif types are not fully interchangeable, suggesting that the ChIP-derived consensus (CABVTG) includes sites having distinct regulatory outputs. Further analysis of position, frequency of occurrence, and sequence conservation revealed significant enrichment and conservation of CABVTG E-box motifs near Twist ChIP-seq signal summits, preferential conservation of ±150 bp surrounding Twist occupied summits, and enrichment of GA- and CA-repeat sequences near Twist occupied summits. Our results show that high resolution in vivo occupancy data can be used to drive efficient discovery and dissection of global and local cis-regulatory logic.

  1. High-resolution melting analysis of the spa locus reveals significant diversity within sequence type 93 methicillin-resistant Staphylococcus aureus from northern Australia.

    PubMed

    Tong, S Y C; Lilliebridge, R A; Holt, D C; McDonald, M I; Currie, B J; Giffard, P M

    2009-12-01

    High-resolution melting analysis is an inherently robust, easy and inexpensive approach to the examination of genomic regions containing single-nucleotide polymorphisms and hypervariable loci. Staphylococcus aureus sequence type (ST) 93 is a singleton, Panton-Valentine leukocidin-positive clone unique to Australia. A high-resolution melting-based method for the identification of ST93 was developed, and a similar approach was used to reveal diversity within the spa locus of this lineage. Statistical and graphical methods that account for instrumental and operator-dependent variation in high-resolution melting curves were developed, to allow greater confidence and reproducibility in deciding whether another curve is truly different from the baseline curve of an amplicon with known sequence. The data support a very early acquisition, or multiple independent acquisitions, of SCCmec by ST93 methicillin-susceptible S. aureus (MSSA), and the coexistence of MSSA and methicillin-resistant S. aureus versions of the same lineage within northern Australia.

  2. Counterintuitive DNA Sequence Dependence in Supercoiling-Induced DNA Melting

    PubMed Central

    Vlijm, Rifka; v.d. Torre, Jaco; Dekker, Cees

    2015-01-01

    The metabolism of DNA in cells relies on the balance between hybridized double-stranded DNA (dsDNA) and local de-hybridized regions of ssDNA that provide access to binding proteins. Traditional melting experiments, in which short pieces of dsDNA are heated up until the point of melting into ssDNA, have determined that AT-rich sequences have a lower binding energy than GC-rich sequences. In cells, however, the double-stranded backbone of DNA is destabilized by negative supercoiling, and not by temperature. To investigate what the effect of GC content is on DNA melting induced by negative supercoiling, we studied DNA molecules with a GC content ranging from 38% to 77%, using single-molecule magnetic tweezer measurements in which the length of a single DNA molecule is measured as a function of applied stretching force and supercoiling density. At low force (<0.5pN), supercoiling results into twisting of the dsDNA backbone and loop formation (plectonemes), without inducing any DNA melting. This process was not influenced by the DNA sequence. When negative supercoiling is introduced at increasing force, local melting of DNA is introduced. We measured for the different DNA molecules a characteristic force Fchar, at which negative supercoiling induces local melting of the dsDNA. Surprisingly, GC-rich sequences melt at lower forces than AT-rich sequences: Fchar = 0.56pN for 77% GC but 0.73pN for 38% GC. An explanation for this counterintuitive effect is provided by the realization that supercoiling densities of a few percent only induce melting of a few percent of the base pairs. As a consequence, denaturation bubbles occur in local AT-rich regions and the sequence-dependent effect arises from an increased DNA bending/torsional energy associated with the plectonemes. This new insight indicates that an increased GC-content adjacent to AT-rich DNA regions will enhance local opening of the double-stranded DNA helix. PMID:26513573

  3. Counterintuitive DNA Sequence Dependence in Supercoiling-Induced DNA Melting.

    PubMed

    Vlijm, Rifka; V D Torre, Jaco; Dekker, Cees

    2015-01-01

    The metabolism of DNA in cells relies on the balance between hybridized double-stranded DNA (dsDNA) and local de-hybridized regions of ssDNA that provide access to binding proteins. Traditional melting experiments, in which short pieces of dsDNA are heated up until the point of melting into ssDNA, have determined that AT-rich sequences have a lower binding energy than GC-rich sequences. In cells, however, the double-stranded backbone of DNA is destabilized by negative supercoiling, and not by temperature. To investigate what the effect of GC content is on DNA melting induced by negative supercoiling, we studied DNA molecules with a GC content ranging from 38% to 77%, using single-molecule magnetic tweezer measurements in which the length of a single DNA molecule is measured as a function of applied stretching force and supercoiling density. At low force (<0.5pN), supercoiling results into twisting of the dsDNA backbone and loop formation (plectonemes), without inducing any DNA melting. This process was not influenced by the DNA sequence. When negative supercoiling is introduced at increasing force, local melting of DNA is introduced. We measured for the different DNA molecules a characteristic force Fchar, at which negative supercoiling induces local melting of the dsDNA. Surprisingly, GC-rich sequences melt at lower forces than AT-rich sequences: Fchar = 0.56pN for 77% GC but 0.73pN for 38% GC. An explanation for this counterintuitive effect is provided by the realization that supercoiling densities of a few percent only induce melting of a few percent of the base pairs. As a consequence, denaturation bubbles occur in local AT-rich regions and the sequence-dependent effect arises from an increased DNA bending/torsional energy associated with the plectonemes. This new insight indicates that an increased GC-content adjacent to AT-rich DNA regions will enhance local opening of the double-stranded DNA helix.

  4. A new method for simultaneous detection and discrimination of Bovine herpesvirus types 1 (BoHV-1) and 5 (BoHV-5) using real time PCR with high resolution melting (HRM) analysis.

    PubMed

    Marin, M S; Quintana, S; Leunda, M R; Recavarren, M; Pagnuco, I; Späth, E; Pérez, S; Odeón, A

    2016-01-01

    Bovine herpesvirus types 1 (BoHV-1) and 5 (BoHV-5) are antigenically and genetically similar. The aim of this study was to develop a simple and reliable one-step real time PCR assay with high resolution melting (HRM) analysis for the simultaneous detection and differentiation of BoHV-1 and BoHV-5. Optimization of assay conditions was performed with DNA from reference strains. Then, DNA from field isolates, clinical samples and tissue samples of experimentally infected animals were studied by real time PCR-HRM. An efficient amplification of real time PCR products was obtained, and a clear melting curve and appropriate melting peaks for both viruses were achieved in the HRM curve analysis for BoHV type identification. BoHV was identified in all of the isolates and clinical samples, and BoHV types were properly differentiated. Furthermore, viral DNA was detected in 12/18 and 7/18 samples from BoHV-1- and BoHV-5-infected calves, respectively. Real time PCR-HRM achieved a higher sensitivity compared with virus isolation or conventional PCR. In this study, HRM was used as a novel procedure. This method provides rapid, sensitive, specific and simultaneous detection of bovine alpha-herpesviruses DNA. Thus, this technique is an excellent tool for diagnosis, research and epidemiological studies of these viruses in cattle. PMID:26478539

  5. A Rapid Multiplex Real-Time PCR High-Resolution Melt Curve Assay for the Simultaneous Detection of Bacillus cereus, Listeria monocytogenes, and Staphylococcus aureus in Food.

    PubMed

    Forghani, Fereidoun; Wei, Shuai; Oh, Deog-Hwan

    2016-05-01

    Three important foodborne pathogens, Bacillus cereus, Listeria monocytogenes, and Staphylococcus aureus, are of great concern for food safety. They may also coexist in food matrices and, in the case of B. cereus and S. aureus, the resulting illnesses can resemble each other owing to similar symptoms. Therefore, their simultaneous detection may have advantages in terms of cost savings and rapidity. Given this context, a rapid multiplex real-time PCR high-resolution melt curve assay for the simultaneous detection of these three pathogens in food was developed. The assay successfully detected B. cereus (gyrB), L. monocytogenes (hly), and S. aureus (nuc) in a single reaction, and the average melting temperatures were 76.23, 80.19, and 74.01°C, respectively. The application of SYTO9 dye and a slow melt curve analysis ramp rate (0.1°C/s) enabled the production of sharp, high-resolution melt curve peaks that were easily distinguishable from each other. The detection limit in food (milk, rice, and lettuce) was 3.7 × 10(3) CFU/g without an enrichment step and 3.7 × 10(1) CFU/g following the 10-h enrichment. Hence, the assay developed here is specific and sensitive, providing an efficient tool for implementation in food for the simultaneous detection of B. cereus, L. monocytogenes, and S. aureus . PMID:27296430

  6. Identification and Evaluation of Single-Nucleotide Polymorphisms in Allotetraploid Peanut (Arachis hypogaea L.) Based on Amplicon Sequencing Combined with High Resolution Melting (HRM) Analysis.

    PubMed

    Hong, Yanbin; Pandey, Manish K; Liu, Ying; Chen, Xiaoping; Liu, Hong; Varshney, Rajeev K; Liang, Xuanqiang; Huang, Shangzhi

    2015-01-01

    The cultivated peanut (Arachis hypogaea L.) is an allotetraploid (AABB) species derived from the A-genome (Arachis duranensis) and B-genome (Arachis ipaensis) progenitors. Presence of two versions of a DNA sequence based on the two progenitor genomes poses a serious technical and analytical problem during single nucleotide polymorphism (SNP) marker identification and analysis. In this context, we have analyzed 200 amplicons derived from expressed sequence tags (ESTs) and genome survey sequences (GSS) to identify SNPs in a panel of genotypes consisting of 12 cultivated peanut varieties and two diploid progenitors representing the ancestral genomes. A total of 18 EST-SNPs and 44 genomic-SNPs were identified in 12 peanut varieties by aligning the sequence of A. hypogaea with diploid progenitors. The average frequency of sequence polymorphism was higher for genomic-SNPs than the EST-SNPs with one genomic-SNP every 1011 bp as compared to one EST-SNP every 2557 bp. In order to estimate the potential and further applicability of these identified SNPs, 96 peanut varieties were genotyped using high resolution melting (HRM) method. Polymorphism information content (PIC) values for EST-SNPs ranged between 0.021 and 0.413 with a mean of 0.172 in the set of peanut varieties, while genomic-SNPs ranged between 0.080 and 0.478 with a mean of 0.249. Total 33 SNPs were used for polymorphism detection among the parents and 10 selected lines from mapping population Y13Zh (Zhenzhuhei × Yueyou13). Of the total 33 SNPs, nine SNPs showed polymorphism in the mapping population Y13Zh, and seven SNPs were successfully mapped into five linkage groups. Our results showed that SNPs can be identified in allotetraploid peanut with high accuracy through amplicon sequencing and HRM assay. The identified SNPs were very informative and can be used for different genetic and breeding applications in peanut.

  7. Identification and Evaluation of Single-Nucleotide Polymorphisms in Allotetraploid Peanut (Arachis hypogaea L.) Based on Amplicon Sequencing Combined with High Resolution Melting (HRM) Analysis

    PubMed Central

    Hong, Yanbin; Pandey, Manish K.; Liu, Ying; Chen, Xiaoping; Liu, Hong; Varshney, Rajeev K.; Liang, Xuanqiang; Huang, Shangzhi

    2015-01-01

    The cultivated peanut (Arachis hypogaea L.) is an allotetraploid (AABB) species derived from the A-genome (Arachis duranensis) and B-genome (Arachis ipaensis) progenitors. Presence of two versions of a DNA sequence based on the two progenitor genomes poses a serious technical and analytical problem during single nucleotide polymorphism (SNP) marker identification and analysis. In this context, we have analyzed 200 amplicons derived from expressed sequence tags (ESTs) and genome survey sequences (GSS) to identify SNPs in a panel of genotypes consisting of 12 cultivated peanut varieties and two diploid progenitors representing the ancestral genomes. A total of 18 EST-SNPs and 44 genomic-SNPs were identified in 12 peanut varieties by aligning the sequence of A. hypogaea with diploid progenitors. The average frequency of sequence polymorphism was higher for genomic-SNPs than the EST-SNPs with one genomic-SNP every 1011 bp as compared to one EST-SNP every 2557 bp. In order to estimate the potential and further applicability of these identified SNPs, 96 peanut varieties were genotyped using high resolution melting (HRM) method. Polymorphism information content (PIC) values for EST-SNPs ranged between 0.021 and 0.413 with a mean of 0.172 in the set of peanut varieties, while genomic-SNPs ranged between 0.080 and 0.478 with a mean of 0.249. Total 33 SNPs were used for polymorphism detection among the parents and 10 selected lines from mapping population Y13Zh (Zhenzhuhei × Yueyou13). Of the total 33 SNPs, nine SNPs showed polymorphism in the mapping population Y13Zh, and seven SNPs were successfully mapped into five linkage groups. Our results showed that SNPs can be identified in allotetraploid peanut with high accuracy through amplicon sequencing and HRM assay. The identified SNPs were very informative and can be used for different genetic and breeding applications in peanut. PMID:26697032

  8. A real-time ARMS PCR/high-resolution melt curve assay for the detection of the three primary mitochondrial mutations in Leber’s hereditary optic neuropathy

    PubMed Central

    Ryan, Fergus; O’Dwyer, Veronica; Neylan, Derek

    2016-01-01

    Purpose Approximately 95% of patients who are diagnosed with Leber’s hereditary optic neuropathy (LHON) have one of three mitochondrial point mutations responsible for the disease, G3460A, G11778A, and T14484C. The purpose of this study was to develop a novel multiplex real-time amplification-refractory mutation system (ARMS) PCR combined with high-resolution melt curves to identify the individual mutations involved. The study aimed to provide a more robust, cost- and time-effective mutation detection strategy than that offered with currently available methods. The assay reported in this study will allow diagnostic laboratories to avoid costly next-generation sequencing (NGS) assays for most patients with LHON and to focus resources on patients with unknown mutations that require further analysis. Methods The test uses a combination of multiplex allele-specific PCR (ARMS PCR) in combination with a high-resolution melt curve analysis to detect the presence of the mutations in G3460A, G11778A, and T14484C. PCR primer sets were designed to produce a control PCR product and PCR products only in the presence of the mutations in 3460A, 11778A, and 14484C in a multiplex single tube format. Products produce discrete well-separated melt curves to clearly detect the mutations. Results This novel real-time ARMS PCR/high-resolution melt curve assay accurately detected 95% of the mutations that cause LHON. The test has proved to be robust, cost- and time-effective with the real-time closed tube system taking approximately 1 h to complete. Conclusions A novel real-time ARMS PCR/high-resolution melt curve assay is described for the detection of the three primary mitochondrial mutations in LHON. This test provides a simple, robust, easy-to-read output that is cost- and time-effective, thus providing an alternative method to individual endpoint PCR-restriction fragment length polymorphism (RFLP), PCR followed by Sanger sequencing or pyrosequencing, and next-generation sequencing

  9. High-resolution profiling of Drosophila replication start sites reveals a DNA shape and chromatin signature of metazoan origins.

    PubMed

    Comoglio, Federico; Schlumpf, Tommy; Schmid, Virginia; Rohs, Remo; Beisel, Christian; Paro, Renato

    2015-05-01

    At every cell cycle, faithful inheritance of metazoan genomes requires the concerted activation of thousands of DNA replication origins. However, the genetic and chromatin features defining metazoan replication start sites remain largely unknown. Here, we delineate the origin repertoire of the Drosophila genome at high resolution. We address the role of origin-proximal G-quadruplexes and suggest that they transiently stall replication forks in vivo. We dissect the chromatin configuration of replication origins and identify a rich spatial organization of chromatin features at initiation sites. DNA shape and chromatin configurations, not strict sequence motifs, mark and predict origins in higher eukaryotes. We further examine the link between transcription and origin firing and reveal that modulation of origin activity across cell types is intimately linked to cell-type-specific transcriptional programs. Our study unravels conserved origin features and provides unique insights into the relationship among DNA topology, chromatin, transcription, and replication initiation across metazoa.

  10. High-resolution FISH of the entire integrated Epstein-Barr virus genome on extended human DNA.

    PubMed

    Lestou, V S; Strehl, S; Lion, T; Gadner, H; Ambros, P F

    1996-01-01

    Here we report a high-resolution fluorescence in situ hybridization (FISH) analysis of the integrated Epstein-Barr virus (EBV) genome in chromosomes, decondensed interphase nuclear chromatin, and linearly extended chromatin fibers. We analyzed the EBV DNA integrated into the human genome in the well-characterized Burkitt's lymphoma cell line Namalwa, which contains two complete EBV genomes. The integration occurs via the terminal repeats of the virus and was always detectable at chromosome band 1p35. Using the biotinylated BamHIW fragment of the viral DNA, we observed distinct pairs of signals or small nuclear RNA "tracks" within interphase nuclei. FISH to stretched DNA fibers has a higher resolving power and; therefore, enables analysis of the structural organization of DNA. Application of this methodology to linearly extended chromatin of Namalwa cells using different EBV fragments allowed us to visualize the ordered arrangement of the integrated virus. Based on the predicted span of 0.34 nm per base pair for relaxed DNA, length measurements of 30 images showed a good correlation between the mean physical length of hybridized EBV DNA of 52.8 microns (158 kb) without the terminal repeats, and the EBV genomic length of 172 kb, including the terminal repeats. This DNA mapping procedure represents a useful tool for studying the structural organization of integrated viral genomes, and its application will have implications for the understanding of integration processes. PMID:8941376

  11. Strong thermodynamic coupling between sub-ice-shelf melting and sea ice in a high-resolution global sea-ice-ocean isopycnal model

    NASA Astrophysics Data System (ADS)

    Sergienko, O. V.

    2015-12-01

    Sub-ice-shelf melting(freezing) of the Antarctic ice shelves acts as a source(sink) of freshwater, therefore, affects ocean water properties and circulation. In its turn, sub-ice-shelf melting/freezing is controlled by the ocean water properties that reach the sub-ice-shelf cavities. The properties of these water masses are determined by heat and fresh-water exchange with sea ice and atmosphere. Simulations of a high-resolution (1/8 deg) global sea-ice-ocean isopycnal model capable to resolve the ocean circulation in sub-ice-shelf cavities of Antarctic ice shelves and account for the thermodynamic interaction of the circulation with ice shelves show that melting/freezing rates have a strong seasonal cycle with highest melting rates observed in the Austral Fall. On the continental shelf, subsurface ocean temperatures (100-300 m) have a similar seasonal cycle which is lagged with respect to the surface. Shelf temperatures peak in the summertime, followed by rapid cooling towards the freezing point as seasonal ice cover increases. The lagged warming in the subsurface is attributable to reduced heat loss to the atmosphere in the presence of seasonal sea ice. This suggests that the seasonal cycle in melt rates is controlled by the phasing of subsurface temperatures on the continental shelf, which is in turn dominated by sea ice. The outflowing fresh, cold and light meltwater formed in sub-ice-shelf cavities remains in the mixed layer and promotes formation of sea ice and its longer persistence into the Austral Summer. These processes suggest the presence of strong mutual feedbacks between sub-ice-shelf melting and sea ice formation around Antarctic ice shelves.

  12. Preliminary validation of a novel high-resolution melt-based typing method based on the multilocus sequence typing scheme of Streptococcus pyogenes.

    PubMed

    Richardson, L J; Tong, S Y C; Towers, R J; Huygens, F; McGregor, K; Fagan, P K; Currie, B J; Carapetis, J R; Giffard, P M

    2011-09-01

    The major limitation of current typing methods for Streptococcus pyogenes, such as emm sequence typing and T typing, is that these are based on regions subject to considerable selective pressure. Multilocus sequence typing (MLST) is a better indicator of the genetic backbone of a strain but is not widely used due to high costs. The objective of this study was to develop a robust and cost-effective alternative to S. pyogenes MLST. A 10-member single nucleotide polymorphism (SNP) set that provides a Simpson's Index of Diversity (D) of 0.99 with respect to the S. pyogenes MLST database was derived. A typing format involving high-resolution melting (HRM) analysis of small fragments nucleated by each of the resolution-optimized SNPs was developed. The fragments were 59-119 bp in size and, based on differences in G+C content, were predicted to generate three to six resolvable HRM curves. The combination of curves across each of the 10 fragments can be used to generate a melt type (MelT) for each sequence type (ST). The 525 STs currently in the S. pyogenes MLST database are predicted to resolve into 298 distinct MelTs and the method is calculated to provide a D of 0.996 against the MLST database. The MelTs are concordant with the S. pyogenes population structure. To validate the method we examined clinical isolates of S. pyogenes of 70 STs. Curves were generated as predicted by G+C content discriminating the 70 STs into 65 distinct MelTs.

  13. Inferring coarse-grain histone-DNA interaction potentials from high-resolution structures of the nucleosome

    NASA Astrophysics Data System (ADS)

    Meyer, Sam; Everaers, Ralf

    2015-02-01

    The histone-DNA interaction in the nucleosome is a fundamental mechanism of genomic compaction and regulation, which remains largely unknown despite increasing structural knowledge of the complex. In this paper, we propose a framework for the extraction of a nanoscale histone-DNA force-field from a collection of high-resolution structures, which may be adapted to a larger class of protein-DNA complexes. We applied the procedure to a large crystallographic database extended by snapshots from molecular dynamics simulations. The comparison of the structural models first shows that, at histone-DNA contact sites, the DNA base-pairs are shifted outwards locally, consistent with locally repulsive forces exerted by the histones. The second step shows that the various force profiles of the structures under analysis derive locally from a unique, sequence-independent, quadratic repulsive force-field, while the sequence preferences are entirely due to internal DNA mechanics. We have thus obtained the first knowledge-derived nanoscale interaction potential for histone-DNA in the nucleosome. The conformations obtained by relaxation of nucleosomal DNA with high-affinity sequences in this potential accurately reproduce the experimental values of binding preferences. Finally we address the more generic binding mechanisms relevant to the 80% genomic sequences incorporated in nucleosomes, by computing the conformation of nucleosomal DNA with sequence-averaged properties. This conformation differs from those found in crystals, and the analysis suggests that repulsive histone forces are related to local stretch tension in nucleosomal DNA, mostly between adjacent contact points. This tension could play a role in the stability of the complex.

  14. Population diversity of B-locus alleles observed by high-resolution DNA typing.

    PubMed

    Fernandez-Viña, M; Lazaro, A M; Sun, Y; Miller, S; Forero, L; Stastny, P

    1995-03-01

    HLA B-locus typing by group-specific PCR and hybridization with SSOP was performed in 81 10th IHWS B cell lines and 334 selected subjects of our local panel, from four ethnic groups. Most of the B-locus serological specificities were well defined. However, some antigens like B41, B58, B56, the splits of B14, and some subtypes of B5, were not accurately assigned by serology. In the panel studied, we found 17 hybridization patterns that corresponded to probable new alleles. New patterns occurred in the four ethnic groups examined. Multiple subtypes of B35, B5, B15, B41, B44, B57, B58, B70, B14, B40, B22 were found in subjects of the same ethnic group. In view of the poor serological definition of some alleles, and the occurrence of multiple subtypes in the same ethnic population, it appears that high resolution B-locus typing may be an important addition for detection of potentially relevant HLA incompatibilities in transplantation. It should also be valuable for population studies and for the investigation of HLA associations with diseases.

  15. High-resolution crystal structure of Z-DNA in complex with Cr(3+) cations.

    PubMed

    Drozdzal, Pawel; Gilski, Miroslaw; Kierzek, Ryszard; Lomozik, Lechoslaw; Jaskolski, Mariusz

    2015-04-01

    This work is part of our project aimed at characterizing metal-binding properties of left-handed Z-DNA helices. The three Cr(3+) cations found in the asymmetric unit of the d(CGCGCG)2-Cr(3+) crystal structure do not form direct coordination bonds with atoms of the Z-DNA molecule. Instead, the hydrated Cr(3+) ions are engaged in outer-sphere interactions with phosphate groups and O6 and N7 guanine atoms of the DNA. The Cr(3+)(1) and Cr(3+)(2) ions have disordered coordination spheres occupied by six water molecules each. These partial-occupancy chromium cations are 2.354(15) Å apart and are bridged by three water molecules from their hydration spheres. The Cr(3+)(3) cation has distorted square pyramidal geometry. In addition to the high degree of disorder of the DNA backbone, alternate conformations are also observed for the deoxyribose and base moieties of the G2 nucleotide. Our work illuminates the question of conformational flexibility of Z-DNA and its interaction mode with transition-metal cations.

  16. Different Z DNA forming sequences are revealed in phi X174 RFI by high resolution darkfield immuno-electron microscopy.

    PubMed Central

    Revet, B; Zarling, D A; Jovin, T M; Delain, E

    1984-01-01

    The specific interaction between left-handed Z DNA sequences in negatively supercoiled bacteriophage phi X174 replicative form I (RFI) DNA and anti-Z DNA immunoglobulin G (IgG) was investigated by high resolution darkfield immuno-electron microscopy. DNA-antibody complexes were formed and maintained under optimal binding conditions, purified by column chromatography, and visualized after uranyl acetate staining without using aldehyde fixation, shadowing, or second antibody. Bivalent anti-Z DNA IgGs bound to RFI molecules, thus forming intramolecular bridges. They could also oligomerize separate molecules by intermolecular linking of Z DNA sequences. At relatively low ionic strength and low temperature, high affinity anti-Z IgG was retained at certain loci even after restriction endonuclease cleavage of the DNA. In these cleaved molecules some superhelices could be visualized in the loops generated by the bivalent IgG. To our knowledge this is the first example of polypeptide stabilization of local superhelical strain in a cut molecule. Z DNA sequences in phi X174 RFI DNA were mapped. Alternating tracts of purines and pyrimidines starting at nucleotides 763, 1027, 1714, 2146, 2363, 3504, 4161, 4911 and 5345 occur within the nine different anti-Z IgG binding sites which were expressed with varying frequencies (53-3%) on the molecules. Usually, a limited number of sites (generally less than or equal to 2) exists on any one molecule. The formation of multiple Z sites (at the extracted superhelix density) in a given molecule is probably non-cooperative due to relaxation of torsional stress by the B----Z transition. Z sites occur in several different genes, including regions where transcription is attenuated and, in one case, in front of a promoter of transcription. Images Fig. 1. PMID:6241150

  17. Amundsen Sea sector ice shelf thickness, melt rates, and inland response from annual high-resolution DEM mosaics

    NASA Astrophysics Data System (ADS)

    Shean, D. E.; Joughin, I. R.; Smith, B. E.; Alexandrov, O.; Moratto, Z.; Porter, C. C.; Morin, P. J.

    2014-12-01

    Significant grounding line retreat, acceleration, and thinning have occurred along the Amundsen Sea sector of West Antarctica in recent decades. These changes are driven primarily by ice-ocean interaction beneath ice shelves, but existing observations of the spatial distribution, timing, and magnitude of ice shelf melt are limited. Using the NASA Ames Stereo Pipeline, we generated digital elevation models (DEMs) with ~2 m posting from all ~450 available WorldView-1/2 along-track stereopairs for the Amundsen Sea sector. A novel iterative closest point algorithm was used to coregister DEMs to filtered Operation IceBridge ATM/LVIS data and ICESat-1 GLAS data, offering optimal sub-meter horizontal/vertical accuracy. The corrected DEMs were used to produce annual mosaics for the entire ~500x700 km region with focused, sub-annual products for ice shelves and grounding zones. These mosaics provide spatially-continuous measurements of ice shelf topography with unprecedented detail. Using these data, we derive estimates of ice shelf thickness for regions in hydrostatic equilibrium and map networks of sub-shelf melt channels for the Pine Island (PIG), Thwaites, Crosson, and Dotson ice shelves. We also document the break-up of the Thwaites ice shelf and PIG rift evolution leading up to the 2013 calving event. Eulerian difference maps document 2010-2014 thinning over fast-flowing ice streams and adjacent grounded ice. These data reveal the greatest thinning rates over the Smith Glacier ice plain and slopes beyond the margins of the fast-flowing PIG trunk. Difference maps also highlight the filling of at least two subglacial lakes ~30 km upstream of the PIG grounding line in 2011. Lagrangian difference maps reveal the spatial distribution of ice shelf thinning, which can primarily be attributed to basal melt. Preliminary results show focused ice shelf thinning within troughs and large basal channels, especially along the western margin of the Dotson ice shelf. These new data

  18. Quantitative High-Resolution Sensing of DNA Hybridization Using Magnetic Tweezers with Evanescent Illumination

    PubMed Central

    Oliver, Piercen M.; Park, Jin Seon; Vezenov, Dmitri

    2012-01-01

    We applied the combined approach of evanescent nanometry and force spectroscopy using magnetic tweezers to quantify the degree of hybridization of a single synthetic single-stranded DNA oligomer to a resolution approaching a single-base. In this setup, the 200 nucleotide long DNA was covalently attached to the surface of an optically transparent solid support at one end and to the surface of a superparamagnetic fluorescent microsphere (force probe) at the other end. The force was applied to the probes using an electromagnet. The end-to-end molecular distance (i.e. out-of-image-plane position of the force probe) was determined from the intensity of the probe fluorescent image observed with total-internal reflectance microscopy. An equation of state for single stranded DNA molecules under tension (extensible freely jointed chain) was used to derive the penetration depth of the evanescent field and to calibrate the magnetic properties of the force probes. The parameters of the magnetic response of the force probes obtained from the equation of state remained constant when changing the penetration depth, indicating a robust calibration procedure. The results of such a calibration were also confirmed using independently measured probe-surface distances for probes mounted onto cantilevers of an atomic force microscope. Upon hybridization of the complementary 50 nucleotide-long oligomer to the surface-bound 200-mer, the changes in the force-distance curves were consistent with the quantitative conversion of 25% of the original single-stranded DNA to its double-stranded form, which was modeled as an elastic rod. The method presented here for quantifying the hybridization state of the single DNA molecules has potential for determining the degree of hybridization of individual molecules in a single molecule array with high accuracy. PMID:21103547

  19. Dialysis purification of integrase-DNA complexes provides high-resolution atomic force microscopy images: dimeric recombinant HIV-1 integrase binding and specific looping on DNA.

    PubMed

    Tsuruyama, Tatsuaki; Nakai, Tonau; Ohmori, Rei; Ozeki, Munetaka; Tamaki, Keiji; Yoshikawa, Kenichi

    2013-01-01

    It remains difficult to obtain high-resolution atomic force microscopy images of HIV-1 integrase bound to DNA in a dimeric or tetrameric fashion. We therefore constructed specific target DNAs to assess HIV-1 integrase binding and purified the complex by dialysis prior to analysis. Our resulting atomic force microscopy analyses indicated precise size of binding human immunodeficiency virus type 1 (HIV-1) recombinant integrase in a tetrameric manner, inducing formation of a loop-like or figure-eight-like secondary structure in the target DNA. Our findings regarding the target DNA secondary structure provide new insights into the intermediate states of retroviral integration.

  20. Rapid detection and identification of Wuchereria bancrofti, Brugia malayi, B. pahangi, and Dirofilaria immitis in mosquito vectors and blood samples by high resolution melting real-time PCR.

    PubMed

    Thanchomnang, Tongjit; Intapan, Pewpan M; Tantrawatpan, Chairat; Lulitanond, Viraphong; Chungpivat, Sudchit; Taweethavonsawat, Piyanan; Kaewkong, Worasak; Sanpool, Oranuch; Janwan, Penchom; Choochote, Wej; Maleewong, Wanchai

    2013-12-01

    A simple, rapid, and high-throughput method for detection and identification of Wuchereria bancrofti, Brugia malayi, Brugia pahangi, and Dirofilaria immitis in mosquito vectors and blood samples was developed using a real-time PCR combined with high-resolution melting (HRM) analysis. Amplicons of the 4 filarial species were generated from 5S rRNA and spliced leader sequences by the real-time PCR and their melting temperatures were determined by the HRM method. Melting of amplicons from W. bancrofti, B. malayi, D. immitis, and B. pahangi peaked at 81.5±0.2℃, 79.0±0.3℃, 76.8±0.1℃, and 79.9±0.1℃, respectively. This assay is relatively cheap since it does not require synthesis of hybridization probes. Its sensitivity and specificity were 100%. It is a rapid and technically simple approach, and an important tool for population surveys as well as molecular xenomonitoring of parasites in vectors. PMID:24516268

  1. Method for site-specific detection of m6A nucleoside presence in RNA based on high-resolution melting (HRM) analysis.

    PubMed

    Golovina, Anna Y; Dzama, Margarita M; Petriukov, Kirill S; Zatsepin, Timofei S; Sergiev, Petr V; Bogdanov, Alexey A; Dontsova, Olga A

    2014-02-01

    Chemical landscape of natural RNA species is decorated with the large number of modified nucleosides. Some of those could easily be detected by reverse transcription, while others permit only high-performance liquid chromatography or mass-spectrometry detection. Presence of m(6)A nucleoside at a particular position of long RNA molecule is challenging to observe. Here we report an easy and high-throughput method for detection of m(6)A nucleosides in RNA based on high-resolution melting analysis. The method relies on the previous knowledge of the modified nucleoside position at a particular place of RNA and allows rapid screening for conditions or genes necessary for formation of that modification. PMID:24265225

  2. Discovery of common Asian copy number variants using integrated high-resolution array CGH and massively parallel DNA sequencing.

    PubMed

    Park, Hansoo; Kim, Jong-Il; Ju, Young Seok; Gokcumen, Omer; Mills, Ryan E; Kim, Sheehyun; Lee, Seungbok; Suh, Dongwhan; Hong, Dongwan; Kang, Hyunseok Peter; Yoo, Yun Joo; Shin, Jong-Yeon; Kim, Hyun-Jin; Yavartanoo, Maryam; Chang, Young Wha; Ha, Jung-Sook; Chong, Wilson; Hwang, Ga-Ram; Darvishi, Katayoon; Kim, Hyeran; Yang, Song Ju; Yang, Kap-Seok; Kim, Hyungtae; Hurles, Matthew E; Scherer, Stephen W; Carter, Nigel P; Tyler-Smith, Chris; Lee, Charles; Seo, Jeong-Sun

    2010-05-01

    Copy number variants (CNVs) account for the majority of human genomic diversity in terms of base coverage. Here, we have developed and applied a new method to combine high-resolution array comparative genomic hybridization (CGH) data with whole-genome DNA sequencing data to obtain a comprehensive catalog of common CNVs in Asian individuals. The genomes of 30 individuals from three Asian populations (Korean, Chinese and Japanese) were interrogated with an ultra-high-resolution array CGH platform containing 24 million probes. Whole-genome sequencing data from a reference genome (NA10851, with 28.3x coverage) and two Asian genomes (AK1, with 27.8x coverage and AK2, with 32.0x coverage) were used to transform the relative copy number information obtained from array CGH experiments into absolute copy number values. We discovered 5,177 CNVs, of which 3,547 were putative Asian-specific CNVs. These common CNVs in Asian populations will be a useful resource for subsequent genetic studies in these populations, and the new method of calling absolute CNVs will be essential for applying CNV data to personalized medicine.

  3. High-Resolution Specificity from DNA Sequencing Highlights Alternative Modes of Lac Repressor Binding

    PubMed Central

    Zuo, Zheng; Stormo, Gary D.

    2014-01-01

    Knowing the specificity of transcription factors is critical to understanding regulatory networks in cells. The lac repressor–operator system has been studied for many years, but not with high-throughput methods capable of determining specificity comprehensively. Details of its binding interaction and its selection of an asymmetric binding site have been controversial. We employed a new method to accurately determine relative binding affinities to thousands of sequences simultaneously, requiring only sequencing of bound and unbound fractions. An analysis of 2560 different DNA sequence variants, including both base changes and variations in operator length, provides a detailed view of lac repressor sequence specificity. We find that the protein can bind with nearly equal affinities to operators of three different lengths, but the sequence preference changes depending on the length, demonstrating alternative modes of interaction between the protein and DNA. The wild-type operator has an odd length, causing the two monomers to bind in alternative modes, making the asymmetric operator the preferred binding site. We tested two other members of the LacI/GalR protein family and find that neither can bind with high affinity to sites with alternative lengths or shows evidence of alternative binding modes. A further comparison with known and predicted motifs suggests that the lac repressor may be unique in this ability and that this may contribute to its selection. PMID:25209146

  4. A mitochondrial species identification assay for Australian blacktip sharks (Carcharhinus tilstoni, C. limbatus and C. amblyrhynchoides) using real-time PCR and high-resolution melt analysis.

    PubMed

    Morgan, Jess A T; Welch, David J; Harry, Alistair V; Street, Raewyn; Broderick, Damien; Ovenden, Jennifer R

    2011-09-01

    Tropical Australian shark fisheries target two morphologically indistinguishable blacktip sharks, the Australian blacktip (Carcharhinus tilstoni) and the common blacktip (C. limbatus). Their relative contributions to northern and eastern Australian coastal fisheries are unclear because of species identification difficulties. The two species differ in their number of precaudal vertebrae, which is difficult and time consuming to obtain in the field. But, the two species can be distinguished genetically with diagnostic mutations in their mitochondrial DNA ND4 gene. A third closely related sister species, the graceful shark C. amblyrhynchoides, can also be distinguished by species-specific mutations in this gene. DNA sequencing is an effective diagnostic tool, but is relatively expensive and time consuming. In contrast, real-time high-resolution melt (HRM) PCR assays are rapid and relatively inexpensive. These assays amplify regions of DNA with species-specific genetic mutations that result in PCR products with unique melt profiles. A real-time HRM PCR species-diagnostic assay (RT-HRM-PCR) has been developed based on the mtDNA ND4 gene for rapid typing of C. tilstoni, C. limbatus and C. amblyrhynchoides. The assay was developed using ND4 sequences from 66 C. tilstoni, 33. C. limbatus and five C. amblyrhynchoides collected from Indonesia and Australian states and territories; Western Australia, the Northern Territory, Queensland and New South Wales. The assay was shown to be 100% accurate on 160 unknown blacktip shark tissue samples by full mtDNA ND4 sequencing. PMID:21565127

  5. A High-Resolution Map of Segmental DNA Copy Number Variation in the Mouse Genome

    PubMed Central

    Graubert, Timothy A; Selzer, Rebecca R; Richmond, Todd A; Eis, Peggy S; Shannon, William D; Li, Xia; McLeod, Howard L; Cheverud, James M; Ley, Timothy J

    2007-01-01

    Submicroscopic (less than 2 Mb) segmental DNA copy number changes are a recently recognized source of genetic variability between individuals. The biological consequences of copy number variants (CNVs) are largely undefined. In some cases, CNVs that cause gene dosage effects have been implicated in phenotypic variation. CNVs have been detected in diverse species, including mice and humans. Published studies in mice have been limited by resolution and strain selection. We chose to study 21 well-characterized inbred mouse strains that are the focus of an international effort to measure, catalog, and disseminate phenotype data. We performed comparative genomic hybridization using long oligomer arrays to characterize CNVs in these strains. This technique increased the resolution of CNV detection by more than an order of magnitude over previous methodologies. The CNVs range in size from 21 to 2,002 kb. Clustering strains by CNV profile recapitulates aspects of the known ancestry of these strains. Most of the CNVs (77.5%) contain annotated genes, and many (47.5%) colocalize with previously mapped segmental duplications in the mouse genome. We demonstrate that this technique can identify copy number differences associated with known polymorphic traits. The phenotype of previously uncharacterized strains can be predicted based on their copy number at these loci. Annotation of CNVs in the mouse genome combined with sequence-based analysis provides an important resource that will help define the genetic basis of complex traits. PMID:17206864

  6. High-resolution analysis of DNA synthesis start sites and nucleosome architecture at efficient mammalian replication origins.

    PubMed

    Lombraña, Rodrigo; Almeida, Ricardo; Revuelta, Isabel; Madeira, Sofia; Herranz, Gonzalo; Saiz, Néstor; Bastolla, Ugo; Gómez, María

    2013-10-01

    DNA replication origins are poorly characterized genomic regions that are essential to recruit and position the initiation complex to start DNA synthesis. Despite the lack of specific replicator sequences, initiation of replication does not occur at random sites in the mammalian genome. This has lead to the view that DNA accessibility could be a major determinant of mammalian origins. Here, we performed a high-resolution analysis of nucleosome architecture and initiation sites along several origins of different genomic location and firing efficiencies. We found that mammalian origins are highly variable in nucleosome conformation and initiation patterns. Strikingly, initiation sites at efficient CpG island-associated origins always occur at positions of high-nucleosome occupancy. Origin recognition complex (ORC) binding sites, however, occur at adjacent but distinct positions marked by labile nucleosomes. We also found that initiation profiles mirror nucleosome architecture, both at endogenous origins and at a transgene in a heterologous system. Our studies provide a unique insight into the relationship between chromatin structure and initiation sites in the mammalian genome that has direct implications for how the replication programme can be accommodated to diverse epigenetic scenarios.

  7. High-resolution profiling of γH2AX around DNA double strand breaks in the mammalian genome

    PubMed Central

    Iacovoni, Jason S; Caron, Pierre; Lassadi, Imen; Nicolas, Estelle; Massip, Laurent; Trouche, Didier; Legube, Gaëlle

    2010-01-01

    Chromatin acts as a key regulator of DNA-related processes such as DNA damage repair. Although ChIP-chip is a powerful technique to provide high-resolution maps of protein–genome interactions, its use to study DNA double strand break (DSB) repair has been hindered by the limitations of the available damage induction methods. We have developed a human cell line that permits induction of multiple DSBs randomly distributed and unambiguously positioned within the genome. Using this system, we have generated the first genome-wide mapping of γH2AX around DSBs. We found that all DSBs trigger large γH2AX domains, which spread out from the DSB in a bidirectional, discontinuous and not necessarily symmetrical manner. The distribution of γH2AX within domains is influenced by gene transcription, as parallel mappings of RNA Polymerase II and strand-specific expression showed that γH2AX does not propagate on active genes. In addition, we showed that transcription is accurately maintained within γH2AX domains, indicating that mechanisms may exist to protect gene transcription from γH2AX spreading and from the chromatin rearrangements induced by DSBs. PMID:20360682

  8. Precise Detection of IDH1/2 and BRAF Hotspot Mutations in Clinical Glioma Tissues by a Differential Calculus Analysis of High-Resolution Melting Data.

    PubMed

    Hatae, Ryusuke; Hata, Nobuhiro; Yoshimoto, Koji; Kuga, Daisuke; Akagi, Yojiro; Murata, Hideki; Suzuki, Satoshi O; Mizoguchi, Masahiro; Iihara, Koji

    2016-01-01

    High resolution melting (HRM) is a simple and rapid method for screening mutations. It offers various advantages for clinical diagnostic applications. Conventional HRM analysis often yields equivocal results, especially for surgically obtained tissues. We attempted to improve HRM analyses for more effective applications to clinical diagnostics. HRM analyses were performed for IDH1R132 and IDH2R172 mutations in 192 clinical glioma samples in duplicate and these results were compared with sequencing results. BRAFV600E mutations were analyzed in 52 additional brain tumor samples. The melting profiles were used for differential calculus analyses. Negative second derivative plots revealed additional peaks derived from heteroduplexes in PCR products that contained mutations; this enabled unequivocal visual discrimination of the mutations. We further developed a numerical expression, the HRM-mutation index (MI), to quantify the heteroduplex-derived peak of the mutational curves. Using this expression, all IDH1 mutation statuses matched those ascertained by sequencing, with the exception of three samples. These discordant results were all derived from the misinterpretation of sequencing data. The effectiveness of our approach was further validated by analyses of IDH2R172 and BRAFV600E mutations. The present analytical method enabled an unequivocal and objective HRM analysis and is suitable for reliable mutation scanning in surgically obtained glioma tissues. This approach could facilitate molecular diagnostics in clinical environments. PMID:27529619

  9. Rapid and Reliable Detection of Glucose-6-Phosphate Dehydrogenase (G6PD) Gene Mutations in Han Chinese Using High-Resolution Melting Analysis

    PubMed Central

    Yan, Jing-bin; Xu, Hong-ping; Xiong, Can; Ren, Zhao-rui; Tian, Guo-li; Zeng, Fanyi; Huang, Shu-zhen

    2010-01-01

    Glucose-6-phosphate dehydrogenase (G6PD) deficiency, an X-linked inherited disease, is one of the most common enzymopathies and affects over 400 million people worldwide. In China at least 21 distinct point mutations have been identified so far. In this study high-resolution melting (HRM) analysis was used to screen for G6PD mutations in 260 unrelated Han Chinese individuals, and the rapidity and reliability of this method was investigated. The mutants were readily differentiated by using HRM analysis, which produced distinct melting curves for each tested mutation. Interestingly, G1388A and G1376T, the two most common variants accounting for 50% to 60% of G6PD deficiency mutations in the Chinese population, could be differentiated in a single reaction. Further, two G6PD mutations not previously reported in the Chinese population were identified in this study. One of these mutations, designated “G6PD Jiangxi G1340T,” involved a G1340T substitution in exon 11, predicting a Gly447Val change in the protein. The other mutation involved a C406T substitution in exon 5. The frequencies of the common polymorphism site C1311T/IVS (intervening sequence) XI t93c between patients with G6PD and healthy volunteers were not significantly different. Thus, HRM analysis will be a useful alternative for screening G6PD mutations. PMID:20203002

  10. Rapid and reliable detection of glucose-6-phosphate dehydrogenase (G6PD) gene mutations in Han Chinese using high-resolution melting analysis.

    PubMed

    Yan, Jing-bin; Xu, Hong-ping; Xiong, Can; Ren, Zhao-rui; Tian, Guo-li; Zeng, Fanyi; Huang, Shu-zhen

    2010-05-01

    Glucose-6-phosphate dehydrogenase (G6PD) deficiency, an X-linked inherited disease, is one of the most common enzymopathies and affects over 400 million people worldwide. In China at least 21 distinct point mutations have been identified so far. In this study high-resolution melting (HRM) analysis was used to screen for G6PD mutations in 260 unrelated Han Chinese individuals, and the rapidity and reliability of this method was investigated. The mutants were readily differentiated by using HRM analysis, which produced distinct melting curves for each tested mutation. Interestingly, G1388A and G1376T, the two most common variants accounting for 50% to 60% of G6PD deficiency mutations in the Chinese population, could be differentiated in a single reaction. Further, two G6PD mutations not previously reported in the Chinese population were identified in this study. One of these mutations, designated "G6PD Jiangxi G1340T," involved a G1340T substitution in exon 11, predicting a Gly447Val change in the protein. The other mutation involved a C406T substitution in exon 5. The frequencies of the common polymorphism site C1311T/IVS (intervening sequence) XI t93c between patients with G6PD and healthy volunteers were not significantly different. Thus, HRM analysis will be a useful alternative for screening G6PD mutations. PMID:20203002

  11. Rapid and specific detection of Salmonella in water samples using real-time PCR and High Resolution Melt (HRM) curve analysis.

    PubMed

    van Blerk, G N; Leibach, L; Mabunda, A; Chapman, A; Louw, D

    2011-01-01

    A real-time PCR assay combined with a pre-enrichment step for the specific and rapid detection of Salmonella in water samples is described. Following amplification of the invA gene target, High Resolution Melt (HRM) curve analysis was used to discriminate between products formed and to positively identify invA amplification. The real-time PCR assay was evaluated for specificity and sensitivity. The assay displayed 100% specificity for Salmonella and combined with a 16-18 h non-selective pre-enrichment step, the assay proved to be highly sensitive with a detection limit of 1.0 CFU/ml for surface water samples. The detection assay also demonstrated a high intra-run and inter-run repeatability with very little variation in invA amplicon melting temperature. When applied to water samples received routinely by the laboratory, the assay showed the presence of Salmonella in particularly surface water and treated effluent samples. Using the HRM based assay, the time required for Salmonella detection was drastically shortened to less than 24 h compared to several days when using standard culturing methods. This assay provides a useful tool for routine water quality monitoring as well as for quick screening during disease outbreaks.

  12. Precise Detection of IDH1/2 and BRAF Hotspot Mutations in Clinical Glioma Tissues by a Differential Calculus Analysis of High-Resolution Melting Data.

    PubMed

    Hatae, Ryusuke; Hata, Nobuhiro; Yoshimoto, Koji; Kuga, Daisuke; Akagi, Yojiro; Murata, Hideki; Suzuki, Satoshi O; Mizoguchi, Masahiro; Iihara, Koji

    2016-01-01

    High resolution melting (HRM) is a simple and rapid method for screening mutations. It offers various advantages for clinical diagnostic applications. Conventional HRM analysis often yields equivocal results, especially for surgically obtained tissues. We attempted to improve HRM analyses for more effective applications to clinical diagnostics. HRM analyses were performed for IDH1R132 and IDH2R172 mutations in 192 clinical glioma samples in duplicate and these results were compared with sequencing results. BRAFV600E mutations were analyzed in 52 additional brain tumor samples. The melting profiles were used for differential calculus analyses. Negative second derivative plots revealed additional peaks derived from heteroduplexes in PCR products that contained mutations; this enabled unequivocal visual discrimination of the mutations. We further developed a numerical expression, the HRM-mutation index (MI), to quantify the heteroduplex-derived peak of the mutational curves. Using this expression, all IDH1 mutation statuses matched those ascertained by sequencing, with the exception of three samples. These discordant results were all derived from the misinterpretation of sequencing data. The effectiveness of our approach was further validated by analyses of IDH2R172 and BRAFV600E mutations. The present analytical method enabled an unequivocal and objective HRM analysis and is suitable for reliable mutation scanning in surgically obtained glioma tissues. This approach could facilitate molecular diagnostics in clinical environments.

  13. Application of the High Resolution Melting analysis for genetic mapping of Sequence Tagged Site markers in narrow-leafed lupin (Lupinus angustifolius L.).

    PubMed

    Kamel, Katarzyna A; Kroc, Magdalena; Święcicki, Wojciech

    2015-01-01

    Sequence tagged site (STS) markers are valuable tools for genetic and physical mapping that can be successfully used in comparative analyses among related species. Current challenges for molecular markers genotyping in plants include the lack of fast, sensitive and inexpensive methods suitable for sequence variant detection. In contrast, high resolution melting (HRM) is a simple and high-throughput assay, which has been widely applied in sequence polymorphism identification as well as in the studies of genetic variability and genotyping. The present study is the first attempt to use the HRM analysis to genotype STS markers in narrow-leafed lupin (Lupinus angustifolius L.). The sensitivity and utility of this method was confirmed by the sequence polymorphism detection based on melting curve profiles in the parental genotypes and progeny of the narrow-leafed lupin mapping population. Application of different approaches, including amplicon size and a simulated heterozygote analysis, has allowed for successful genetic mapping of 16 new STS markers in the narrow-leafed lupin genome.

  14. Precise Detection of IDH1/2 and BRAF Hotspot Mutations in Clinical Glioma Tissues by a Differential Calculus Analysis of High-Resolution Melting Data

    PubMed Central

    Hatae, Ryusuke; Yoshimoto, Koji; Kuga, Daisuke; Akagi, Yojiro; Murata, Hideki; Suzuki, Satoshi O.; Mizoguchi, Masahiro; Iihara, Koji

    2016-01-01

    High resolution melting (HRM) is a simple and rapid method for screening mutations. It offers various advantages for clinical diagnostic applications. Conventional HRM analysis often yields equivocal results, especially for surgically obtained tissues. We attempted to improve HRM analyses for more effective applications to clinical diagnostics. HRM analyses were performed for IDH1R132 and IDH2R172 mutations in 192 clinical glioma samples in duplicate and these results were compared with sequencing results. BRAFV600E mutations were analyzed in 52 additional brain tumor samples. The melting profiles were used for differential calculus analyses. Negative second derivative plots revealed additional peaks derived from heteroduplexes in PCR products that contained mutations; this enabled unequivocal visual discrimination of the mutations. We further developed a numerical expression, the HRM-mutation index (MI), to quantify the heteroduplex-derived peak of the mutational curves. Using this expression, all IDH1 mutation statuses matched those ascertained by sequencing, with the exception of three samples. These discordant results were all derived from the misinterpretation of sequencing data. The effectiveness of our approach was further validated by analyses of IDH2R172 and BRAFV600E mutations. The present analytical method enabled an unequivocal and objective HRM analysis and is suitable for reliable mutation scanning in surgically obtained glioma tissues. This approach could facilitate molecular diagnostics in clinical environments. PMID:27529619

  15. Identification and Differentiation of Monilinia Species Causing Brown Rot of Pome and Stone Fruit using High-Resolution Melting (HRM) Analysis.

    PubMed

    Papavasileiou, Antonios; Madesis, Panagiotis B; Karaoglanidis, George S

    2016-09-01

    Brown rot is a devastating disease of stone fruit caused by Monilinia spp. Among these species, Monilinia fructicola is a quarantine pathogen in Europe but has recently been detected in several European countries. Identification of brown rot agents relies on morphological differences or use of molecular methods requiring fungal isolation. The current study was initiated to develop and validate a high-resolution melting (HRM) method for the identification of the Monilinia spp. and for the detection of M. fructicola among other brown rot pathogens. Based on the sequence of the cytb intron from M. laxa, M. fructicola, M. fructigena, M. mumecola, M. linhartiana, and M. yunnanensis isolates originating from several countries, a pair of universal primers for species identification and a pair of primers specific to M. fructicola were designed. The specificity of the primers was verified to ensure against cross-reaction with other fungal species. The melting curve analysis using the universal primers generated six different HRM curve profiles, each one specific for each species. Τhe HRM analysis primers specific to M. fructicola amplified a 120-bp region with a distinct melt profile corresponding to the presence of M. fructicola, regardless of the presence of other species. HRM analysis can be a useful tool for rapid identification and differentiation of the six Monilinia spp. using a single primer pair. This novel assay has the potential for simultaneous identification and differentiation of the closely related Monilinia spp. as well as for the differentiation of M. fructicola from other common pathogens or saprophytes that may occur on the diseased fruit. PMID:27247082

  16. Probing the structural disorder of basalts and slab-driven andesite melts: Insights from high-resolution solid-state NMR study

    NASA Astrophysics Data System (ADS)

    Park, S.; Lee, S.

    2012-12-01

    Whereas the structure of multi-component silicate melts has strong implication for the properties of natural silicate melts and relevant magmatic processes in Earth's mantle and crust, little is known about their atomic structures due to lack of suitable experimental probes of multi-component amorphous oxides. Although most of the progress in melt structure has been made for relatively simple binary and ternary silicate glasses, recent advances in high-resolution solid-state NMR (nuclear magnetic resonance) unveil previously unknown structural details of multi-component silicate melts (Lee, S. K. and Sung, S., Chem. Geol., 2008, 256, 326; Lee et al., P. Natl. Acad. Sci. USA., 2011, 108, 6847; Park and Lee, Geochim. Cosmochim. Acta, 2012, 80, 125). In this study we report experimental results on the effects of composition. atomic structure of CaO-MgO-Al_{2} O_{3} -SiO_{2} (CMAS) glasses in diopside (CaMgSi_{2}O_{6}) and Ca-tschermakite (CaAl_{2}SiO_{6}) join and glass in the diopside-anorthite eutectic composition (Di_{64}An_{36})—model systems for basaltic melts—using solid-state NMR. We also report the first high-resolution experimental results on the atomic structure of CaO-MgO-Na_{2}O-Al_{2}O_{3}-SiO_{2} (CMNAS) glasses in diopside and jadeite (NaAlSi_{2}O_{6}) join, and glass in the natural phonolite composition (CaO: MgO: Na_{2}O: K_{2}O: Al_{2}O_{3}: SiO_{2}= 1.4: 8.0: 9.0: 3.8: 13: 64 mol%), a model system for slab driven andesite melts. The Al-27 3QMAS (triple quantum magic angle spinning) NMR spectra of CMAS glasses in diopside-Ca-tschermakite join show predominant ^{[4]}Al and a non-negligible fraction of ^{[5]}Al. Approximately 3.3% of ^{[5]}Al is observed for Di_{64}An_{36} glass. The Al-27 3QMAS NMR spectra of CMNAS glasses in diopside and jadeite join show mostly ^{[4]}Al and a non-negligible fraction of ^{[5]}Al (X_{Diopside}=0.75, the mole fraction of diopside content). While the C_{q} (quadrupolar coupling constant) of ^{[4]}Al for glasses in

  17. Fast Prediction of DNA Melting Bubbles Using DNA Thermodynamic Stability.

    PubMed

    Zrimec, Jan; Lapanje, Ales

    2015-01-01

    DNA melting bubbles are the basis of many DNA-protein interactions, such as those in regulatory DNA regions driving gene expression, DNA replication and bacterial horizontal gene transfer. Bubble formation is affected by DNA duplex stability and thermally induced duplex destabilization (TIDD). Although prediction of duplex stability with the nearest neighbor (NN) method is much faster than prediction of TIDD with the Peyrard-Bishop-Dauxois (PBD) model, PBD predicted TIDD defines regulatory DNA regions with higher accuracy and detail. Here, we considered that PBD predicted TIDD is inherently related to the intrinsic duplex stabilities of destabilization regions. We show by regression modeling that NN duplex stabilities can be used to predict TIDD almost as accurately as is predicted with PBD. Predicted TIDD is in fact ascribed to non-linear transformation of NN duplex stabilities in destabilization regions as well as effects of neighboring regions relative to destabilization size. Since the prediction time of our models is over six orders of magnitude shorter than that of PBD, the models present an accessible tool for researchers. TIDD can be predicted on our webserver at http://tidd.immt.eu.

  18. Unraveling Host-Vector-Arbovirus Interactions by Two-Gene High Resolution Melting Mosquito Bloodmeal Analysis in a Kenyan Wildlife-Livestock Interface

    PubMed Central

    Omondi, David; Masiga, Daniel K.; Ajamma, Yvonne Ukamaka; Fielding, Burtram C.; Njoroge, Laban; Villinger, Jandouwe

    2015-01-01

    The blood-feeding patterns of mosquitoes are directly linked to the spread of pathogens that they transmit. Efficient identification of arthropod vector bloodmeal hosts can identify the diversity of vertebrate species potentially involved in disease transmission cycles. While molecular bloodmeal analyses rely on sequencing of cytochrome b (cyt b) or cytochrome oxidase 1 gene PCR products, recently developed bloodmeal host identification based on high resolution melting (HRM) analyses of cyt b PCR products is more cost-effective. To resolve the diverse vertebrate hosts that mosquitoes may potentially feed on in sub-Saharan Africa, we utilized HRM profiles of both cyt b and 16S ribosomal RNA genes. Among 445 blood-fed Aedeomyia, Aedes, Anopheles, Culex, Mansonia, and Mimomyia mosquitoes from Kenya’s Lake Victoria and Lake Baringo regions where many mosquito-transmitted pathogens are endemic, we identified 33 bloodmeal hosts including humans, eight domestic animal species, six peridomestic animal species and 18 wildlife species. This resolution of vertebrate host species was only possible by comparing profiles of both cyt b and 16S markers, as melting profiles of some pairs of species were similar for either marker but not both. We identified mixed bloodmeals in a Culex pipiens from Mbita that had fed on a goat and a human and in two Mansonia africana mosquitoes from Baringo that each had fed on a rodent (Arvicanthis niloticus) in addition to a human or baboon. We further detected Sindbis and Bunyamwera viruses in blood-fed mosquito homogenates by Vero cell culture and RT-PCR in Culex, Aedeomyia, Anopheles and Mansonia mosquitoes from Baringo that had fed on humans and livestock. The observed mosquito feeding on both arbovirus amplifying hosts (including sheep and goats) and possible arbovirus reservoirs (birds, porcupine, baboons, rodents) informs arbovirus disease epidemiology and vector control strategies. PMID:26230507

  19. Unraveling Host-Vector-Arbovirus Interactions by Two-Gene High Resolution Melting Mosquito Bloodmeal Analysis in a Kenyan Wildlife-Livestock Interface.

    PubMed

    Omondi, David; Masiga, Daniel K; Ajamma, Yvonne Ukamaka; Fielding, Burtram C; Njoroge, Laban; Villinger, Jandouwe

    2015-01-01

    The blood-feeding patterns of mosquitoes are directly linked to the spread of pathogens that they transmit. Efficient identification of arthropod vector bloodmeal hosts can identify the diversity of vertebrate species potentially involved in disease transmission cycles. While molecular bloodmeal analyses rely on sequencing of cytochrome b (cyt b) or cytochrome oxidase 1 gene PCR products, recently developed bloodmeal host identification based on high resolution melting (HRM) analyses of cyt b PCR products is more cost-effective. To resolve the diverse vertebrate hosts that mosquitoes may potentially feed on in sub-Saharan Africa, we utilized HRM profiles of both cyt b and 16S ribosomal RNA genes. Among 445 blood-fed Aedeomyia, Aedes, Anopheles, Culex, Mansonia, and Mimomyia mosquitoes from Kenya's Lake Victoria and Lake Baringo regions where many mosquito-transmitted pathogens are endemic, we identified 33 bloodmeal hosts including humans, eight domestic animal species, six peridomestic animal species and 18 wildlife species. This resolution of vertebrate host species was only possible by comparing profiles of both cyt b and 16S markers, as melting profiles of some pairs of species were similar for either marker but not both. We identified mixed bloodmeals in a Culex pipiens from Mbita that had fed on a goat and a human and in two Mansonia africana mosquitoes from Baringo that each had fed on a rodent (Arvicanthis niloticus) in addition to a human or baboon. We further detected Sindbis and Bunyamwera viruses in blood-fed mosquito homogenates by Vero cell culture and RT-PCR in Culex, Aedeomyia, Anopheles and Mansonia mosquitoes from Baringo that had fed on humans and livestock. The observed mosquito feeding on both arbovirus amplifying hosts (including sheep and goats) and possible arbovirus reservoirs (birds, porcupine, baboons, rodents) informs arbovirus disease epidemiology and vector control strategies.

  20. Unraveling Host-Vector-Arbovirus Interactions by Two-Gene High Resolution Melting Mosquito Bloodmeal Analysis in a Kenyan Wildlife-Livestock Interface.

    PubMed

    Omondi, David; Masiga, Daniel K; Ajamma, Yvonne Ukamaka; Fielding, Burtram C; Njoroge, Laban; Villinger, Jandouwe

    2015-01-01

    The blood-feeding patterns of mosquitoes are directly linked to the spread of pathogens that they transmit. Efficient identification of arthropod vector bloodmeal hosts can identify the diversity of vertebrate species potentially involved in disease transmission cycles. While molecular bloodmeal analyses rely on sequencing of cytochrome b (cyt b) or cytochrome oxidase 1 gene PCR products, recently developed bloodmeal host identification based on high resolution melting (HRM) analyses of cyt b PCR products is more cost-effective. To resolve the diverse vertebrate hosts that mosquitoes may potentially feed on in sub-Saharan Africa, we utilized HRM profiles of both cyt b and 16S ribosomal RNA genes. Among 445 blood-fed Aedeomyia, Aedes, Anopheles, Culex, Mansonia, and Mimomyia mosquitoes from Kenya's Lake Victoria and Lake Baringo regions where many mosquito-transmitted pathogens are endemic, we identified 33 bloodmeal hosts including humans, eight domestic animal species, six peridomestic animal species and 18 wildlife species. This resolution of vertebrate host species was only possible by comparing profiles of both cyt b and 16S markers, as melting profiles of some pairs of species were similar for either marker but not both. We identified mixed bloodmeals in a Culex pipiens from Mbita that had fed on a goat and a human and in two Mansonia africana mosquitoes from Baringo that each had fed on a rodent (Arvicanthis niloticus) in addition to a human or baboon. We further detected Sindbis and Bunyamwera viruses in blood-fed mosquito homogenates by Vero cell culture and RT-PCR in Culex, Aedeomyia, Anopheles and Mansonia mosquitoes from Baringo that had fed on humans and livestock. The observed mosquito feeding on both arbovirus amplifying hosts (including sheep and goats) and possible arbovirus reservoirs (birds, porcupine, baboons, rodents) informs arbovirus disease epidemiology and vector control strategies. PMID:26230507

  1. Characterization of Genetic Diversity of Bacillus anthracis in France by Using High-Resolution Melting Assays and Multilocus Variable-Number Tandem-Repeat Analysis ▿ †

    PubMed Central

    Derzelle, S.; Laroche, S.; Le Flèche, P.; Hauck, Y.; Thierry, S.; Vergnaud, G.; Madani, N.

    2011-01-01

    Using high-resolution melting (HRM) analysis, we developed a cost-effective method to genotype a set of 13 phylogenetically informative single-nucleotide polymorphisms (SNPs) within the genome of Bacillus anthracis. SNP discrimination assays were performed in monoplex or duplex and applied to 100 B. anthracis isolates collected in France from 1953 to 2009 and a few reference strains. HRM provided a reliable and cheap alternative to subtype B. anthracis into one of the 12 major sublineages or subgroups. All strains could be correctly positioned on the canonical SNP (canSNP) phylogenetic tree, except the divergent Pasteur vaccine strain ATCC 4229. We detected the cooccurrence of three canSNP subgroups in France. The dominant B.Br.CNEVA sublineage was found to be prevalent in the Alps, the Pyrenees, the Auvergne region, and the Saône-et-Loire department. Strains affiliated with the A.Br.008/009 subgroup were observed throughout most of the country. The minor A.Br.001/002 subgroup was restricted to northeastern France. Multiple-locus variable-number tandem-repeat analysis using 24 markers further resolved French strains into 60 unique profiles and identified some regional patterns. Diversity found within the A.Br.008/009 and B.Br.CNEVA subgroups suggests that these represent old, ecologically established clades in France. Phylogenetic relationships with strains from other parts of the world are discussed. PMID:21998431

  2. Rapid Detection and Simultaneous Genotyping of Cronobacter spp. (formerly Enterobacter sakazakii) in Powdered Infant Formula Using Real-time PCR and High Resolution Melting (HRM) Analysis

    PubMed Central

    Cai, Xian-Quan; Yu, Hai-Qiong; Ruan, Zhou-Xi; Yang, Lei-Liang; Bai, Jian-Shan; Qiu, De-Yi; Jian, Zhi-Hua; Xiao, Yi-Qian; Yang, Jie-Yang; Le, Thanh Hoa; Zhu, Xing-Quan

    2013-01-01

    Cronobacter spp. is an emerging pathogen that causes meningitis, sepsis, bacteremia, and necrotizing enterocolitis in neonates and children. The present study developed an assay integrating real-time PCR and high resolution melting (HRM) analysis targeting the OmpA gene for the specific detection and rapid identification of Cronobacter spp. (formerly Enterobacter sakazakii) in powdered infant formula. Eleven Cronobacter field isolates and 25 reference strains were examined using one pair of primers, having the accuracy of 100% in reference to conventional methods. The assay was proved to be highly sensitive with a detection limit of 102 CFU/ml without pre-enrichment, and highly concordant (100%) when compared with ISO-IDF 22964 in 89 actual samples. The method performed for Cronobacter spp. detection was less than 24 h, drastically shortened, compared to several days using standard culturing method, it is probe-free and reduces a risk of PCR carryover. Moreover, all Cronobacter strains examined in this study were genotyped into two species according to their HRM profiles. The established method should provide a molecular tool for direct detection and simultaneous genotyping of Cronobacter spp. in powdered infant formula. PMID:23825624

  3. Rapid detection and non-subjective characterisation of infectious bronchitis virus isolates using high-resolution melt curve analysis and a mathematical model.

    PubMed

    Hewson, Kylie; Noormohammadi, Amir H; Devlin, Joanne M; Mardani, Karim; Ignjatovic, Jagoda

    2009-01-01

    Infectious bronchitis virus (IBV) is a coronavirus that causes upper respiratory, renal and/or reproductive diseases with high morbidity in poultry. Classification of IBV is important for implementation of vaccination strategies to control the disease in commercial poultry. Currently, the lengthy process of sequence analysis of the IBV S1 gene is considered the gold standard for IBV strain identification, with a high nucleotide identity (e.g. > or =95%) indicating related strains. However, this gene has a high propensity to mutate and/or undergo recombination, and alone it may not be reliable for strain identification. A real-time polymerase chain reaction (RT-PCR) combined with high-resolution melt (HRM) curve analysis was developed based on the 3'UTR of IBV for rapid detection and classification of IBV from commercial poultry. HRM curves generated from 230 to 435-bp PCR products of several IBV strains were subjected to further analysis using a mathematical model also developed during this study. It was shown that a combination of HRM curve analysis and the mathematical model could reliably group 189 out of 190 comparisons of pairs of IBV strains in accordance with their 3'UTR and S1 gene identities. The newly developed RT-PCR/HRM curve analysis model could detect and rapidly identify novel and vaccine-related IBV strains, as confirmed by S1 gene and 3'UTR nucleotide sequences. This model is a rapid, reliable, accurate and non-subjective system for detection of IBVs in poultry flocks.

  4. Molecular identification of Mycobacterium avium subsp. silvaticum by duplex high-resolution melt analysis and subspecies-specific real-time PCR.

    PubMed

    Rónai, Zsuzsanna; Csivincsik, Ágnes; Dán, Ádám

    2015-05-01

    Accurate identification of mycobacterial species and subspecies is essential to evaluate their significance and to perform epidemiological studies. The subspecies of Mycobacterium avium have different attributes but coincide in their zoonotic potential. Our knowledge about M. avium subsp. silvaticum is limited, since its identification is uncertain. Mycobacterium avium subsp. avium and M. avium subsp. silvaticum can be discriminated from each other based only on phenotypic characteristics, as they have almost identical genome sequences. Here we describe the development of a diagnostic method which enables the molecular identification of M. avium subsp. silvaticum and discrimination from M. avium subsp. avium based on genomic differences in a duplex high-resolution melt and M. avium subsp. silvaticum-specific mismatch real-time PCR. The developed assay was tested on reference strains and 199 field isolates, which were analyzed by phenotypic methods previously. This assay not only identified all 63 M. avium subsp. silvaticum and 138 M. avium subsp. avium strains correctly but also enabled the detection of mixed M. avium subsp. avium-M. avium subsp. silvaticum cultures. This is the first time that such a large panel of strains has been analyzed, and we also report the first isolation of M. avium subsp. silvaticum from red fox, red deer, wild boar, cattle, and badger. This assay is reliable, rapid, simple, inexpensive, and robust. It eliminates the long-existing problem of ambiguous phenotypic identification and opens up the possibility for detailed and comprehensive strain studies.

  5. Integrated Microfluidic Card with TaqMan Probes and High-Resolution Melt Analysis To Detect Tuberculosis Drug Resistance Mutations across 10 Genes

    PubMed Central

    Pholwat, Suporn; Liu, Jie; Stroup, Suzanne; Gratz, Jean; Banu, Sayera; Rahman, S. M. Mazidur; Ferdous, Sara Sabrina; Foongladda, Suporn; Boonlert, Duangjai; Ogarkov, Oleg; Zhdanova, Svetlana; Kibiki, Gibson; Heysell, Scott

    2015-01-01

    ABSTRACT Genotypic methods for drug susceptibility testing of Mycobacterium tuberculosis are desirable to speed the diagnosis and proper therapy of tuberculosis (TB). However, the numbers of genes and polymorphisms implicated in resistance have proliferated, challenging diagnostic design. We developed a microfluidic TaqMan array card (TAC) that utilizes both sequence-specific probes and high-resolution melt analysis (HRM), providing two layers of detection of mutations. Twenty-seven primer pairs and 40 probes were designed to interrogate 3,200 base pairs of critical regions of the inhA, katG, rpoB, embB, rpsL, rrs, eis, gyrA, gyrB, and pncA genes. The method was evaluated on 230 clinical M. tuberculosis isolates from around the world, and it yielded 96.1% accuracy (2,431/2,530) in comparison to that of Sanger sequencing and 87% accuracy in comparison to that of the slow culture-based susceptibility testing. This TAC-HRM method integrates assays for 10 genes to yield fast, comprehensive, and accurate drug susceptibility results for the 9 major antibiotics used to treat TB and could be deployed to improve treatment outcomes. PMID:25714709

  6. High resolution melting technique for molecular epidemiological studies of cystic echinococcosis: differentiating G1, G3, and G6 genotypes of Echinococcus granulosus sensu lato.

    PubMed

    Rostami, Sima; Talebi, Saeed; Babaei, Zahra; Sharbatkhori, Mitra; Ziaali, Naser; Rostami, Habib; Harandi, Majid Fasihi

    2013-10-01

    Reliable and rapid genotyping of large number of Echinococcus granulosus sensu lato isolates is crucial for understanding the epidemiology and transmission of cystic echinococcosis. We have developed a method for distinguishing and discriminating common genotypes of E. granulosus s.l. (G1, G3, and G6) in Iran. This method is based on polymerase chain reaction coupled with high resolution melting curve (HRM), ramping from 70 to 86 °C with fluorescence data acquisition set at 0.1 °C increments and continuous fluorescence monitoring. Consistency of this technique was assessed by inter- and intra-assays. Assessment of intra- and inter-assay variability showed low and acceptable coefficient of variations ranging from 0.09 to 0.17 %. Two hundred and eighty E. granulosus s.l. isolates from sheep, cattle, and camel were used to evaluate the applicability and accuracy of the method. The isolates were categorized as G1 (93, 94, and 25%), G3 (7, 4, and 4%), and G6 (0, 2, and 71%) for sheep, cattle, and camel, respectively. HRM results were completely compatible with those obtained from sequencing and rostellar hook measurement. This method proved to be a valuable screening tool for large-scale molecular epidemiological studies. PMID:23832641

  7. Genotyping of Salmonella enterica serovar Typhimurium isolates by multilocus variable number of tandem repeat high-resolution melting analysis (MLV-HRMA).

    PubMed

    Keeratipibul, Suwimon; Silamat, Panusanun; Phraephaisarn, Chirapiphat; Srisitthinam, Daranee; Takahashi, Hajime; Chaturongkasumrit, Yuphakhun; Vesaratchavest, Mongkol

    2015-01-01

    Salmonella enterica serovar Typhimurium (Salmonella Typhimurium) is one of the most important virulent foodborne pathogens in industrialized countries. The ability to type bacterial strains is essential for surveillance, investigation of outbreaks, and epidemiological studies. Multilocus variable number tandem repeat combined with high-resolution melting analysis (MLV-HRMA) is a fast, cost-efficient, and easy sample genotyping method. In this study, MLV-HRMA and multilocus variable number tandem repeat analysis (MLVA) were used to differentiate between the allelic variants in 5 tandem repeat (TR) loci in 117 Salmonella Typhimurium isolates derived from various farms, slaughterhouses, market, and humans in Thailand. Both MLV-HRMA and MLVA analyses resulted in the identification of a total of 43 different genotypes, but slight differences were observed in cluster analysis results between the 2 methods. The unweighted pair-group method with arithmetic mean-based cluster analysis showed the same core clades; some small differences in the placement of sister-clades and subgrouping were observed due to the inability to reliably type the polymorphic STTR3 locus in the MLV-HRMA. The results of this study show that the MLV-HRMA, following the selection of suitable TR loci, is a relatively reliable and rapid screening method capable of differentiating between Salmonella Typhimurium isolates on the basis of allelic diversity at TR loci. As such, MLV-HRMA can be potentially used to investigate and track sources of contamination in order to effectively control Salmonella contamination in the food supply chain. PMID:25457374

  8. Melting transition of directly linked gold nanoparticle DNA assembly

    NASA Astrophysics Data System (ADS)

    Sun, Y.; Harris, N. C.; Kiang, C.-H.

    2005-05-01

    DNA melting and hybridization is a fundamental biological process as well as a crucial step in many modern biotechnology applications. DNA confined on surfaces exhibits a behavior different from that in free solutions. The system of DNA-capped gold nanoparticles exhibits unique phase transitions and represents a new class of complex fluids. Depending on the sequence of the DNA, particles can be linked to each other through direct complementary DNA sequences or via a ‘linker’ DNA, whose sequence is complementary to the sequence attached to the gold nanoparticles. We observed different melting transitions for these two distinct systems.

  9. High regularity of Z-DNA revealed by ultra high-resolution crystal structure at 0.55 ņ

    PubMed Central

    Brzezinski, Krzysztof; Brzuszkiewicz, Anna; Dauter, Miroslawa; Kubicki, Maciej; Jaskolski, Mariusz; Dauter, Zbigniew

    2011-01-01

    The crystal structure of a Z-DNA hexamer duplex d(CGCGCG)2 determined at ultra high resolution of 0.55 Å and refined without restraints, displays a high degree of regularity and rigidity in its stereochemistry, in contrast to the more flexible B-DNA duplexes. The estimations of standard uncertainties of all individually refined parameters, obtained by full-matrix least-squares optimization, are comparable with values that are typical for small-molecule crystallography. The Z-DNA model generated with ultra high-resolution diffraction data can be used to revise the stereochemical restraints applied in lower resolution refinements. Detailed comparisons of the stereochemical library values with the present accurate Z-DNA parameters, shows in general a good agreement, but also reveals significant discrepancies in the description of guanine-sugar valence angles and in the geometry of the phosphate groups. PMID:21459852

  10. Thiazole orange as the fluorescent intercalator in a high resolution fid assay for determining DNA binding affinity and sequence selectivity of small molecules.

    PubMed

    Boger, D L; Tse, W C

    2001-09-01

    The viability of using thiazole orange as an alternative to ethidium bromide in a fluorescent intercalator displacement (FID) assay is explored by profiling the DNA binding affinity and sequence selectivity of netropsin. Utilizing a library of hairpin deoxyoligonucleotides containing all possible four base-pair sequences, the method provides a high resolution profile of the DNA binding properties of small molecules in a high throughput format.

  11. Probing the microscopic flexibility of DNA from melting temperatures

    NASA Astrophysics Data System (ADS)

    Weber, Gerald; Essex, Jonathan W.; Neylon, Cameron

    2009-10-01

    The microscopic flexibility of DNA is a key ingredient for understanding its interaction with proteins and drugs but is still poorly understood and technically challenging to measure. Several experimental methods probe very long DNA samples, but these miss local flexibility details. Others mechanically disturb or modify short molecules and therefore do not obtain flexibility properties of unperturbed and pristine DNA. Here, we show that it is possible to extract very detailed flexibility information about unmodified DNA from melting temperatures with statistical physics models. We were able to retrieve, from published melting temperatures, several established flexibility properties such as the presence of highly flexible TATA regions of genomic DNA and support recent findings that DNA is very flexible at short length scales. New information about the nanoscale Na+ concentration dependence of DNA flexibility was determined and we show the key role of ApT and TpA steps when it comes to ion-dependent flexibility and melting temperatures.

  12. Dinuclear osmium(II) probes for high-resolution visualisation of cellular DNA structure using electron microscopy.

    PubMed

    Wragg, Ashley; Gill, Martin R; Hill, Christopher J; Su, Xiaodi; Meijer, Anthony J H M; Smythe, Carl; Thomas, Jim A

    2014-12-01

    Two dinuclear osmium polypyridyl complexes function as convenient, easy to handle TEM contrast agents and facilitate the high-resolution visualisation of intracellular structure, particularly sub-nuclear detail.

  13. Structure and disorder in iron-bearing sodium silicate glasses and melts: High-resolution 29Si and 17O solid-state NMR study

    NASA Astrophysics Data System (ADS)

    Kim, H.; Lee, S.

    2012-12-01

    Understanding of the effect of iron content on the structure (Si coordination environment and the degree of polymerization) of iron-bearing silicate melts and glasses is essential for studying their macroscopic properties and diverse geological processes in Earth's interior. Although the recent advances in high-resolution solid-state NMR techniques provide detailed structural information of a diverse iron-free oxide glasses with varying composition (e.g., Lee, P. Natl. Acad. Sci. USA., 2011, 108, 6847; Lee and Sung, Chem. Geol., 2008, 256, 326; Park and Lee, Geochim. Cosmochim. Acta, 2012, 80, 125; Lee et al., Phys. Rev., 103, 095501, 2009), their application to iron-bearing silicate glasses has a limited usefulness in resolving atomic configurations due to the effect of paramagnetic cation (i.e., Fe) on the NMR spectra. Here, we report the first ^{29}Si and ^{17}O NMR spectra for sodium-iron silicate glasses with varying iron content (Na_{2}O-Fe_{2}O_{3}-SiO_{2} glasses, up to 34.60 wt% Fe_{2}O_{3}), revealing previously unknown details of iron-induced changes in structure and disorder. While signal intensity decreases and peak width increases exponentially with increasing iron content [=Fe_{2}O_{3}/(Na_{2}O+Fe_{2}O_{3})], ^{29}Si MAS NMR spectra for sodium-iron silicate glasses present the slight peak shift and an asymmetrical peak broadening toward higher Q^{n} species with increasing iron content. This result implies an increase in the degree of polymerization with increasing iron content. Additionally, ^{29}Si spin-relaxation time (T_{1}) for the glasses decreases with increasing of iron content by several orders of magnitude. ^{17}O 3QMAS NMR spectra for the glasses show well-resolved non-bridging oxygen (NBO, Na-O-Si) and bridging oxygen (BO, Si-O-Si) even at relatively high iron content, providing the first direct experimental estimation of the degree of polymerization. In sodium-iron silicate glasses, the fraction of NBO decreases with increasing iron

  14. Bartonellae in domestic and stray cats from Israel: comparison of bacterial cultures and high-resolution melt real-time PCR as diagnostic methods.

    PubMed

    Gutiérrez, Ricardo; Morick, Danny; Gross, Ifat; Winkler, Ronen; Abdeen, Ziad; Harrus, Shimon

    2013-12-01

    To determine the occurrence of feline bartonellosis in Israel, blood samples were collected from 179 stray and 155 domestic cats from 18 cities or villages in central and northcentral Israel. Samples were screened for Bartonella infection by culture isolation and molecular detection using high-resolution melt (HRM) real-time PCR assay targeting the 16S-23S rRNA internal transcribed spacer (ITS). All positive samples were confirmed by two additional HRM real-time PCR assays targeting two fragments of the β-subunit of RNA polymerase (rpoB) and the 16S rRNA genes. The prevalence of Bartonella spp. infection in the general tested population was 25.1% (84/334). A higher prevalence was detected in the stray (30.7%; 55/179) than the domestic cats (18.7%; 29/155). Bartonella henselae, Bartonella clarridgeiae, and Bartonella koehlerae were highly prevalent in both cat populations, however their distribution among the two populations varied significantly (p=0.016). B. clarridgeiae and B. koehlerae were found to be more prevalent in stray than domestic cats, whereas B. henselae was evenly distributed. Co-infection with two or more different Bartonella spp. was determined in 2.1% (7) of the cats. The ITS HRM real-time PCR assay used in this study was shown to have a greater screening power than bacterial isolation, detecting 94.0% (79/84) compared to 35.7% (30/84), respectively, of all positive samples. The high prevalence of these zoonotic Bartonella species, coupled with the overpopulation of stray cats, and increased numbers of domestic cats in the major urban centers in Israel represent a significant threat for the public health in this country.

  15. Rapid detection of multidrug-resistant Mycobacterium tuberculosis by use of real-time PCR and high-resolution melt analysis.

    PubMed

    Ramirez, Melissa V; Cowart, Kelley C; Campbell, Patricia J; Morlock, Glenn P; Sikes, David; Winchell, Jonas M; Posey, James E

    2010-11-01

    The current study describes the development of a unique real-time PCR assay for the detection of mutations conferring drug resistance in Mycobacterium tuberculosis. The rifampicin resistance determinant region (RRDR) of rpoB and specific regions of katG and the inhA promoter were targeted for the detection of rifampin (RIF) and isoniazid (INH) resistance, respectively. Additionally, this assay was multiplexed to discriminate Mycobacterium tuberculosis complex (MTC) strains from nontuberculous Mycobacteria (NTM) strains by targeting the IS6110 insertion element. High-resolution melting (HRM) analysis following real-time PCR was used to identify M. tuberculosis strains containing mutations at the targeted loci, and locked nucleic acid (LNA) probes were used to enhance the detection of strains containing specific single-nucleotide polymorphism (SNP) transversion mutations. This method was used to screen 252 M. tuberculosis clinical isolates, including 154 RIF-resistant strains and 174 INH-resistant strains based on the agar proportion method of drug susceptibility testing (DST). Of the 154 RIF-resistant strains, 148 were also resistant to INH and therefore classified as multidrug resistant (MDR). The assay demonstrated sensitivity and specificity of 91% and 98%, respectively, for the detection of RIF resistance and 87% and 100% for the detection of INH resistance. Overall, this assay showed a sensitivity of 85% and a specificity of 98% for the detection of MDR strains. This method provides a rapid, robust, and inexpensive way to detect the dominant mutations known to confer MDR in M. tuberculosis strains and offers several advantages over current molecular and culture-based techniques.

  16. DETECTION OF DNA DAMAGE USING MELTING ANALYSIS TECHNIQUES

    EPA Science Inventory

    A rapid and simple fluorescence screening assay for UV radiation-, chemical-, and enzyme-induced DNA damage is reported. This assay is based on a melting/annealing analysis technique and has been used with both calf thymus DNA and plasmid DNA (puc 19 plasmid from E. coli). DN...

  17. Association between the p73 gene G4C14-to-A4T14 single nucleotide polymorphism and risk of cervical cancer by high resolution melting and PCR with confronting two-pair primers in a Chinese population

    PubMed Central

    GUO, HAIYAN; YANG, SHAODI; XU, LIJIAN; LI, DING; TANG, JIANXIN; WANG, SHUANGSHAUNG; WEI, BENJIE; LIU, ZHENGCHUN

    2016-01-01

    As a member of the p53 gene family, the p73 gene can affect an individual's susceptibility to cancer through a p53-like manner. DNA sequence variation in the p73 gene has been reported to be associated with cancer risk. The present study aimed to identify whether the p73 gene G4C14-to-A4T14 single nucleotide polymorphism (SNP) is associated with risk of cervical cancer in a Chinese population. The p73 G4C14-to-A4T14 polymorphism was genotyped in 175 cervical cancer and 189 healthy control peripheral blood DNA samples using high resolution melting, polymerase chain reaction with confronting two-pair primers and direct DNA sequencing. The results demonstrated that carriers of the AT/AT genotype were associated with a significantly increased risk of cervical cancer (P=0.042; χ2=4.122; odds ratio = 2.241; 95% confidence interval = 1.013–4.956) compared with the GC/GC genotype carriers. In addition, there was a significant association between p73 genotypes and tumor size in patients with cervical cancer (P=0.014; χ2=8.607). However, no association was identified between p73 genotypes and tumor stage, histological type or lymph node metastasis in patients with cervical cancer. These results suggest that the p73 G4C14-to-A4T14 SNP may function as a marker of genetic susceptibility to cervical cancer in the Chinese population. PMID:27347206

  18. A comparison of Direct sequencing, Pyrosequencing, High resolution melting analysis, TheraScreen DxS, and the K-ras StripAssay for detecting KRAS mutations in non small cell lung carcinomas

    PubMed Central

    2012-01-01

    Background It is mandatory to confirm the absence of mutations in the KRAS gene before treating metastatic colorectal cancers with epidermal growth factor receptor inhibitors, and similar regulations are being considered for non-small cell lung carcinomas (NSCLC) and other tumor types. Routine diagnosis of KRAS mutations in NSCLC is challenging because of compromised quantity and quality of biological material. Although there are several methods available for detecting mutations in KRAS, there is little comparative data regarding their analytical performance, economic merits, and workflow parameters. Methods We compared the specificity, sensitivity, cost, and working time of five methods using 131 frozen NSCLC tissue samples. We extracted genomic DNA from the samples and compared the performance of Sanger cycle sequencing, Pyrosequencing, High-resolution melting analysis (HRM), and the Conformité Européenne (CE)-marked TheraScreen DxS and K-ras StripAssay kits. Results and conclusions Our results demonstrate that TheraScreen DxS and the StripAssay, in that order, were most effective at diagnosing mutations in KRAS. However, there were still unsatisfactory disagreements between them for 6.1% of all samples tested. Despite this, our findings are likely to assist molecular biologists in making rational decisions when selecting a reliable, efficient, and cost-effective method for detecting KRAS mutations in heterogeneous clinical tumor samples. PMID:22995035

  19. Differentiation between pathogenic serotype 1 isolates of Marek's disease virus and the Rispens CVI988 vaccine in Australia using real-time PCR and high resolution melt curve analysis.

    PubMed

    Renz, K G; Cheetham, B F; Walkden-Brown, S W

    2013-01-01

    Two real-time PCR assays were developed which enable quantitation and differentiation between pathogenic Australian isolates of Marek's disease virus (MDV) serotype 1 and the serotype 1 vaccine strain Rispens CVI988. The assays are based on a DNA sequence variation in the meq gene between pathogenic and vaccinal MDV1 which has been confirmed by sequencing of 20 Australian field strains of MDV. Complete specificity has been demonstrated in samples containing pathogenic MDV (n=20), Rispens (3 commercial vaccine strains), or both. The limit of detection of both the Rispens-specific and the pathogenic MDV1-specific assays was 10 viral copies/reaction. The tests successfully differentiated and quantified MDV in mixtures of pathogenic and vaccinal Rispens virus. A high resolution melt curve analysis targeting the same SNP used for the real-time PCR assays was also developed which successfully detected sequence variation between Md5, six Australian MDV1 isolates and the three Rispens vaccines. However it was ineffective at differentiating mixtures of pathogenic and vaccinal MDV1. The real-time PCR assays have both diagnostic and epidemiological applications as they enable differentiation and quantitation of Rispens CVI988 and pathogenic MDV1 in co-infected chickens in Australia.

  20. Structural correlations and melting of B-DNA fibers

    SciTech Connect

    Wildes, Andrew; Theodorakopoulos, Nikos; Valle-Orero, Jessica; Cuesta-Lopez, Santiago; Peyrard, Michel; Garden, Jean-Luc

    2011-06-15

    Despite numerous attempts, understanding the thermal denaturation of DNA is still a challenge due to the lack of structural data on the transition since standard experimental approaches to DNA melting are made in solution and do not provide spatial information. We report a measurement using neutron scattering from oriented DNA fibers to determine the size of the regions that stay in the double-helix conformation as the melting temperature is approached from below. A Bragg peak from the B form of DNA is observed as a function of temperature and its width and integrated intensity are measured. These results, complemented by a differential calorimetry study of the melting of B-DNA fibers as well as electrophoresis and optical observation data, are analyzed in terms of a one-dimensional mesoscopic model of DNA.

  1. THE MELTING MECHANISM OF DNA TETHERED TO A SURFACE

    PubMed Central

    QAMHIEH, KHAWLA; WONG, KA-YIU; LYNCH, GILLIAN C.; PETTITT, B. MONTGOMERY

    2009-01-01

    The details of melting of DNA immobilized on a chip or nanoparticle determines the sensitivity and operating characteristics of many analytical and synthetic biotechnological devices. Yet, little is known about the differences in how the DNA melting occurs between a homogeneous solution and that on a chip. We used molecular dynamics simulations to explore possible pathways for DNA melting on a chip. Simulation conditions were chosen to ensure that melting occurred in a submicrosecond timescale. The temperature was set to 400 K and the NaCl concentration was set to 0.1 M. We found less symmetry than in the solution case where for oligomeric double-stranded nucleic acids both ends melted with roughly equal probability. On a prepared silica surface we found melting is dominated by fraying from the end away from the surface. Strand separation was hindered by nonspecific surface adsorption at this temperature. At elevated temperatures the melted DNA was attracted to even uncharged organically coated surfaces demonstrating surface fouling. While hybridization is not the simple reverse of melting, this simulation has implications for the kinetics of hybridization. PMID:19802357

  2. Hydration of nucleic acid fragments: comparison of theory and experiment for high-resolution crystal structures of RNA, DNA, and DNA-drug complexes.

    PubMed

    Hummer, G; García, A E; Soumpasis, D M

    1995-05-01

    A computationally efficient method to describe the organization of water around solvated biomolecules is presented. It is based on a statistical mechanical expression for the water-density distribution in terms of particle correlation functions. The method is applied to analyze the hydration of small nucleic acid molecules in the crystal environment, for which high-resolution x-ray crystal structures have been reported. Results for RNA [r(ApU).r(ApU)] and DNA [d(CpG).d(CpG) in Z form and with parallel strand orientation] and for DNA-drug complexes [d(CpG).d(CpG) with the drug proflavine intercalated] are described. A detailed comparison of theoretical and experimental data shows positional agreement for the experimentally observed water sites. The presented method can be used for refinement of the water structure in x-ray crystallography, hydration analysis of nuclear magnetic resonance structures, and theoretical modeling of biological macromolecules such as molecular docking studies. The speed of the computations allows hydration analyses of molecules of almost arbitrary size (tRNA, protein-nucleic acid complexes, etc.) in the crystal environment and in aqueous solution.

  3. Estimation of the diversity between DNA calorimetric profiles, differential melting curves and corresponding melting temperatures.

    PubMed

    Chang, Chun-Ling; Fridman, Alexander S; Grigoryan, Inessa E; Galyuk, Elena N; Murashko, Oleg N; Hu, Chin-Kun; Lando, Dmitri Y

    2016-11-01

    The Poland-Fixman-Freire formalism was adapted for modeling of calorimetric DNA melting profiles, and applied to plasmid pBR 322 and long random sequences. We studied the influence of the difference (HGC -HAT ) between the helix-coil transition enthalpies of AT and GC base pairs on the calorimetric melting profile and on normalized calorimetric melting profile. A strong alteration of DNA calorimetrical profile with HGC -HAT was demonstrated. In contrast, there is a relatively slight change in the normalized profiles and in corresponding ordinary (optical) normalized differential melting curves (DMCs). For fixed HGC -HAT , the average relative deviation (S) between DMC and normalized calorimetric profile, and the difference between their melting temperatures (Tcal -Tm ) are weakly dependent on peculiarities of the multipeak fine structure of DMCs. At the same time, both the deviation S and difference (Tcal -Tm ) enlarge with the temperature melting range of the helix-coil transition. It is shown that the local deviation between DMC and normalized calorimetric profile increases in regions of narrow peaks distant from the melting temperature. PMID:27422497

  4. High-resolution genomic assays provide insight into the division of labor between TLS and HDR in mammalian replication of damaged DNA.

    PubMed

    Livneh, Zvi; Cohen, Isadora S; Paz-Elizur, Tamar; Davidovsky, Dana; Carmi, Dalit; Swain, Umakanta; Mirlas-Neisberg, Nataly

    2016-08-01

    The multitude of DNA lesions that continuously form in DNA cannot all be detected and removed prior to replication. Thus, encounters of the replication fork with DNA damage become inevitable. Such encounters inhibit fork progression, leading to replication fork arrest or to replication re-priming downstream of the damage site. Either of these events will result in the formation of gap-lesion structures, in which a damaged base is located in a single stranded stretch of DNA, that is vulnerable to subsequent nicking. The double strand break that would ensue if ssDNA becomes nicked constitutes escalation of the damage from nucleotide(s)-specific to chromosomal scale. Cells employ two universal DNA damage tolerance (DDT) strategies to resolve these situations, by converting the gap-lesion structures into dsDNA without repairing the damage. The first is translesion DNA synthesis (TLS), in which a specialized low-fidelity DNA polymerase inserts a nucleotide opposite the damaged one. TLS is inherently mutagenic, due to the miscoding nature of most damaged nucleotides. The second strategy is homology-dependent repair (HDR), which relies on the presence of an identical intact sister chromatid. The molecular mechanisms that regulate the division of labor between these pathways are poorly understood. This review focuses on the balance between TLS and HDR in mammalian cells, discussing recent findings that were made possible thanks to newly developed high resolution genomic assays, and highlighting the role of the DNA lesion's properties in DDT pathway choice.

  5. High-resolution genomic assays provide insight into the division of labor between TLS and HDR in mammalian replication of damaged DNA.

    PubMed

    Livneh, Zvi; Cohen, Isadora S; Paz-Elizur, Tamar; Davidovsky, Dana; Carmi, Dalit; Swain, Umakanta; Mirlas-Neisberg, Nataly

    2016-08-01

    The multitude of DNA lesions that continuously form in DNA cannot all be detected and removed prior to replication. Thus, encounters of the replication fork with DNA damage become inevitable. Such encounters inhibit fork progression, leading to replication fork arrest or to replication re-priming downstream of the damage site. Either of these events will result in the formation of gap-lesion structures, in which a damaged base is located in a single stranded stretch of DNA, that is vulnerable to subsequent nicking. The double strand break that would ensue if ssDNA becomes nicked constitutes escalation of the damage from nucleotide(s)-specific to chromosomal scale. Cells employ two universal DNA damage tolerance (DDT) strategies to resolve these situations, by converting the gap-lesion structures into dsDNA without repairing the damage. The first is translesion DNA synthesis (TLS), in which a specialized low-fidelity DNA polymerase inserts a nucleotide opposite the damaged one. TLS is inherently mutagenic, due to the miscoding nature of most damaged nucleotides. The second strategy is homology-dependent repair (HDR), which relies on the presence of an identical intact sister chromatid. The molecular mechanisms that regulate the division of labor between these pathways are poorly understood. This review focuses on the balance between TLS and HDR in mammalian cells, discussing recent findings that were made possible thanks to newly developed high resolution genomic assays, and highlighting the role of the DNA lesion's properties in DDT pathway choice. PMID:27262613

  6. Assessment of high resolution melt analysis feasibility for evaluation of beta-globin gene mutations as a reproducible, cost-efficient and fast alternative to the present conventional method

    PubMed Central

    Ramezanzadeh, Mahboubeh; Salehi, Mansour; Salehi, Rasoul

    2016-01-01

    Background: Beta-thalassemia is the most prevalent monogenic disease throughout the world. It was the first genetic disorder nominated for nation-wide prevention programs involving population screening for heterozygotes and prenatal diagnosis (PND) in Iran. Due to the high prevalence of beta-thalassemia, the shift from conventional mutation detection methods to more recently developed techniques based on novel innovative technologies are essential. We aimed to develop a real-time polymerase chain reaction (PCR) based protocol using high resolution melting (HRM) analysis for diagnosis of common beta-thalassemia mutations. Materials and Methods: Forty DNA samples extracted from peripheral blood of suspected beta-thalassemia carriers participated in this study were subjected to amplification refractory mutation system (ARMS). We then used 20 of these samples for HRM optimization. When 100% sensitivity and specificity was obtained with HRM procedure, we applied the technique for mutation detection on another remaining 20 samples as thalassemia cases with unknown mutations (detected mutations with ARMS-PCR kept confidential). Finally, the HRM procedure applied on 2 chorionic villous sample (CVS) biopsied from 12 weeks gestational age pregnant women for routine PND analysis. Results: In the first step of study, Fr 8/9 (+G), IVSI-1 (G > A), IVSI-5 (G > C), IVSI-110 (G > A), and CD44 (−C) mutations were diagnosed in samples under study using ARMS-PCR technique. Finally, the HRM procedure applied on 20 unknown samples and 2 CVS The results of HRM were in complete concordance with ARMS and confirmed by sequencing. Conclusions: The advantages of HRM analysis over conventional methods is high throughput, rapid, accurate, cost-effective, and reproducible. PMID:27169102

  7. Comparison of high resolution melting analysis, pyrosequencing, next generation sequencing and immunohistochemistry to conventional Sanger sequencing for the detection of p.V600E and non-p.V600E BRAF mutations

    PubMed Central

    2014-01-01

    Background The approval of vemurafenib in the US 2011 and in Europe 2012 improved the therapy of not resectable or metastatic melanoma. Patients carrying a substitution of valine to glutamic acid at codon 600 (p.V600E) or a substitution of valine to leucine (p.V600K) in BRAF show complete or partial response. Therefore, the precise identification of the underlying somatic mutations is essential. Herein, we evaluate the sensitivity, specificity and feasibility of six different methods for the detection of BRAF mutations. Methods Samples harboring p.V600E mutations as well as rare mutations in BRAF exon 15 were compared to wildtype samples. DNA was extracted from formalin-fixed paraffin-embedded tissues by manual micro-dissection and automated extraction. BRAF mutational analysis was carried out by high resolution melting (HRM) analysis, pyrosequencing, allele specific PCR, next generation sequencing (NGS) and immunohistochemistry (IHC). All mutations were independently reassessed by Sanger sequencing. Due to the limited tumor tissue available different numbers of samples were analyzed with each method (82, 72, 60, 72, 49 and 82 respectively). Results There was no difference in sensitivity between the HRM analysis and Sanger sequencing (98%). All mutations down to 6.6% allele frequency could be detected with 100% specificity. In contrast, pyrosequencing detected 100% of the mutations down to 5% allele frequency but exhibited only 90% specificity. The allele specific PCR failed to detect 16.3% of the mutations eligible for therapy with vemurafenib. NGS could analyze 100% of the cases with 100% specificity but exhibited 97.5% sensitivity. IHC showed once cross-reactivity with p.V600R but was a good amendment to HRM. Conclusion Therefore, at present, a combination of HRM and IHC is recommended to increase sensitivity and specificity for routine diagnostic to fulfill the European requirements concerning vemurafenib therapy of melanoma patients. PMID:24410877

  8. Ultra-fast high-resolution agarose electrophoresis of DNA and RNA using low-molarity conductive media.

    PubMed

    Brody, Jonathan R; Calhoun, Eric S; Gallmeier, Eike; Creavalle, Talisa D; Kern, Scott E

    2004-10-01

    Current DNA electrophoretic solutions employ high ionic concentrations and require long electrophoretic run times. Here we demonstrate that high and low molecular weight double-stranded DNA, single-stranded DNA (ssDNA), and RNA can be separated rapidly in agarose-based low-molarity conductive media. Separation of small DNA fragments was optimized by substituting 1-mM solutions of alkali metals or a nonbiological amine that distributed voltage with a minute current. These ultra-dilute solutions can separate DNA at least 15-fold faster Low-molarity media at 5-10 mM adequately separated RNA and larger DNA fragments as well. These novel media reduce the Joule heating of the electrophoretic system and allow for easy-to-use, ultra-fast separation of DNA fragments.

  9. A DNA Melting Exercise for a Large Laboratory Class

    ERIC Educational Resources Information Center

    Levine, Lauren A.; Junker, Matthew; Stark, Myranda; Greenleaf, Dustin

    2015-01-01

    A simple and economical experimental setup is described that enables multiple individuals or groups within a laboratory class to measure the thermal melting of double stranded DNA simultaneously. The setup utilizes a basic spectrophotometer capable of measuring absorbance at 260 nm, UV plastic cuvettes, and a stirring hot plate. Students measure…

  10. Validation of High Resolution Melting Analysis (HRM) of the Amplified ITS2 Region for the Detection and Identification of Yeasts from Clinical Samples: Comparison with Culture and MALDI-TOF Based Identification

    PubMed Central

    Duyvejonck, Hans; Cools, Piet; Decruyenaere, Johan; Roelens, Kristien; Noens, Lucien; Vermeulen, Stefan; Claeys, Geert; Decat, Ellen; Van Mechelen, Els; Vaneechoutte, Mario

    2015-01-01

    Aim Candida species are known as opportunistic pathogens, and a possible cause of invasive infections. Because of their species-specific antimycotic resistance patterns, reliable techniques for their detection, quantification and identification are needed. We validated a DNA amplification method for direct detection of Candida spp. from clinical samples, namely the ITS2-High Resolution Melting Analysis (direct method), by comparing it with a culture and MALDI-TOF Mass Spectrometry based method (indirect method) to establish the presence of Candida species in three different types of clinical samples. Materials and Methods A total of 347 clinical samples, i.e. throat swabs, rectal swabs and vaginal swabs, were collected from the gynaecology/obstetrics, intensive care and haematology wards at the Ghent University Hospital, Belgium. For the direct method, ITS2-HRM was preceded by NucliSENS easyMAG DNA extraction, directly on the clinical samples. For the indirect method, clinical samples were cultured on Candida ID and individual colonies were identified by MALDI-TOF. Results For 83.9% of the samples there was complete concordance between both techniques, i.e. the same Candida species were detected in 31.1% of the samples or no Candida species were detected in 52.8% of the samples. In 16.1% of the clinical samples, discrepant results were obtained, of which only 6.01% were considered as major discrepancies. Discrepancies occurred mostly when overall numbers of Candida cells in the samples were low and/or when multiple species were present in the sample. Discussion Most of the discrepancies could be decided in the advantage of the direct method. This is due to samples in which no yeast could be cultured whereas low amounts could be detected by the direct method and to samples in which high quantities of Candida robusta according to ITS2-HRM were missed by culture on Candida ID agar. It remains to be decided whether the diagnostic advantages of the direct method

  11. High resolution-sensitivity characterization of polycyclic aromatic hydrocarbon-DNA adducts using fluorescence line narrowing spectrometry

    SciTech Connect

    Cooper, R.S.

    1988-07-01

    The application of fluorescence line narrowing spectrometry (FLNS) to the investigation of polar polycyclic aromatic hydrocarbon (PAH) metabolites and their corresponding DNA adducts is demonstrated. The selectivity is shown through the successful resolution of all components in separate mixtures of similar but distinct derivatives of benzo(a)pyrene, benz(a)anthracene, and chrysene. The separate mixtures were composed of six metabolites, five DNA adducts, each metabolite and its corresponding DNA adduct, and six metabolites and two DNA adducts. The broad applicability of FLNS is demonstrated through applications to the analysis of globin adducts, PAH metabolites in urine, and real samples, and to the investigation of carcinogenic metabolic pathways. 98 refs., 31 figs., 5 tabs.

  12. Mapping DNA quantity into electrophoretic mobility through quantum dot nanotethers for high-resolution genetic and epigenetic analysis.

    PubMed

    Zhang, Yi; Liu, Kelvin J; Wang, Tian-Li; Shih, Ie-Ming; Wang, Tza-Huei

    2012-01-24

    Newly discovered nanoparticle properties have driven the development of novel applications and uses. We report a new observation where the electrophoretic mobility of a quantum dot/DNA nanoassembly can be precisely modulated by the degree of surface DNA conjugation. By using streptavidin-coated quantum dots (QDs) as nanotethers to gather biotin-labeled DNA into electrophoretic nanoassemblies, the QD surface charge is modulated and transformed into electrophoretic mobility shifts using standard agarose gel electrophoresis. Typical fluorescent assays quantify based on relative intensity. However, this phenomenon uses a novel approach that accurately maps DNA quantity into shifts in relative band position. This property was applied in a QD-enabled nanoassay called quantum dot electrophoretic mobility shift assay (QEMSA) that enables accurate quantification of DNA targets down to 1.1-fold (9%) changes in quantity, beyond what is achievable in qPCR. In addition to these experimental findings, an analytical model is presented to explain this behavior. Finally, QEMSA was applied to both genetic and epigenetic analysis of cancer. First, it was used to analyze copy number variation (CNV) of the RSF1/HBXAP gene, where conventional approaches for CNV analysis based on comparative genomic hybridization (CGH), microarrays, and qPCR are unable to reliably differentiate less than 2-fold changes in copy number. Then, QEMSA was used for DNA methylation analysis of the p16/CDK2A tumor suppressor gene, where its ability to detect subtle changes in methylation was shown to be superior to that of qPCR.

  13. DNA melting and genotoxicity induced by silver nanoparticles and graphene.

    PubMed

    Ivask, Angela; Voelcker, Nicolas H; Seabrook, Shane A; Hor, Maryam; Kirby, Jason K; Fenech, Michael; Davis, Thomas P; Ke, Pu Chun

    2015-05-18

    We have revealed a connection between DNA-nanoparticle (NP) binding and in vitro DNA damage induced by citrate- and branched polyethylenimine-coated silver nanoparticles (c-AgNPs and b-AgNPs) as well as graphene oxide (GO) nanosheets. All three types of nanostructures triggered an early onset of DNA melting, where the extent of the melting point shift depends upon both the type and concentration of the NPs. Specifically, at a DNA/NP weight ratio of 1.1/1, the melting temperature of lambda DNA dropped from 94 °C down to 76 °C, 60 °C, and room temperature for GO, c-AgNPs and b-AgNPs, respectively. Consistently, dynamic light scattering revealed that the largest changes in DNA hydrodynamic size were also associated with the binding of b-AgNPs. Upon introduction to cells, b-AgNPs also exhibited the highest cytotoxicity, at the half-maximal inhibitory (IC50) concentrations of 3.2, 2.9, and 5.2 mg/L for B and T-lymphocyte cell lines and primary lymphocytes, compared to the values of 13.4, 12.2, and 12.5 mg/L for c-AgNPs and 331, 251, and 120 mg/L for GO nanosheets, respectively. At cytotoxic concentrations, all NPs elicited elevated genotoxicities via the increased number of micronuclei in the lymphocyte cells. However, b-AgNPs also induced micronuclei at subtoxic concentrations starting from 0.1 mg/L, likely due to their stronger cellular adhesion and internalization, as well as their subsequent interference with normal DNA synthesis or chromosome segregation during the cell cycle. This study facilitates our understanding of the effects of NP chemical composition, surface charge, and morphology on DNA stability and genotoxicity, with implications ranging from nanotoxicology to nanobiotechnology and nanomedicine. PMID:25781053

  14. Comprehensive High-Resolution Mass Spectrometric Analysis of DNA Phosphate Adducts Formed by the Tobacco-Specific Lung Carcinogen 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone

    PubMed Central

    2015-01-01

    The tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK, 1) is a potent lung carcinogen in laboratory animals and is believed to play a key role in the development of lung cancer in smokers. Metabolic activation of NNK leads to the formation of pyridyloxobutyl DNA adducts, a critical step in its mechanism of carcinogenesis. In addition to DNA nucleobase adducts, DNA phosphate adducts can be formed by pyridyloxobutylation of the oxygen atoms of the internucleotidic phosphodiester linkages. We report the use of a liquid chromatography–nanoelectrospray ionization–high-resolution tandem mass spectrometry technique to characterize 30 novel pyridyloxobutyl DNA phosphate adducts in calf thymus DNA (CT-DNA) treated with 4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanone (NNKOAc, 2), a regiochemically activated form of NNK. A 15N3-labeled internal standard was synthesized for one of the most abundant phosphate adducts, dCp[4-oxo-4-(3-pyridyl)butyl]dC (CpopC), and this standard was used to quantify CpopC and to estimate the levels of other adducts in the NNKOAc-treated CT-DNA. Formation of DNA phosphate adducts by NNK in vivo was further investigated in rats treated with NNK acutely (0.1 mmol/kg once daily for 4 days by subcutaneous injection) and chronically (5 ppm in drinking water for 10, 30, 50, and 70 weeks). This study provides the first comprehensive structural identification and quantitation of a panel of DNA phosphate adducts of a structurally complex carcinogen and chemical support for future mechanistic studies of tobacco carcinogenesis in humans. PMID:26398225

  15. A conformational transition in the structure of a 2'-thiomethyl-modified DNA visualized at high resolution

    SciTech Connect

    Pallan, Pradeep S.; Prakash, Thazha P.; Li, Feng; Eoff, Robert L.; Manoharan, Muthiah; Egli, Martin

    2009-06-17

    Crystal structures of A-form and B-form DNA duplexes containing 2'-S-methyl-uridines reveal that the modified residues adopt a RNA-like C3'-endo pucker, illustrating that the replacement of electronegative oxygen at the 2'-carbon of RNA by sulfur does not appear to fundamentally alter the conformational preference of the sugar in the oligonucleotide context and sterics trump stereoelectronics.

  16. High-resolution copy number profiling by array CGH using DNA isolated from formalin-fixed, paraffin-embedded tissues.

    PubMed

    van Essen, Hendrik F; Ylstra, Bauke

    2012-01-01

    We describe protocols to acquire high-quality DNA from formalin-fixed, paraffin-embedded (FFPE) tissues for the use in array comparative genome hybridization (CGH). Formalin fixation combined with paraffin embedding is routine procedure for solid malignancies in the diagnostic practice of the pathologist. As a consequence, large archives of FFPE tissues are available in pathology institutes across the globe. This archival material is for many research questions an invaluable resource, with long-term clinical follow-up and survival data available. FFPE is, thus, highly attractive for large genomics studies, including experiments requiring samples for test/learning and validation. Most larger array CGH studies have, therefore, made use of FFPE material and show that CNAs have tumor- and tissue-specific traits (Chin et al. Cancer Cell 10: 529-541, 2006; Fridlyand et al. BMC Cancer 6: 96, 2006; Weiss et al. Oncogene 22: 1872-1879, 2003; Jong et al. Oncogene 26: 1499-1506, 2007). The protocols described are tailored to array CGH of FFPE solid malignancies: from sectioning FFPE blocks to specific cynosures for pathological revisions of sections, DNA isolation, quality testing, and amplification. The protocols are technical in character and elaborate up to the labeling of isolated DNA while further processes and interpretation and data analysis are beyond the scope.

  17. Oxygen-aromatic contacts in intra-strand base pairs: analysis of high-resolution DNA crystal structures and quantum chemical calculations.

    PubMed

    Jain, Alok; Krishna Deepak, R N V; Sankararamakrishnan, Ramasubbu

    2014-07-01

    Three-dimensional structures of biomolecules are stabilized by a large number of non-covalent interactions and some of them such as van der Waals, electrostatic and hydrogen bond interactions are well characterized. Delocalized π-electron clouds of aromatic residues are known to be involved in cation-π, CH-π, OH-π and π-π interactions. In proteins, many examples have been found in which the backbone carbonyl oxygen of one residue makes close contact with the aromatic center of aromatic residues. Quantum chemical calculations suggest that such contacts may provide stability to the protein secondary structures. In this study, we have systematically analyzed the experimentally determined high-resolution DNA crystal structures and identified 91 examples in which the aromatic center of one base is in close contact (<3.5Ǻ) with the oxygen atom of preceding (Group-I) or succeeding base (Group-II). Examples from Group-I are overwhelmingly observed and cytosine or thymine is the preferred base contributing oxygen atom in Group-I base pairs. A similar analysis of high-resolution RNA structures surprisingly did not yield many examples of oxygen-aromatic contact of similar type between bases. Ab initio quantum chemical calculations on compounds based on DNA crystal structures and model compounds show that interactions between the bases in base pairs with oxygen-aromatic contacts are energetically favorable. Decomposition of interaction energies indicates that dispersion forces are the major cause for energetically stable interaction in these base pairs. We speculate that oxygen-aromatic contacts in intra-strand base pairs in a DNA structure may have biological significance.

  18. High-resolution detection of DNA binding sites of the global transcriptional regulator GlxR in Corynebacterium glutamicum.

    PubMed

    Jungwirth, Britta; Sala, Claudia; Kohl, Thomas A; Uplekar, Swapna; Baumbach, Jan; Cole, Stewart T; Pühler, Alfred; Tauch, Andreas

    2013-01-01

    The transcriptional regulator GlxR has been characterized as a global hub within the gene-regulatory network of Corynebacterium glutamicum. Chromatin immunoprecipitation with a specific anti-GlxR antibody and subsequent high-throughput sequencing (ChIP-seq) was applied to C. glutamicum to get new in vivo insights into the gene composition of the GlxR regulon. In a comparative approach, C. glutamicum cells were grown with either glucose or acetate as the sole carbon source prior to immunoprecipitation. High-throughput sequencing resulted in 69 million reads and 2.6 Gb of genomic information. After mapping of these data on the genome sequence of C. glutamicum, 107 enriched DNA fragments were detected from cells grown with glucose as carbon source. GlxR binding sites were identified in the sequence of 79 enriched DNA fragments, of which 21 sites were not previously reported. Electrophoretic mobility shift assays with 40-mer oligomers covering the GlxR binding sites were performed for validation of the in vivo results. The detection of new binding sites confirmed the role of GlxR as a regulator of carbon source metabolism and energy conversion, but additionally revealed binding of GlxR in front of the 6C non-coding RNA gene and to non-canonical DNA binding sites within protein-coding regions. The present study underlines the dynamics within the GlxR regulon by identifying in vivo targets during growth on glucose and contributes to the expansion of knowledge of this important transcriptional regulator.

  19. Calculating Freshwater Input from Iceberg Melt in Greenlandic Fjords by Combining In Situ Observations of Iceberg Movement with High Resolution Satellite Imagery

    NASA Astrophysics Data System (ADS)

    Sulak, D. J.; Sutherland, D.; Stearns, L. A.; Hamilton, G. S.

    2015-12-01

    Understanding fjord circulation in Greenland's outlet glacial fjords is crucial to explaining recent temporal and spatial variability in glacier dynamics, as well as freshwater transport on the continental shelf. The fjords are commonly assumed to exhibit a plume driven circulation that draws in warmer and saltier Atlantic-origin water toward the glacier at depth. Freshwater input at glacier termini directly drives this circulation and significantly influences water column stratification, which indirectly feeds back on the plume driven circulation. Previous work has focused on freshwater inputs from surface runoff and submarine melting, but the contribution from iceberg melt, a potentially important freshwater source, has not been quantified. Here, we develop a new technique combining in situ observations of movement from iceberg-mounted GPS units with multispectral satellite imagery from Landsat 8. The combination of datasets allows us to examine the details of iceberg movement and quantify mean residence times in a given fjord. We then use common melt rate parameterizations to estimate freshwater input for a given iceberg, utilizing novel satellite-derived iceberg distributions to scale up to a fjord-wide freshwater contribution. We apply this technique to Rink Isbræ and Kangerlussuup Sermia in west Greenland, and Helheim Glacier in southeast Greenland. The analysis can be rapidly expanded to look at other systems as well as seasonal and interannual changes in how icebergs affect the circulation and stratification of Greenland's outlet glacial fjords. Ultimately, this work will lead to a more complete understanding of the wide range of factors that control the observed regional variability in Greenland's glaciers.

  20. Comprehensive and high-resolution typing of swine leukocyte antigen DQA from genomic DNA and determination of 25 new SLA class II haplotypes.

    PubMed

    Le, M T; Choi, H; Choi, M-K; Nguyen, D T; Kim, J-H; Seo, H G; Cha, S-Y; Seo, K; Chun, T; Schook, L B; Park, C

    2012-12-01

    We previously reported the development of genomic-DNA-based high-resolution genotyping methods for SLA-DQB1 and DRB1. Here, we report the successful typing of SLA-DQA using similar methodological principles. We designed a method for comprehensive genotyping of SLA-DQA using intronic sequence information of SLA-DQA exon 2 that we had obtained from 12 animals with different SLA-DQB1 genotypes. We expanded our typing to 76 selected animals with diverse DQB1 and DRB1 genotypes, 140 random animals from 7 pig breeds, and 3 wild boars. This resulted in the identification of 17 DQA alleles with 49 genotypes. Two new alleles were identified from wild boars. Combine with SLA-DQB1, and DRB1 typing results, we identified 34 SLA class II haplotypes including 25 that were previously unreported.

  1. The trans-Saharan slave trade - clues from interpolation analyses and high-resolution characterization of mitochondrial DNA lineages

    PubMed Central

    2010-01-01

    Background A proportion of 1/4 to 1/2 of North African female pool is made of typical sub-Saharan lineages, in higher frequencies as geographic proximity to sub-Saharan Africa increases. The Sahara was a strong geographical barrier against gene flow, at least since 5,000 years ago, when desertification affected a larger region, but the Arab trans-Saharan slave trade could have facilitate enormously this migration of lineages. Till now, the genetic consequences of these forced trans-Saharan movements of people have not been ascertained. Results The distribution of the main L haplogroups in North Africa clearly reflects the known trans-Saharan slave routes: West is dominated by L1b, L2b, L2c, L2d, L3b and L3d; the Center by L3e and some L3f and L3w; the East by L0a, L3h, L3i, L3x and, in common with the Center, L3f and L3w; while, L2a is almost everywhere. Ages for the haplogroups observed in both sides of the Saharan desert testify the recent origin (holocenic) of these haplogroups in sub-Saharan Africa, claiming a recent introduction in North Africa, further strengthened by the no detection of local expansions. Conclusions The interpolation analyses and complete sequencing of present mtDNA sub-Saharan lineages observed in North Africa support the genetic impact of recent trans-Saharan migrations, namely the slave trade initiated by the Arab conquest of North Africa in the seventh century. Sub-Saharan people did not leave traces in the North African maternal gene pool for the time of its settlement, some 40,000 years ago. PMID:20459715

  2. High-resolution `Fiber-FISH`: The generation of barcodes along large genes and the study of replication structures in human DNA

    SciTech Connect

    Dunnen, J.T.D.; Rosenberg, C.; Blonden, L.A.J.

    1994-09-01

    Fluorescent In Situ Hybridization (FISH) is a powerful technique enabling the simultaneous study of multiple targets. FISH covers a wide range of genomic resolutions and recently developed spreading techniques bring the resolution of FISH mapping to the level of the DNA double helix itself. We have successfully used these `Fiber-FISH` approaches, in combination with digital image analysis, as tools to assess cosmid contigs, YAC maps, YAC chimerism, the sizing of gaps/overlaps and study of replication structures in human DNA. The size estimates obtained are highly accurate and reproducible down to 0.3 {mu}m or 1 kb, the theoretical light microscopic resolution. Fiber-FISH on human cells allowed mapping and ordering of sequences at distances ranging from 1 to 400 kb. For two large genes, thyroglobin (330 kb) and dystrophin (2.3 Mb), linear, multi-color bar codes were derived using adjacent and overlapping cosmids. This approach should ultimately enable the rapid identification of gene rearrangements. Furthermore, we developed a Fiber-FISH protocol using cosmid probes on YACs in decondensed yeast DNA. The high resolution, combined with the high efficiency and strong signals obtained, enabled both the establishment of detailed linear maps and the direct visualization of replication `forks` and `bubbles`. The multi-color patterns directionally orient the replication process and identify sequences in cloned human DNA which function as replication origins in yeast. Replication intermediates were also observed in unsynchronized human cells, suggesting that Fiber-FISH allows mapping of replication origins along megabase regions of DNA. This opens exciting possibilities to study mammalian replication, its timing, its delineation in replication zones and its potential relation with processes as X-inactivation and genomic imprinting.

  3. Salt Concentration Effects on Equilibrium Melting Curves from DNA Microarrays

    PubMed Central

    Fuchs, J.; Fiche, J.-B.; Buhot, A.; Calemczuk, R.; Livache, T.

    2010-01-01

    DNA microarrays find applications in an increasing number of domains where more quantitative results are required. DNA being a charged polymer, the repulsive interactions between the surface of the microarray and the targets in solution are increasing upon hybridization. Such electrostatic penalty is generally reduced by increasing the salt concentration. In this article, we present equilibrium-melting curves obtained from dedicated physicochemical experiments on DNA microarrays in order to get a better understanding of the electrostatic penalty incurred during the hybridization reaction at the surface. Various salt concentrations have been considered and deviations from the commonly used Langmuir adsorption model are experimentally quantified for the first time in agreement with theoretical predictions. PMID:20858434

  4. dnaMATE: a consensus melting temperature prediction server for short DNA sequences.

    PubMed

    Panjkovich, Alejandro; Norambuena, Tomás; Melo, Francisco

    2005-07-01

    An accurate and robust large-scale melting temperature prediction server for short DNA sequences is dispatched. The server calculates a consensus melting temperature value using the nearest-neighbor model based on three independent thermodynamic data tables. The consensus method gives an accurate prediction of melting temperature, as it has been recently demonstrated in a benchmark performed using all available experimental data for DNA sequences within the length range of 16-30 nt. This constitutes the first web server that has been implemented to perform a large-scale calculation of melting temperatures in real time (up to 5000 DNA sequences can be submitted in a single run). The expected accuracy of calculations carried out by this server in the range of 50-600 mM monovalent salt concentration is that 89% of the melting temperature predictions will have an error or deviation of <5 degrees C from experimental data. The server can be freely accessed at http://dna.bio.puc.cl/tm.html. The standalone executable versions of this software for LINUX, Macintosh and Windows platforms are also freely available at the same web site. Detailed further information supporting this server is available at the same web site referenced above.

  5. A new method for high-resolution imaging of Ku foci to decipher mechanisms of DNA double-strand break repair

    PubMed Central

    Britton, Sébastien; Coates, Julia

    2013-01-01

    DNA double-strand breaks (DSBs) are the most toxic of all genomic insults, and pathways dealing with their signaling and repair are crucial to prevent cancer and for immune system development. Despite intense investigations, our knowledge of these pathways has been technically limited by our inability to detect the main repair factors at DSBs in cells. In this paper, we present an original method that involves a combination of ribonuclease- and detergent-based preextraction with high-resolution microscopy. This method allows direct visualization of previously hidden repair complexes, including the main DSB sensor Ku, at virtually any type of DSB, including those induced by anticancer agents. We demonstrate its broad range of applications by coupling it to laser microirradiation, super-resolution microscopy, and single-molecule counting to investigate the spatial organization and composition of repair factories. Furthermore, we use our method to monitor DNA repair and identify mechanisms of repair pathway choice, and we show its utility in defining cellular sensitivities and resistance mechanisms to anticancer agents. PMID:23897892

  6. High-resolution melting (HRM) of the cytochrome B gene: a powerful approach to identify blood-meal sources in Chagas disease Vectors.

    PubMed

    Peña, Victor H; Fernández, Geysson J; Gómez-Palacio, Andrés M; Mejía-Jaramillo, Ana M; Cantillo, Omar; Triana-Chávez, Omar

    2012-01-01

    Methods to determine blood-meal sources of hematophagous Triatominae bugs (Chagas disease vectors) are serological or based on PCR employing species-specific primers or heteroduplex analysis, but these are expensive, inaccurate, or problematic when the insect has fed on more than one species. To solve those problems, we developed a technique based on HRM analysis of the mitochondrial gene cytochrome B (Cyt b). This technique recognized 14 species involved in several ecoepidemiological cycles of the transmission of Trypanosoma cruzi and it was suitable with DNA extracted from intestinal content and feces 30 days after feeding, revealing a resolution power that can display mixed feedings. Field samples were analyzed showing blood meal sources corresponding to domestic, peridomiciliary and sylvatic cycles. The technique only requires a single pair of primers that amplify the Cyt b gene in vertebrates and no other standardization, making it quick, easy, relatively inexpensive, and highly accurate.

  7. DNA melting temperature assay for assessing the stability of DNA polyplexes intended for nonviral gene delivery.

    PubMed

    Schallon, Anja; Synatschke, Christopher V; Pergushov, Dmitry V; Jérôme, Valérie; Müller, Axel H E; Freitag, Ruth

    2011-10-01

    Many synthetic polycations have the ability to form complexes with the polyanion DNA, yet only a few, most notably poly(ethylene imine) (PEI), are efficient gene-delivery vehicles. Although a common explanation of this observation relies on the buffering capacity of the polycation, the intracellular stability of the complex may also play a role and should not be neglected. Assays typically used to follow complex formation, however, often do not provide the required information on stability. In this article, we propose the change in the DNA melting temperature observable after complex formation to be a significant indicator of complex stability. For a given DNA/polycation ratio, changes in the melting temperature are shown to depend on the polycation chemistry but not on the DNA topology or the polycation architecture. Effects of changes in the DNA/polycation ratio as well as the effect of polycation quaternization can be interpreted using the melting temperature assay. Finally, the assay was used to follow the displacement of DNA from the complexes by poly(methacrylic acid) or short single-stranded DNA sequences as competing polyanions.

  8. Entropy-driven denaturation and bubble nucleation in DNA melting

    NASA Astrophysics Data System (ADS)

    Singha Roy, Subhamoy; Bandyopadhyay, Pratul

    2015-02-01

    The conformational properties of a DNA molecule when mapped onto a Heisenberg spin system denaturation transition can be formulated in terms of quantum phase transition induced by a quench where the temperature effect is incorporated in the quench time. Here torsion takes on the role of the external field. The denaturation transition occurs when the entanglement entropy of the spin system vanishes. As the critical region corresponds to a two-limit behaviour the entanglement entropy gradually decreases with the gradual increase in the fraction of open base pairs and when the entropy vanishes the two strands are separated. The sequence heterogeneity interplays with the entropy effects and we have the onset of denaturation bubbles. We have studied the melting profiles for different sequence-specific DNA molecules and the results are found to be in excellent agreement with experiment.

  9. The effect of local melting of DNA on DNA loop formation

    NASA Astrophysics Data System (ADS)

    Jeong, Jiyoun; Kim, Harold

    Statistical mechanics of double-stranded DNA (dsDNA) is well described by the wormlike chain model (WLC) which assumes a harmonic bending potential. Such smooth bending potential may no longer be valid for large bending angles to form small loops (<100 bp). Instead, DNA may rely on rare structural transitions such as local melting (opening) of base pairs to lower the energetic cost. In theory, open base pairs called bubbles can increase the looping probability of short DNA molecules by a few orders of magnitude, but a robust experimental validation of this theoretical prediction is lacking. Here, we investigated the correlation between local melting probability and looping dynamics of dsDNA using single-molecule fluorescence resonance energy transfer (FRET). We designed two groups of short DNA molecules with low and high melting probabilities around their center and measured their looping and unlooping rates in equilibrium. Our data allow rigorous tests of meltable wormlike chain (MWLC) models at short length scales for setting ranges of acceptable free energy cost of bubble formation and flexibility values of a bubble.

  10. High-resolution headlamp

    NASA Astrophysics Data System (ADS)

    Gut, Carsten; Cristea, Iulia; Neumann, Cornelius

    2016-04-01

    The following article shall describe how human vision by night can be influenced. At first, front lighting systems that are already available on the market will be described, followed by their analysis with respect to the positive effects on traffic safety. Furthermore, how traffic safety by night can be increased since the introduction of high resolution headlamps shall be discussed.

  11. High-Resolution Global Analysis of the Influences of Bas1 and Ino4 Transcription Factors on Meiotic DNA Break Distributions in Saccharomyces cerevisiae

    PubMed Central

    Zhu, Xuan; Keeney, Scott

    2015-01-01

    Meiotic recombination initiates with DNA double-strand breaks (DSBs) made by Spo11. In Saccharomyces cerevisiae, many DSBs occur in “hotspots” coinciding with nucleosome-depleted gene promoters. Transcription factors (TFs) stimulate DSB formation in some hotspots, but TF roles are complex and variable between locations. Until now, available data for TF effects on global DSB patterns were of low spatial resolution and confined to a single TF. Here, we examine at high resolution the contributions of two TFs to genome-wide DSB distributions: Bas1, which was known to regulate DSB activity at some loci, and Ino4, for which some binding sites were known to be within strong DSB hotspots. We examined fine-scale DSB distributions in TF mutant strains by deep sequencing oligonucleotides that remain covalently bound to Spo11 as a byproduct of DSB formation, mapped Bas1 and Ino4 binding sites in meiotic cells, evaluated chromatin structure around DSB hotspots, and measured changes in global messenger RNA levels. Our findings show that binding of these TFs has essentially no predictive power for DSB hotspot activity and definitively support the hypothesis that TF control of DSB numbers is context dependent and frequently indirect. TFs often affected the fine-scale distributions of DSBs within hotspots, and when seen, these effects paralleled effects on local chromatin structure. In contrast, changes in DSB frequencies in hotspots did not correlate with quantitative measures of chromatin accessibility, histone H3 lysine 4 trimethylation, or transcript levels. We also ruled out hotspot competition as a major source of indirect TF effects on DSB distributions. Thus, counter to prevailing models, roles of these TFs on DSB hotspot strength cannot be simply explained via chromatin “openness,” histone modification, or compensatory interactions between adjacent hotspots. PMID:26245832

  12. Comparison of experimental to MELTSIM calculated DNA melting of the (A+T) rich Dictyostelium discoideum genome: denaturation maps distinguish exons from introns.

    PubMed

    Marx, K A; Assil, I Q; Bizzaro, J W; Blake, R D

    1998-10-01

    The slime mold, Dictyostelium discoideum, possesses an (A+T) rich eukaryotic genome that is being sequenced in the Human Genome Project. High resolution melting curves of isolated total and fractionated nuclear D. discoideum DNA(AX3 strain) were determined experimentally and are compared to melting curves calculated from GENBANK sequences (1.59% of genome) by the statistical thermodynamics program MELTSIM (1), parameterized for long DNA sequences (2,3). The lower and upper temperature limits of calculated melting agree well with the observed melting of total DNA. The experimental curve is unusual in that it contains a number of sharp peaks. MELTSIM allowed us to calculate positional denaturation maps of D. discoideum GENBANK sequence documents containing the 26S, 5.8S and 17S rDNA gene sequences, a major satellite DNA and repetitive sequence family present in 100-200 copies/nucleus. These denaturation maps contain subtransitions that correspond with a number of the experimentally observed peaks, some of which we show to correspond with rDNA gene enriched CsCl gradient fractions of D. discoideum DNA. MELTSIM calculated curves of coding, intron and flanking sequences indicate that both intron and flanking sequences are extremely (A+T) rich and account for most of the low temperature melting. There is no temperature overlap between thermal stabilities of these sequence domains and those of coding DNA. The latter must satisfy triplet codon constraints of higher (G+C) content. These large stability property differences enable a denaturation mapping feature of MELTSIM to clearly distinguish exon positions from those of introns and flanking DNA in long D. discoideum gene containing sequences.

  13. High resolution melting analysis (HRM) as a new tool for the identification of species belonging to the Lactobacillus casei group and comparison with species-specific PCRs and multiplex PCR.

    PubMed

    Iacumin, Lucilla; Ginaldi, Federica; Manzano, Marisa; Anastasi, Veronica; Reale, Anna; Zotta, Teresa; Rossi, Franca; Coppola, Raffaele; Comi, Giuseppe

    2015-04-01

    The correct identification and characterisation of bacteria is essential for several reasons: the classification of lactic acid bacteria (LAB) has changed significantly over the years, and it is important to distinguish and define them correctly, according to the current nomenclature, avoiding problems in the interpretation of literature, as well as mislabelling when probiotic are used in food products. In this study, species-specific PCR and HRM (high-resolution melting) analysis were developed to identify strains belonging to the Lactobacillus casei group and to classify them into L. casei, Lactobacillus paracasei and Lactobacillus rhamnosus. HRM analysis confirmed to be a potent, simple, fast and economic tool for microbial identification. In particular, 201 strains, collected from International collections and attributed to the L. casei group, were examined using these techniques and the results were compared with consolidated molecular methods, already published. Seven of the tested strains don't belong to the L. casei group. Among the remaining 194 strains, 6 showed inconsistent results, leaving identification undetermined. All the applied techniques were congruent for the identification of the vast majority of the tested strains (188). Notably, for 46 of the strains, the identification differed from the previous attribution. PMID:25475306

  14. MECP2 mutations in Czech patients with Rett syndrome and Rett-like phenotypes: novel mutations, genotype-phenotype correlations and validation of high-resolution melting analysis for mutation scanning.

    PubMed

    Zahorakova, Daniela; Lelkova, Petra; Gregor, Vladimir; Magner, Martin; Zeman, Jiri; Martasek, Pavel

    2016-07-01

    Rett syndrome (RTT) is an X-linked neurodevelopmental disorder characterized by developmental regression with loss of motor, communication and social skills, onset of stereotypic hand movements and often seizures. RTT is primarily caused by de novo mutations in the methyl-CpG-binding protein 2 gene (MECP2). We established a high-resolution melting (HRM) technique for mutation scanning of the MECP2 gene and performed analyses in Czech patients with RTT, autism spectrum conditions and intellectual disability with Rett-like features. In the cases with confirmed MECP2 mutations, we determined X-chromosome inactivation (XCI), examined the relationships between genotype and clinical severity and evaluated the modifying influence of XCI. Our results demonstrate that HRM analysis is a reliable method for the detection of point mutations, small deletions and duplications in the MECP2 gene. We identified 29 pathogenic mutations in 75 girls, including four novel mutations: c.155_1189del1035;909_932inv;insC, c.573delC, c.857_858dupAA and c.1163_1200del38. Skewed XCI (ratio >75%) was found in 19.3% of the girls, but no gross divergence in clinical severity was observed. Our findings confirm a high mutation frequency in classic RTT (92%) and a correlation between the MECP2 mutation type and clinical severity. We also demonstrate limitations of XCI in explaining all of the phenotypic differences in RTT.

  15. Detection of EGFR mutation in supernatant, cell pellets of pleural effusion and tumor tissues from non-small cell lung cancer patients by high resolution melting analysis and sequencing.

    PubMed

    Lin, Jie; Gu, Ye; Du, Rui; Deng, Min; Lu, Yaodan; Ding, Yanqing

    2014-01-01

    To determine epidermal growth factor receptor (EGFR) mutation in advanced non-small cell lung cancer (NSCLC) patients and compare the detection efficiency between different sample resources, both high resolution melting (HRM) analysis and direct sequencing method were used to analyze 36 pleural effusion samples and 22 matched biopsy tumor tissues collected from NSCLC patients. For each pleural effusion sample, the supernatant and the cell pellets were examined separately. Among all the 36 cases of pleural effusion samples, 18 mutations of EGFR were found in cell-free supernatant while 13 mutations were found in the cell pellets as detected by HRM analysis. In the 22 matched samples, 13 cases of EGFR mutations were identified in paraffin-embedded biopsy tissue samples, 12 cases in the cell-free supernatant and 9 cases in the cell pellets of pleural effusion. EGFR mutations in 15 cases out of the total 36 pleural effusion samples detected by direct sequencing were also identified by HRM analysis, giving 100% efficiency for HRM method. The results established the important role of HRM as a reliable and efficient method to determine EGFR mutation status and indicated the feasibility of using pleural effusion in replacement of biopsy tissues in particular clinical cases. Furthermore, the cell-free supernatant of pleural effusion might be a better resource for mutation detection than cell pellets.

  16. A screening method for the detection of the 35S promoter and the nopaline synthase terminator in genetically modified organisms in a real-time multiplex polymerase chain reaction using high-resolution melting-curve analysis.

    PubMed

    Akiyama, Hiroshi; Nakamura, Fumi; Yamada, Chihiro; Nakamura, Kosuke; Nakajima, Osamu; Kawakami, Hiroshi; Harikai, Naoki; Furui, Satoshi; Kitta, Kazumi; Teshima, Reiko

    2009-11-01

    To screen for unauthorized genetically modified organisms (GMO) in the various crops, we developed a multiplex real-time polymerase chain reaction high-resolution melting-curve analysis method for the simultaneous qualitative detection of 35S promoter sequence of cauliflower mosaic virus (35SP) and the nopaline synthase terminator (NOST) in several crops. We selected suitable primer sets for the simultaneous detection of 35SP and NOST and designed the primer set for the detection of spiked ColE1 plasmid to evaluate the validity of the polymerase chain reaction (PCR) analyses. In addition, we optimized the multiplex PCR conditions using the designed primer sets and EvaGreen as an intercalating dye. The contamination of unauthorized GMO with single copy similar to NK603 maize can be detected as low as 0.1% in a maize sample. Furthermore, we showed that the present method would be applicable in identifying GMO in various crops and foods like authorized GM soybean, authorized GM potato, the biscuit which is contaminated with GM soybeans and the rice which is contaminated with unauthorized GM rice. We consider this method to be a simple and reliable assay for screening for unauthorized GMO in crops and the processing food products.

  17. Comparative analysis of two broad-range PCR assays for pathogen detection in positive-blood-culture bottles: PCR-high-resolution melting analysis versus PCR-mass spectrometry.

    PubMed

    Jeng, Kevin; Gaydos, Charlotte A; Blyn, Lawrence B; Yang, Samuel; Won, Helen; Matthews, Heather; Toleno, Donna; Hsieh, Yu-Hsiang; Carroll, Karen C; Hardick, Justin; Masek, Billy; Kecojevic, Alexander; Sampath, Rangarajan; Peterson, Stephen; Rothman, Richard E

    2012-10-01

    Detection of pathogens in bloodstream infections is important for directing antimicrobial treatment, but current culture-based approaches can be problematic. Broad-range PCR assays which target conserved genomic motifs for postamplification amplicon analysis permit detection of sepsis-causing pathogens. Comparison of different broad-range assays is important for informing future implementation strategies. In this study, we compared positive-blood-culture bottles processed by PCR coupled to high-resolution melting curve analysis (PCR/HRMA) and PCR coupled to electrospray ionization-mass spectrometry (PCR/ESI-MS) to microbiology culture results. Genus-level concordance was 90% (confidence interval [CI], 80 to 96%) for PCR/HRMA and 94% (CI, 85 to 98%) for PCR/ESI-MS. Species-level concordance was 90% (CI, 80 to 96%) for PCR/HRMA and 86% (CI, 75 to 93%) for PCR/ESI-MS. Unlike PCR/HRMA, PCR/ESI-MS was able to resolve polymicrobial samples. Our results demonstrated that the two assays have similar overall concordance rates but may have different roles as potential adjunctive tests with standard blood culture, since each method has different capabilities, advantages, and disadvantages.

  18. High resolution data acquisition

    DOEpatents

    Thornton, Glenn W.; Fuller, Kenneth R.

    1993-01-01

    A high resolution event interval timing system measures short time intervals such as occur in high energy physics or laser ranging. Timing is provided from a clock (38) pulse train (37) and analog circuitry (44) for generating a triangular wave (46) synchronously with the pulse train (37). The triangular wave (46) has an amplitude and slope functionally related to the time elapsed during each clock pulse in the train. A converter (18, 32) forms a first digital value of the amplitude and slope of the triangle wave at the start of the event interval and a second digital value of the amplitude and slope of the triangle wave at the end of the event interval. A counter (26) counts the clock pulse train (37) during the interval to form a gross event interval time. A computer (52) then combines the gross event interval time and the first and second digital values to output a high resolution value for the event interval.

  19. High resolution data acquisition

    DOEpatents

    Thornton, G.W.; Fuller, K.R.

    1993-04-06

    A high resolution event interval timing system measures short time intervals such as occur in high energy physics or laser ranging. Timing is provided from a clock, pulse train, and analog circuitry for generating a triangular wave synchronously with the pulse train (as seen in diagram on patent). The triangular wave has an amplitude and slope functionally related to the time elapsed during each clock pulse in the train. A converter forms a first digital value of the amplitude and slope of the triangle wave at the start of the event interval and a second digital value of the amplitude and slope of the triangle wave at the end of the event interval. A counter counts the clock pulse train during the interval to form a gross event interval time. A computer then combines the gross event interval time and the first and second digital values to output a high resolution value for the event interval.

  20. High-Resolution Autoradiography

    NASA Technical Reports Server (NTRS)

    Towe, George C; Gomberg, Henry J; Freemen, J W

    1955-01-01

    This investigation was made to adapt wet-process autoradiography to metallurgical samples to obtain high resolution of segregated radioactive elements in microstructures. Results are confined to development of the technique, which was perfected to a resolution of less than 10 microns. The radioactive samples included carbon-14 carburized iron and steel, nickel-63 electroplated samples, a powder product containing nickel-63, and tungsten-185 in N-155 alloy.

  1. Ultra high resolution tomography

    SciTech Connect

    Haddad, W.S.

    1994-11-15

    Recent work and results on ultra high resolution three dimensional imaging with soft x-rays will be presented. This work is aimed at determining microscopic three dimensional structure of biological and material specimens. Three dimensional reconstructed images of a microscopic test object will be presented; the reconstruction has a resolution on the order of 1000 A in all three dimensions. Preliminary work with biological samples will also be shown, and the experimental and numerical methods used will be discussed.

  2. High-resolution X-ray structure of the DNA-binding protein HU from the hyper-thermophilic Thermotoga maritima and the determinants of its thermostability.

    PubMed

    Christodoulou, Evangelos; Rypniewski, Wojciech R; Vorgias, Constantinos R E

    2003-04-01

    The histone-like DNA-binding proteins (HU) are a convenient model for studying factors affecting thermostability because of their relatively simple, easily comparable structures, their common function, and their presence in organisms of widely differing thermostability. We report the determination of the high-resolution structure (1.53 A) at 273 K and 100 K of the HU protein from the hyper-thermophilic eubacterium Thermotoga maritima(HU Tmar, T(m)=80.5 degrees C). The structural data presented clearly show that the HU Tmar has a fold similar to its thermophilic homologue HU from Bacillus stearothermophilus (HU Bst). Based on primary structure analysis, as well as on the results of mutational analysis of HU Bst ( T(m)=61.6 degrees C) and Bacillus subtilis (HU Bsu, T(m)=39.7 degrees C), we have designed and produced several single and combined mutations to study their effect on the thermostability of the recombinant HU Tmar. Among others, the triplet mutant HU Tmar-G15E/E34D/V42I ( T(m)=35.9 degrees C) has converted the extreme thermophilic protein HU Tmar to mesophilic, like HU Bsu. In an attempt to analyze the various mutants of HU Tmar, we crystallized the point mutation HU Tmar-E34D, in which Glu34 was replaced by Asp, similar to the mesophilic HU Bsu. The mutant has T(m)=72.9 degrees C, as measured by circular dichroism, 7.6 degrees C lower than the wild type. The crystal structure of HU Tmar-E34D was determined at 100 K and refined at 1.72 A resolution. A comparison with the wild-type structures clearly shows that two hydrogen bonds have been disrupted between Glu34 from one subunit and Thr13 from the other subunit, and vice versa. Our analysis points to this as the prime cause of the destabilization compared to the wild type. The three new structures were compared, together with the X-ray structure of a similar protein, HU Bst, with the aim of relating their structural properties and different thermal stability. The presented results show that the HU Tmar

  3. High-resolution solid-state NMR study of the effect of composition on network connectivity and structural disorder in multi-component glasses in the diopside and jadeite join: Implications for structure of andesitic melts

    NASA Astrophysics Data System (ADS)

    Park, Sun Young; Lee, Sung Keun

    2014-12-01

    The structural evolution of andesitic melts with varying compositions remains one of the unsolved questions in high-temperature geochemistry and petrology. In this article, we report the structural details of model andesitic glasses [CaO-MgO-Na2O-Al2O3-SiO2 (CMNAS)] in the diopside (CaMgSi2O6) and jadeite (NaAlSi2O6) join using high-resolution, multi-nuclear, solid-state nuclear magnetic resonance (NMR). The 27Al NMR spectra of CMNAS glasses confirm that [4]Al is dominant. While a minor fraction of [5]Al is observed, its presence is only prevalent in the glasses with higher Ca-Mg content. Topological disorder in the glass network also tends to increase with Ca-Mg content as evidenced by the increase in the quadrupolar coupling constant (Cq) of [4]Al for glasses with increasing diopside contents (XDiopside). Despite the complex nature of the glasses studied here (with five oxide components), the 17O 3QMAS NMR spectra resolve diverse bridging oxygens (BOs) and non-bridging oxygens (NBOs), from which the degree of Al avoidance among framework cations (Si and Al) and preferential proximity among non-network cations (Ca2+, Mg2+, and Na+) and each oxygen site can be estimated: presence of Al-O-Al in jadeite glass implies a violation of the Al-avoidance rule in the glasses and the decrease in the fraction of NBOs with increasing XDiopside is consistent with a decrease in their viscosity. Analysis of the peak position of {Ca, Mg}-mixed NBOs, along with the absence of Na-NBO peak, and the peak shape of Si-O-Al reveals preferential partitioning of Ca2+ and Mg2 into NBOs and the proximity of Na+ to BOs. The fraction of highly coordinated Al has been linked to thermodynamic and transport properties of the melts. Considering all the experimental Al coordination environments available in the literature, together with the current experimental studies, we attempt to establish the relationship between the fractions of highly coordinated Al and composition, particularly average

  4. Comparison of multiple genes and 16S-23S rRNA intergenic space region for their capacity in high resolution melt curve analysis to differentiate Mycoplasma gallisepticum vaccine strain ts-11 from field strains.

    PubMed

    Ghorashi, Seyed A; Bradbury, Janet M; Ferguson-Noel, Naola M; Noormohammadi, Amir H

    2013-12-27

    Mycoplasma gallisepticum (MG) is an important avian pathogen causing significant economic losses in the global poultry industry. In an attempt to compare and evaluate existing genotyping methods for differentiation of MG strains/isolates, high resolution melt (HRM) curve analysis was applied to 5 different PCR methods targeting vlhA, pvpA, gapA, mgc2 genes and 16S-23S rRNA intergenic space region (IGSR). To assess the discriminatory power of PCR-HRM of examined genes and IGSR, MG strains ts-11, F, 6/85 and S6, and, initially, 8 field isolates were tested. All MG strains/isolates were differentiated using PCR-HRM curve analysis and genotype confidence percentage (GCP) values of vlhA and pvpA genes, while only 0, 3 and 4 out of 12 MG strains/isolates were differentiated using gapA, mgc2 genes and IGSR, respectively. The HRM curve analysis of vlhA and pvpA genes was found to be highly correlated with the genetic diversity of the targeted genes confirmed by sequence analysis of amplicons generated from MG strains. The potential of the vlhA and pvpA genes was also demonstrated for genotyping of 12 additional MG strains from Europe and the USA. Results from this study provide a direct comparison between genes previously used in sequencing-based genotyping methods for MG strain identification and highlight the usefulness of vlhA and pvpA HRM curve analyses as rapid and reliable tools specially for diagnosis and differentiation of MG strains used here.

  5. Comparative melting and healing of B-DNA and Z-DNA by an infrared laser pulse.

    PubMed

    Man, Viet Hoang; Pan, Feng; Sagui, Celeste; Roland, Christopher

    2016-04-14

    We explore the use of a fast laser melting simulation approach combined with atomistic molecular dynamics simulations in order to determine the melting and healing responses of B-DNA and Z-DNA dodecamers with the same d(5'-CGCGCGCGCGCG-3')2 sequence. The frequency of the laser pulse is specifically tuned to disrupt Watson-Crick hydrogen bonds, thus inducing melting of the DNA duplexes. Subsequently, the structures relax and partially refold, depending on the field strength. In addition to the inherent interest of the nonequilibrium melting process, we propose that fast melting by an infrared laser pulse could be used as a technique for a fast comparison of relative stabilities of same-sequence oligonucleotides with different secondary structures with full atomistic detail of the structures and solvent. This could be particularly useful for nonstandard secondary structures involving non-canonical base pairs, mismatches, etc.

  6. The energetics of tightly bent DNA: a composite elastica model including local melting

    NASA Astrophysics Data System (ADS)

    Evans, Arthur; Levine, Alex

    2012-02-01

    Melting transitions are well-known to be affected by the application of mechanical stress. Motivated by the experiments of Zocchi and collaborators (Qu and Zocchi 2011, EPL 94 18003), we explore the effect of the application of mechanical stress on DNA melting in a particular composite of a stiff double stranded piece of DNA (dsDNA), shorter than its own persistence length, whose ends are linked by a flexible single stranded piece of DNA (ssDNA). The flexible ssDNA acts as a Gaussian polymer coil bending the stiff dsDNA through an elastic force that is controllable by the length of the ssDNA chain. In this talk we present theoretical predictions for two experimentally accessible features: the degree of local dsDNA melting and the local elastic energy of the dsDNA/ssDNA construct both as a function of the length of the attached ssDNA. We also address the effect of introducing a nick (broken covalent bond) in the dsDNA backbone on these results and discuss the implications of such data on the relative importance of backbone elasticity versus base stacking and base pairing interactions in determining the elasticity of dsDNA. This work also addresses open questions in the nonlinear elasticity of DNA in tightly bent curves.

  7. How nanochannel confinement affects the DNA melting transition within the Poland-Scheraga model

    NASA Astrophysics Data System (ADS)

    Reiter-Schad, Michaela; Werner, Erik; Tegenfeldt, Jonas O.; Mehlig, Bernhard; Ambjörnsson, Tobias

    2015-09-01

    When double-stranded DNA molecules are heated, or exposed to denaturing agents, the two strands are separated. The statistical physics of this process has a long history and is commonly described in terms of the Poland-Scheraga (PS) model. Crucial to this model is the configurational entropy for a melted region (compared to the entropy of an intact region of the same size), quantified by the loop factor. In this study, we investigate how confinement affects the DNA melting transition, by using the loop factor for an ideal Gaussian chain. By subsequent numerical solutions of the PS model, we demonstrate that the melting temperature depends on the persistence lengths of single-stranded and double-stranded DNA. For realistic values of the persistence lengths, the melting temperature is predicted to decrease with decreasing channel diameter. We also demonstrate that confinement broadens the melting transition. These general findings hold for the three scenarios investigated: 1. homo-DNA, i.e., identical basepairs along the DNA molecule, 2. random sequence DNA, and 3. "real" DNA, here T4 phage DNA. We show that cases 2 and 3 in general give rise to broader transitions than case 1. Case 3 exhibits a similar phase transition as case 2 provided the random sequence DNA has the same ratio of AT to GC basepairs (A - adenine, T - thymine, G - guanine, C - cytosine). A simple analytical estimate for the shift in melting temperature is provided as a function of nanochannel diameter. For homo-DNA, we also present an analytical prediction of the melting probability as a function of temperature.

  8. High Resolution Laboratory Spectroscopy

    NASA Astrophysics Data System (ADS)

    Brünken, S.; Schlemmer, S.

    2016-05-01

    In this short review we will highlight some of the recent advancements in the field of high-resolution laboratory spectroscopy that meet the needs dictated by the advent of highly sensitive and broadband telescopes like ALMA and SOFIA. Among these is the development of broadband techniques for the study of complex organic molecules, like fast scanning conventional absorption spectroscopy based on multiplier chains, chirped pulse instrumentation, or the use of synchrotron facilities. Of similar importance is the extension of the accessible frequency range to THz frequencies, where many light hydrides have their ground state rotational transitions. Another key experimental challenge is the production of sufficiently high number densities of refractory and transient species in the laboratory, where discharges have proven to be efficient sources that can also be coupled to molecular jets. For ionic molecular species sensitive action spectroscopic schemes have recently been developed to overcome some of the limitations of conventional absorption spectroscopy. Throughout this review examples demonstrating the strong interplay between laboratory and observational studies will be given.

  9. High Resolution Doppler Imager

    NASA Technical Reports Server (NTRS)

    Hays, Paul B.

    1999-01-01

    This report summarizes the accomplishments of the High Resolution Doppler Imager (HRDI) on UARS spacecraft during the period 4/l/96 - 3/31/99. During this period, HRDI operation, data processing, and data analysis continued, and there was a high level of vitality in the HRDI project. The HRDI has been collecting data from the stratosphere, mesosphere, and lower thermosphere since instrument activation on October 1, 1991. The HRDI team has stressed three areas since operations commenced: 1) operation of the instrument in a manner which maximizes the quality and versatility of the collected data; 2) algorithm development and validation to produce a high-quality data product; and 3) scientific studies, primarily of the dynamics of the middle atmosphere. There has been no significant degradation in the HRDI instrument since operations began nearly 8 years ago. HRDI operations are fairly routine, although we have continued to look for ways to improve the quality of the scientific product, either by improving existing modes, or by designing new ones. The HRDI instrument has been programmed to collect data for new scientific studies, such as measurements of fluorescence from plants, measuring cloud top heights, and lower atmosphere H2O.

  10. New results on the melting thermodynamics of a circular DNA chain

    NASA Astrophysics Data System (ADS)

    Kabakçıoğlu, A.; Orlandini, E.; Mukamel, D.

    2010-08-01

    We investigate the impact of supercoil period and nonzero supercoil formation energy on the thermal denaturation of a circular DNA. Our analysis is based on a recently proposed generalization of the Poland-Scheraga model that allows the DNA melting to be studied for plasmids with circular topology, where denaturation is accompanied by formation of supercoils. We find that the previously obtained first-order melting transition persists under the generalization discussed. The dependence of the size of the order-parameter jump at the transition point and the associated melting temperature are obtained analytically.

  11. High Resolution Formaldehyde Photochemistry

    NASA Astrophysics Data System (ADS)

    Ernest, C. T.; Bauer, D.; Hynes, A. J.

    2010-12-01

    Formaldehyde (HCHO) is the most abundant and most important organic carbonyl compound in the atmosphere. The sources of formaldehyde are the oxidation of methane, isoprene, acetone, and other volatile organic compounds (VOCs); fossil fuel combustion; and biomass burning. The dominant loss mechanism for formaldehyde is photolysis which occurs via two pathways: (R1) HCHO + hv → HCO + H (R2) HCHO + hv → H2 + CO The first pathway (R1) is referred to as the radical channel, while the second pathway (R2) is referred to as the molecular channel. The products of both pathways play a significant role in atmospheric chemistry. The CO that is produced in the molecular channel undergoes further oxidation to produce CO2. Under atmospheric conditions, the H atom and formyl radical that are produced in the radical channel undergo rapid reactions with O2 to produce the hydroperoxyl radical (HO2) via (R3) and (R4). (R3) HCO + O2 → HO2 + CO (R4) H + O2 → HO2 Thus, for every photon absorbed, the photolysis of formaldehyde can contribute one CO2 molecule to the global greenhouse budget or two HO2 radicals to the tropospheric HOx (OH + HO2) cycle. The HO2 radicals produced during formaldehyde photolysis have also been implicated in the formation of photochemical smog. The HO2 radicals act as radical chain carriers and convert NO to NO2, which ultimately results in the catalytic production of O3. Constraining the yield of HO2 produced via HCHO photolysis is essential for improving tropospheric chemistry models. In this study, both the absorption cross section and the quantum yield of the radical channel (R1) were measured at high resolution over the tropospherically relevant wavelength range 304-330 nm. For the cross section measurements a narrow linewidth Nd:YAG pumped dye laser was used with a multi-pass cell. Partial pressures of HCHO were kept below 0.3 torr. Simultaneous measurement of OH LIF in a flame allowed absolute calibration of the wavelength scale. Pressure

  12. Single molecule detection of PARP1 and PARP2 interaction with DNA strand breaks and their poly(ADP-ribosyl)ation using high-resolution AFM imaging

    PubMed Central

    Sukhanova, Maria V.; Abrakhi, Sanae; Joshi, Vandana; Pastre, David; Kutuzov, Mikhail M.; Anarbaev, Rashid O.; Curmi, Patrick A.; Hamon, Loic; Lavrik, Olga I.

    2016-01-01

    PARP1 and PARP2 are implicated in the synthesis of poly(ADP-ribose) (PAR) after detection of DNA damage. The specificity of PARP1 and PARP2 interaction with long DNA fragments containing single- and/or double-strand breaks (SSBs and DSBs) have been studied using atomic force microscopy (AFM) imaging in combination with biochemical approaches. Our data show that PARP1 localizes mainly on DNA breaks and exhibits a slight preference for nicks over DSBs, although the protein has a moderately high affinity for undamaged DNA. In contrast to PARP1, PARP2 is mainly detected at a single DNA nick site, exhibiting a low level of binding to undamaged DNA and DSBs. The enhancement of binding affinity of PARP2 for DNA containing a single nick was also observed using fluorescence titration. AFM studies reveal that activation of both PARPs leads to the synthesis of highly branched PAR whose size depends strongly on the presence of SSBs and DSBs for PARP1 and of SSBs for PARP2. The initial affinity between the PARP1, PARP2 and the DNA damaged site appears to influence both the size of the PAR synthesized and the time of residence of PARylated PARP1 and PARP2 on DNA damages. PMID:26673720

  13. Single molecule detection of PARP1 and PARP2 interaction with DNA strand breaks and their poly(ADP-ribosyl)ation using high-resolution AFM imaging.

    PubMed

    Sukhanova, Maria V; Abrakhi, Sanae; Joshi, Vandana; Pastre, David; Kutuzov, Mikhail M; Anarbaev, Rashid O; Curmi, Patrick A; Hamon, Loic; Lavrik, Olga I

    2016-04-01

    PARP1 and PARP2 are implicated in the synthesis of poly(ADP-ribose) (PAR) after detection of DNA damage. The specificity of PARP1 and PARP2 interaction with long DNA fragments containing single- and/or double-strand breaks (SSBs and DSBs) have been studied using atomic force microscopy (AFM) imaging in combination with biochemical approaches. Our data show that PARP1 localizes mainly on DNA breaks and exhibits a slight preference for nicks over DSBs, although the protein has a moderately high affinity for undamaged DNA. In contrast to PARP1, PARP2 is mainly detected at a single DNA nick site, exhibiting a low level of binding to undamaged DNA and DSBs. The enhancement of binding affinity of PARP2 for DNA containing a single nick was also observed using fluorescence titration. AFM studies reveal that activation of both PARPs leads to the synthesis of highly branched PAR whose size depends strongly on the presence of SSBs and DSBs for PARP1 and of SSBs for PARP2. The initial affinity between the PARP1, PARP2 and the DNA damaged site appears to influence both the size of the PAR synthesized and the time of residence of PARylated PARP1 and PARP2 on DNA damages.

  14. High resolution time interval meter

    DOEpatents

    Martin, A.D.

    1986-05-09

    Method and apparatus are provided for measuring the time interval between two events to a higher resolution than reliability available from conventional circuits and component. An internal clock pulse is provided at a frequency compatible with conventional component operating frequencies for reliable operation. Lumped constant delay circuits are provided for generating outputs at delay intervals corresponding to the desired high resolution. An initiation START pulse is input to generate first high resolution data. A termination STOP pulse is input to generate second high resolution data. Internal counters count at the low frequency internal clock pulse rate between the START and STOP pulses. The first and second high resolution data are logically combined to directly provide high resolution data to one counter and correct the count in the low resolution counter to obtain a high resolution time interval measurement.

  15. Novel high-resolution characterization of ancient DNA reveals C > U-type base modification events as the sole cause of post mortem miscoding lesions

    PubMed Central

    Brotherton, Paul; Endicott, Phillip; Sanchez, Juan J.; Beaumont, Mark; Barnett, Ross; Austin, Jeremy; Cooper, Alan

    2007-01-01

    Ancient DNA (aDNA) research has long depended on the power of PCR to amplify trace amounts of surviving genetic material from preserved specimens. While PCR permits specific loci to be targeted and amplified, in many ways it can be intrinsically unsuited to damaged and degraded aDNA templates. PCR amplification of aDNA can produce highly-skewed distributions with significant contributions from miscoding lesion damage and non-authentic sequence artefacts. As traditional PCR-based approaches have been unable to fully resolve the molecular nature of aDNA damage over many years, we have developed a novel single primer extension (SPEX)-based approach to generate more accurate sequence information. SPEX targets selected template strands at defined loci and can generate a quantifiable redundancy of coverage; providing new insights into the molecular nature of aDNA damage and fragmentation. SPEX sequence data reveals inherent limitations in both traditional and metagenomic PCR-based approaches to aDNA, which can make current damage analyses and correct genotyping of ancient specimens problematic. In contrast to previous aDNA studies, SPEX provides strong quantitative evidence that C > U-type base modifications are the sole cause of authentic endogenous damage-derived miscoding lesions. This new approach could allow ancient specimens to be genotyped with unprecedented accuracy. PMID:17715147

  16. The melting phenomenon in random-walk model of DNA

    SciTech Connect

    Hayrapetyan, G. N.; Mamasakhlisov, E. Sh.; Papoyan, Vl. V.; Poghosyan, S. S.

    2012-10-15

    The melting phenomenon in a double-stranded homopolypeptide is considered. The relative distance between the corresponding monomers of two polymer chains is modeled by the two-dimensional random walk on the square lattice. Returns of the random walk to the origin describe the formation of hydrogen bonds between complementary units. To take into account the two competing interactions of monomers inside the chains, we obtain a completely denatured state at finite temperature T{sub c}.

  17. Roles of DNA polymerase I in leading and lagging-strand replication defined by a high-resolution mutation footprint of ColE1 plasmid replication.

    PubMed

    Allen, Jennifer M; Simcha, David M; Ericson, Nolan G; Alexander, David L; Marquette, Jacob T; Van Biber, Benjamin P; Troll, Chris J; Karchin, Rachel; Bielas, Jason H; Loeb, Lawrence A; Camps, Manel

    2011-09-01

    DNA polymerase I (pol I) processes RNA primers during lagging-strand synthesis and fills small gaps during DNA repair reactions. However, it is unclear how pol I and pol III work together during replication and repair or how extensive pol I processing of Okazaki fragments is in vivo. Here, we address these questions by analyzing pol I mutations generated through error-prone replication of ColE1 plasmids. The data were obtained by direct sequencing, allowing an accurate determination of the mutation spectrum and distribution. Pol I's mutational footprint suggests: (i) during leading-strand replication pol I is gradually replaced by pol III over at least 1.3 kb; (ii) pol I processing of Okazaki fragments is limited to ∼20 nt and (iii) the size of Okazaki fragments is short (∼250 nt). While based on ColE1 plasmid replication, our findings are likely relevant to other pol I replicative processes such as chromosomal replication and DNA repair, which differ from ColE1 replication mostly at the recruitment steps. This mutation footprinting approach should help establish the role of other prokaryotic or eukaryotic polymerases in vivo, and provides a tool to investigate how sequence topology, DNA damage, or interactions with protein partners may affect the function of individual DNA polymerases.

  18. Melting behavior and different bound states in three-stranded DNA models.

    PubMed

    Maji, Jaya; Bhattacharjee, Somendra M; Seno, Flavio; Trovato, Antonio

    2014-01-01

    Thermal denaturation of DNA is often studied with coarse-grained models in which native sequential base pairing is mimicked by the existence of attractive interactions only between monomers at the same position along strands (Poland and Scheraga models). Within this framework, the existence of a three-stranded DNA bound state in conditions where a duplex DNA would be in the denaturated state was recently predicted from a study of three directed polymer models on simplified hierarchical lattices (d>2) and in 1+1 dimensions. Such a phenomenon which is similar to the Efimov effect in nuclear physics was named Efimov-DNA. In this paper we study the melting of the three-stranded DNA on a Sierpinski gasket of dimensions d<2 by assigning extra weight factors to fork openings and closings, to induce a two-strand DNA melting. In such a context we can find again the existence of the Efimov-DNA-like state but quite surprisingly we discover also the presence of a different phase, to be called a mixed state, where the strands are pair-wise bound but without three chain contacts. Whereas the Efimov DNA turns out to be a crossover near melting, the mixed phase is a thermodynamic phase. PMID:24580186

  19. Melting behavior and different bound states in three-stranded DNA models.

    PubMed

    Maji, Jaya; Bhattacharjee, Somendra M; Seno, Flavio; Trovato, Antonio

    2014-01-01

    Thermal denaturation of DNA is often studied with coarse-grained models in which native sequential base pairing is mimicked by the existence of attractive interactions only between monomers at the same position along strands (Poland and Scheraga models). Within this framework, the existence of a three-stranded DNA bound state in conditions where a duplex DNA would be in the denaturated state was recently predicted from a study of three directed polymer models on simplified hierarchical lattices (d>2) and in 1+1 dimensions. Such a phenomenon which is similar to the Efimov effect in nuclear physics was named Efimov-DNA. In this paper we study the melting of the three-stranded DNA on a Sierpinski gasket of dimensions d<2 by assigning extra weight factors to fork openings and closings, to induce a two-strand DNA melting. In such a context we can find again the existence of the Efimov-DNA-like state but quite surprisingly we discover also the presence of a different phase, to be called a mixed state, where the strands are pair-wise bound but without three chain contacts. Whereas the Efimov DNA turns out to be a crossover near melting, the mixed phase is a thermodynamic phase.

  20. Binding of an organo-osmium(II) anticancer complex to guanine and cytosine on DNA revealed by electron-based dissociations in high resolution Top-Down FT-ICR mass spectrometry.

    PubMed

    Wootton, Christopher A; Sanchez-Cano, Carlos; Liu, Hong-Ke; Barrow, Mark P; Sadler, Peter J; O'Connor, Peter B

    2015-02-28

    The Os(II) arene anticancer complex [(η(6)-bip)Os(en)Cl](+) (Os1-Cl; where bip = biphenyl, and en = ethylenediamine) binds strongly to DNA. Here we investigate reactions between Os1-Cl and the self-complementary 12-mer oligonucleotide 5'-TAGTAATTACTA-3' (DNA12) using ultra high resolution Fourier Transform-Ion Cyclotron Resonance Mass Spectrometry (FT-ICR MS). Identification of the specific sites of DNA osmiation with {(η(6)-bip)Os(en)}(2+) was made possible by the use of Electron Detachment Dissociation (EDD) which produced a wide range of assignable osmiated MS/MS fragments. In contrast, the more commonly used CAD and IRMPD techniques produced fragments which lose the bound osmium. These studies reveal that not only is guanine G3 a strong binding site for {(η(6)-bip)Os(en)}(2+) but, unexpectedly, so too is cytosine C10. Interestingly, the G3/C10 di-osmiated adduct of DNA12 also formed readily but did not undergo such facile fragmentation by EDD, perhaps due to folding induced by van der Waal's interactions of the bound osmium arene species. These new insights into osmium arene DNA adducts should prove valuable for the design of new organometallic drugs and contribute to understanding the lack of cross resistance of this organometallic anticancer complex with cisplatin.

  1. Using next-generation sequencing for high resolution multiplex analysis of copy number variation from nanogram quantities of DNA from formalin-fixed paraffin-embedded specimens.

    PubMed

    Wood, Henry M; Belvedere, Ornella; Conway, Caroline; Daly, Catherine; Chalkley, Rebecca; Bickerdike, Melissa; McKinley, Claire; Egan, Phil; Ross, Lisa; Hayward, Bruce; Morgan, Joanne; Davidson, Leslie; MacLennan, Ken; Ong, Thian K; Papagiannopoulos, Kostas; Cook, Ian; Adams, David J; Taylor, Graham R; Rabbitts, Pamela

    2010-08-01

    The use of next-generation sequencing technologies to produce genomic copy number data has recently been described. Most approaches, however, reply on optimal starting DNA, and are therefore unsuitable for the analysis of formalin-fixed paraffin-embedded (FFPE) samples, which largely precludes the analysis of many tumour series. We have sought to challenge the limits of this technique with regards to quality and quantity of starting material and the depth of sequencing required. We confirm that the technique can be used to interrogate DNA from cell lines, fresh frozen material and FFPE samples to assess copy number variation. We show that as little as 5 ng of DNA is needed to generate a copy number karyogram, and follow this up with data from a series of FFPE biopsies and surgical samples. We have used various levels of sample multiplexing to demonstrate the adjustable resolution of the methodology, depending on the number of samples and available resources. We also demonstrate reproducibility by use of replicate samples and comparison with microarray-based comparative genomic hybridization (aCGH) and digital PCR. This technique can be valuable in both the analysis of routine diagnostic samples and in examining large repositories of fixed archival material.

  2. DNA melting properties of the dityrosine cross-linked dimer of Ribonuclease A.

    PubMed

    Dinda, Amit Kumar; Chattaraj, Saparya; Ghosh, Sudeshna; Tripathy, Debi Ranjan; Dasgupta, Swagata

    2016-09-01

    Several DNA binding proteins exist in dimeric form when bound with DNA to be able to exhibit various biological processes such as DNA repair, DNA replication and gene expression. Various dimeric forms of Ribonuclease A (RNase A) and other members of the ribonuclease A superfamily are endowed with a multitude of biological activities such as antitumor and antiviral activity. In the present study, we have compared the DNA binding properties between the RNase A monomer and the dityrosine (DT) cross-linked RNase A dimer, and checked the inhibitory effect of DNA on the ribonucleolytic activity of the dimeric protein. An agarose gel based assay shows that like the monomer, the dimer also binds with DNA. The number of nucleotides bound per monomer unit of the dimer is higher than the number of nucleotides that bind with the each monomer. From fluorescence measurements, the association constant (Ka) values for complexation of the monomer and the dimer with ct-DNA are (4.95±0.45)×10(4)M(-1) and (1.29±0.05)×10(6)M(-1) respectively. Binding constant (Kb) values for the binding of the monomer and the dimer with ct-DNA were determined using UV-vis spectroscopy and were found to be (4.96±1.67)×10(4)M(-1) and (4.32±0.31)×10(5)M(-1) respectively. Circular dichroism studies shows that the dimer possesses significant effect on DNA conformation. The melting profile for the ct-DNA-dimer indicated that the melting temperature (Tm) for the ct-DNA-dimer complex is lower compared to the ct-DNA-monomer complex. The ribonucleolytic activity of the dimer, like the monomer, diminishes upon binding with DNA. PMID:27475778

  3. First- or second-order transition in the melting of repeat sequence DNA.

    PubMed Central

    Chen, Y Z; Prohofsky, E W

    1994-01-01

    Both theoretical analysis and observation of the continuity of the melted fraction of base pairs indicate that the melting transition in DNA is second order. Analysis of the salt dependence of the transition by polyelectrolyte limiting laws, however, has first-order dynamics imbedded in the analysis. This paper proposes that the observation taken to be a latent heat of melting in the limiting law analysis could instead be a specific heat anomaly associated with a second-order transition. The limiting laws can be reconstructed based on a second-order transition with a specific heat anomaly. The T2M dependence of this excess heat is also consistent with its being a specific heat anomaly of a system displaying classical critical behavior. Classical critical behavior indicates that theoretical mean field approaches such as MSPA should be particularly appropriate to helix melting studies. PMID:8130338

  4. Unearthing the ecology of soil microorganisms using a high resolution DNA-SIP approach to explore cellulose and xylose metabolism in soil

    DOE PAGES

    Pepe-Ranney, Charles; Campbell, Ashley N.; Koechli, Chantal N.; Berthrong, Sean; Buckley, Daniel H.

    2016-05-12

    We explored microbial contributions to decomposition using a sophisticated approach to DNA Stable Isotope Probing (SIP). Our experiment evaluated the dynamics and ecological characteristics of functionally defined microbial groups that metabolize labile and structural C in soils. We added to soil a complex amendment representing plant derived organic matter substituted with either 13C-xylose or 13C-cellulose to represent labile and structural C pools derived from abundant components of plant biomass. We found evidence for 13C-incorporation into DNA from 13C-xylose and 13C-cellulose in 49 and 63 operational taxonomic units (OTUs), respectively. The types of microorganisms that assimilated 13C in the 13C-xylose treatmentmore » changed over time being predominantly Firrnicutes at day 1 followed by Bacteroidetes at day 3 and then Actinobacteria at day 7. These 13C-labeling dynamics suggest labile C traveled through different trophic levels. In contrast, microorganisms generally metabolized cellulose-C after 14 days and did not change to the same extent in phylogenetic composition over time. Furthermore, microorganisms that metabolized cellulose-C belonged to poorly characterized but cosmopolitan soil lineages including Verrucomicrobia, Chlorotlexi, and Planctomycetes.« less

  5. Unearthing the Ecology of Soil Microorganisms Using a High Resolution DNA-SIP Approach to Explore Cellulose and Xylose Metabolism in Soil.

    PubMed

    Pepe-Ranney, Charles; Campbell, Ashley N; Koechli, Chantal N; Berthrong, Sean; Buckley, Daniel H

    2016-01-01

    We explored microbial contributions to decomposition using a sophisticated approach to DNA Stable Isotope Probing (SIP). Our experiment evaluated the dynamics and ecological characteristics of functionally defined microbial groups that metabolize labile and structural C in soils. We added to soil a complex amendment representing plant derived organic matter substituted with either (13)C-xylose or (13)C-cellulose to represent labile and structural C pools derived from abundant components of plant biomass. We found evidence for (13)C-incorporation into DNA from (13)C-xylose and (13)C-cellulose in 49 and 63 operational taxonomic units (OTUs), respectively. The types of microorganisms that assimilated (13)C in the (13)C-xylose treatment changed over time being predominantly Firmicutes at day 1 followed by Bacteroidetes at day 3 and then Actinobacteria at day 7. These (13)C-labeling dynamics suggest labile C traveled through different trophic levels. In contrast, microorganisms generally metabolized cellulose-C after 14 days and did not change to the same extent in phylogenetic composition over time. Microorganisms that metabolized cellulose-C belonged to poorly characterized but cosmopolitan soil lineages including Verrucomicrobia, Chloroflexi, and Planctomycetes. PMID:27242725

  6. Unearthing the Ecology of Soil Microorganisms Using a High Resolution DNA-SIP Approach to Explore Cellulose and Xylose Metabolism in Soil

    PubMed Central

    Pepe-Ranney, Charles; Campbell, Ashley N.; Koechli, Chantal N.; Berthrong, Sean; Buckley, Daniel H.

    2016-01-01

    We explored microbial contributions to decomposition using a sophisticated approach to DNA Stable Isotope Probing (SIP). Our experiment evaluated the dynamics and ecological characteristics of functionally defined microbial groups that metabolize labile and structural C in soils. We added to soil a complex amendment representing plant derived organic matter substituted with either 13C-xylose or 13C-cellulose to represent labile and structural C pools derived from abundant components of plant biomass. We found evidence for 13C-incorporation into DNA from 13C-xylose and 13C-cellulose in 49 and 63 operational taxonomic units (OTUs), respectively. The types of microorganisms that assimilated 13C in the 13C-xylose treatment changed over time being predominantly Firmicutes at day 1 followed by Bacteroidetes at day 3 and then Actinobacteria at day 7. These 13C-labeling dynamics suggest labile C traveled through different trophic levels. In contrast, microorganisms generally metabolized cellulose-C after 14 days and did not change to the same extent in phylogenetic composition over time. Microorganisms that metabolized cellulose-C belonged to poorly characterized but cosmopolitan soil lineages including Verrucomicrobia, Chloroflexi, and Planctomycetes. PMID:27242725

  7. Hydrogen-bond breaking by O/sub 2/ and N/sub 2/. II. Melting curves of DNA

    SciTech Connect

    Mathers, T.L.; Schoeffler, G.; McGlynn, S.P.

    1982-01-01

    Evidence for hydrogen bond breaking (HBB) by O/sub 2/ and N/sub 2/ in the denaturation or melting of DNA is presented. It was found that air and oxygen significantly reduce the temperature of the DNA melting process. The possible relationship of this HBB ability of oxygen and nitrogen to phenomena observed in vivo are discussed. (ACR)

  8. High-resolution anion-exchange and partition thin-layer chromatography for complex mixtures of 32P-postlabeled DNA adducts.

    PubMed

    Spencer-Beach, G G; Beach, A C; Gupta, R C

    1996-03-01

    32P-Postlabeling has emerged as a major tool for detecting DNA adducts resulting from exposure to complex carcinogen mixtures. An integral component of this assay is multi-directional PEI-cellulose TLC in which lipophilic 32P-adducts are resolved in high-salt, high-urea solvents following removal of the bulk of non-adduct radioactivity. This TLC system is very effective for adducts formed following exposure to individual carcinogens; however, adducts resulting from exposure to complex mixtures (e.g. cigarette smoke) generally appear in the form of the so-called diagonal radioactive zones. By using mixtures of polycyclic aromatic hydrocarbon- and aromatic amine-DNA adducts as well as adducts in mouse skin treated with cigarette smoke condensate, we have demonstrated that a combination of 0.3-0.4 M NH4OH and isopropanol-4 M NH4OH (1-1.4:1) solvents can provide more sharply defined adduct spots than the commonly used urea solvents. The non-urea solvents also result in excellent resolution of many adducts which otherwise may remain buried in diagonal radioactive zones when using the urea solvents. In addition, the signal-to-noise ratio is increased 2- to 5-fold over the urea solvents enabling detection of discrete adducts at < or = 3 adducts per 10(10) nucleotides. These partition TLC solvents also involve fewer manipulations (e.g. no water washes to remove salt and urea), and are likely to be more informative with regards to the type of individual adducts detected in the biomonitoring of humans than has hitherto been possible. PMID:8704930

  9. Trehalose facilitates DNA melting: a single-molecule optical tweezers study.

    PubMed

    Bezrukavnikov, Sergey; Mashaghi, Alireza; van Wijk, Roeland J; Gu, Chan; Yang, Li Jiang; Gao, Yi Qin; Tans, Sander J

    2014-10-01

    Using optical tweezers, here we show that the overstretching transition force of double-stranded DNA (dsDNA) is lowered significantly by the addition of the disaccharide trehalose as well as certain polyol osmolytes. This effect is found to depend linearly on the logarithm of the trehalose concentration. We propose an entropic driving mechanism for the experimentally observed destabilization of dsDNA that is rooted in the higher affinity of the DNA bases for trehalose than for water, which promotes base exposure and DNA melting. Molecular dynamics simulation reveals the direct interaction of trehalose with nucleobases. Experiments with other osmolytes confirm that the extent of dsDNA destabilization is governed by the ratio between polar and apolar fractions of an osmolyte.

  10. The high-resolution crystal structure of a parallel intermolecular DNA G-4 quadruplex/drug complex employing syn glycosyl linkages

    PubMed Central

    Clark, George R.; Pytel, Patrycja D.; Squire, Christopher J.

    2012-01-01

    We have determined the X-ray structure of the complex between the DNA quadruplex d(5′-GGGG-3′)4 and daunomycin, as a potential model for studying drug–telomere interactions. The structure was solved at 1.08 Å by direct methods in space group I4. The asymmetric unit comprises a linear arrangement of one d(GGGG) strand, four daunomycin molecules, a second d(GGGG) strand facing in the opposite direction to the first, and Na and Mg cations. The crystallographic 4-fold axis generates the biological unit, which is a 12-layered structure comprising two sets of four guanine layers, with four layers each of four daunomycins stacked between the 5′ faces of the two quadruplexes. The daunomycin layers fall into two groups which are novel in their mode of self assembly. The only contacts between daunomycin molecules within any one of these layers are van der Waals interactions, however there is substantial π–π stacking between successive daunomycin layers and also with adjacent guanine layers. The structure differs significantly from all other parallel d(TGGGGT)4 quadruplexes in that the 5′ guanine adopts the unusual syn glycosyl linkage, refuting the widespread belief that such conformations should all be anti. In contrast to the related d(TGGGGT)/daunomycin complex, there are no ligand–quadruplex groove insertion interactions. PMID:22373921

  11. The high-resolution crystal structure of a parallel intermolecular DNA G-4 quadruplex/drug complex employing syn glycosyl linkages.

    PubMed

    Clark, George R; Pytel, Patrycja D; Squire, Christopher J

    2012-07-01

    We have determined the X-ray structure of the complex between the DNA quadruplex d(5'-GGGG-3')(4) and daunomycin, as a potential model for studying drug-telomere interactions. The structure was solved at 1.08 Å by direct methods in space group I4. The asymmetric unit comprises a linear arrangement of one d(GGGG) strand, four daunomycin molecules, a second d(GGGG) strand facing in the opposite direction to the first, and Na and Mg cations. The crystallographic 4-fold axis generates the biological unit, which is a 12-layered structure comprising two sets of four guanine layers, with four layers each of four daunomycins stacked between the 5' faces of the two quadruplexes. The daunomycin layers fall into two groups which are novel in their mode of self assembly. The only contacts between daunomycin molecules within any one of these layers are van der Waals interactions, however there is substantial π-π stacking between successive daunomycin layers and also with adjacent guanine layers. The structure differs significantly from all other parallel d(TGGGGT)(4) quadruplexes in that the 5' guanine adopts the unusual syn glycosyl linkage, refuting the widespread belief that such conformations should all be anti. In contrast to the related d(TGGGGT)/daunomycin complex, there are no ligand-quadruplex groove insertion interactions.

  12. High Resolution DNA Stable Isotope Probing Reveals that Root Exudate Addition to Soil Changes the Identity of the Microbes that Degrade Cellulose but not the Rate of Degradation

    NASA Astrophysics Data System (ADS)

    Campbell, A.; Pepe-Ranney, C. P.; Nguyen, A. V. T.; Buckley, D. H.

    2015-12-01

    Plant roots release compounds, such as root exudates, which can alter soil organic matter (SOM) decomposition and have large impacts on soil carbon (C) retention. The changes in SOM turnover resulting from the addition of organic and/or inorganic substrates are termed 'priming effects'. In this study we examine the effects of root exudates on the priming of cellulose added as particulate organic matter. We amended soil microcosms with 13C-cellulose in the presence or absence of artificial root exudate additions and incubated over time for 45 days. Soils receiving the root exudate (RE) were given either one large dose or multiple, small doses of RE. In each treatment we tracked operational taxonomic units (OTUs) assimilating 13C from cellulose (herein, known as a 'responder') over time using DNA stable isotope probing coupled with next generation sequencing. In all treatments the same amount of cellulose-13C was respired indicating the addition of RE did not result in the priming of cellulose decomposition. However, cellulose responders were different depending on treatment and time of sampling (days 14, 28 and 45). We identified a total of 10,361 OTUs, of which there were 369 cellulose responders in the cellulose only treatment, 273 in the repeated, small dose RE treatment, and 358 in the RE single, large dose treatment. Most of the cellulose responders found in all treatments belonged to phyla Bacteroidetes, Planctomycetes, Proteobacteria, Verrucomicrobia, and Chloroflexi. The response time of phyla varies; for instance, more OTUs in Bacteroidetes were observed on day 14 and diminish with each subsequent sampling time. On the other hand, OTUs in Verrucomicrobia increased in response over time. Our study shows no priming effect resulting from the addition of root exudates, although the identity of the microbial mediators of cellulose decomposition varies in each treatment.

  13. Significant melting point depression of two-dimensional folded-chain crystals of isotactic poly(methyl methacrylate)s observed by high-resolution in situ atomic force microscopy.

    PubMed

    Takanashi, Yuma; Kumaki, Jiro

    2013-05-01

    The properties of polymer ultrathin films are a subject of intense study from both practical and academic viewpoints. Previously, we found that upon compression, an isotactic poly(methyl methacrylate) (it-PMMA) Langmuir monolayer crystallized to form a two-dimensional (2D) folded-chain crystal, and the molecular image of the crystal with chain folding and tie chains was clearly visualized by atomic force microscopy (AFM). In the present study, the melting behaviors of the it-PMMA 2D crystals were successfully observed in situ by high-temperature AFM at the molecular lever for the first time. The chain-chain distances (~1.2 nm) of the crystals were clearly resolved even at temperatures close to the melting temperatures (Tm) of the 2D crystals. We found that the Tm of the 2D crystals was at most 90 °C lower than the bulk crystals. The Tm depression strongly depended on the molecular weight, while the molecular weight dependence of the bulk Tm was negligible in the molecular weight regime studied. The Tm depression also depended on the substrates, a slightly larger depression being observed on a sapphire substrate compared to that on a mica. The large Tm depressions of the 2D crystals could not be explained by a simple Thomson-Gibbs argument, theoretical developments are necessary to understand the melting of the 2D crystals.

  14. ANL high-resolution injector

    SciTech Connect

    Minehara, E.; Kutschera, W.; Hartog, P.D.; Billquist, P.; Liu, Z.

    1986-05-01

    The ANL (Argonne National Laboratory) high-resolution injector has been installed to obtain higher mass resolution and higher preacceleration, and to utilize effectively the full mass range of ATLAS (Argonne tandem linac accelerator system). Preliminary results of the first beam test are reported briefly. The design and performance, in particular a high-mass-resolution magnet with aberration compensation, are discussed.

  15. High resolution digital delay timer

    DOEpatents

    Martin, Albert D.

    1988-01-01

    Method and apparatus are provided for generating an output pulse following a trigger pulse at a time delay interval preset with a resolution which is high relative to a low resolution available from supplied clock pulses. A first lumped constant delay (20) provides a first output signal (24) at predetermined interpolation intervals corresponding to the desired high resolution time interval. Latching circuits (26, 28) latch the high resolution data (24) to form a first synchronizing data set (60). A selected time interval has been preset to internal counters (142, 146, 154) and corrected for circuit propagation delay times having the same order of magnitude as the desired high resolution. Internal system clock pulses (32, 34) count down the counters to generate an internal pulse delayed by an interval which is functionally related to the preset time interval. A second LCD (184) corrects the internal signal with the high resolution time delay. A second internal pulse is then applied to a third LCD (74) to generate a second set of synchronizing data (76) which is complementary with the first set of synchronizing data (60) for presentation to logic circuits (64). The logic circuits (64) further delay the internal output signal (72) to obtain a proper phase relationship of an output signal (80) with the internal pulses (32, 34). The final delayed output signal (80) thereafter enables the output pulse generator (82) to produce the desired output pulse (84) at the preset time delay interval following input of the trigger pulse (10, 12).

  16. Advanced very high resolution radiometer

    NASA Technical Reports Server (NTRS)

    1976-01-01

    The advanced very high resolution radiometer development program is considered. The program covered the design, construction, and test of a breadboard model, engineering model, protoflight model, mechanical structural model, and a life test model. Special bench test and calibration equipment was also developed for use on the program.

  17. On-chip magnetic bead-based DNA melting curve analysis using a magnetoresistive sensor

    NASA Astrophysics Data System (ADS)

    Rizzi, Giovanni; Østerberg, Frederik W.; Henriksen, Anders D.; Dufva, Martin; Hansen, Mikkel F.

    2015-04-01

    We present real-time measurements of DNA melting curves in a chip-based system that detects the amount of surface-bound magnetic beads using magnetoresistive magnetic field sensors. The sensors detect the difference between the amount of beads bound to the top and bottom sensor branches of the differential sensor geometry. The sensor surfaces are functionalized with wild type (WT) and mutant type (MT) capture probes, differing by a single base insertion (a single nucleotide polymorphism, SNP). Complementary biotinylated targets in suspension couple streptavidin magnetic beads to the sensor surface. The beads are magnetized by the field arising from the bias current passed through the sensors. We demonstrate the first on-chip measurements of the melting of DNA hybrids upon a ramping of the temperature. This overcomes the limitation of using a single washing condition at constant temperature. Moreover, we demonstrate that a single sensor bridge can be used to genotype a SNP.

  18. High-Resolution Shadowing of Transfer RNA

    PubMed Central

    Abermann, Reinhard J.; Yoshikami, Doju

    1972-01-01

    High-resolution shadowing with metals that melt at high temperatures was used to study macromolecules. Molecules of transfer RNA shadowed with tantalum-tungsten are readily visualized in an electron microscope. Mounting procedures for tRNA were perfected that reproducibly gave uniform distributions of both monomeric and dimeric tRNA particles, and allowed a statistical assessment of their gross shapes and sizes. Monomeric tRNA yielded a fairly homogeneous population of rod-shaped particles, with axial dimensions of about 40 × 85 Å. Dimers of yeast alanine tRNA held together by hydrogen bonds and dimers constructed by covalent linkage of the amino-acid acceptor (3′-) termini of monomers both gave slightly more heterogeneous populations of particles. Yet, their structures were also basically rod shaped, with their lengths ranging to about twice that of the monomer; this result indicates an end-to-end arrangement of the monomeric units within both dimers. These results suggest that the amino-acid acceptor terminus and the anticodon region are at the ends of the rod-shaped, dehydrated tRNA monomer visible by electron microscopy, consistent with the generally accepted view of tRNA structure in solution suggested by other workers using other methods. This study demonstrates that high-resolution shadowing with tantalum-tungsten provides a means to examine the three-dimensional structures of relatively small biological macromolecules. Images PMID:4504373

  19. High Resolution Imaging Spectrometer (HIRIS)

    NASA Technical Reports Server (NTRS)

    Conley, Joseph M.; Herring, Mark; Norris, David D.

    1988-01-01

    The High Resolution Imaging Spectrometer (HIRIS), related data system, orbit, and mission operations are described. The pushbroom instrument simultaneously images the terrestrial surface in 192 spectral bands from 0.4 to 2.5 microns. The swath width is 30 km and spatial resolution is 30 m. It is planned to be launched with the Earth Observing System aboard the Space Station Polar Platform in 1995. Array detectors allow concurrent integration of the signals at 192,000 detector elements.

  20. Modification of the melting properties of duplex DNA by attachment of a GC-rich DNA sequence as determined by denaturing gradient gel electrophoresis.

    PubMed Central

    Myers, R M; Fischer, S G; Maniatis, T; Lerman, L S

    1985-01-01

    The melting behavior of a DNA fragment carrying the mouse beta maj-globin promoter was investigated as a means of establishing procedures for separating DNA fragments differing by any single base substitution using the denaturing gradient gel electrophoresis procedure of Fischer and Lerman (1,2). We find that attachment of a 300 base pair GC-rich DNA sequence, termed a GC-clamp, to a 135 bp DNA fragment carrying the mouse beta-globin promoter significantly alters the pattern of DNA melting within the promoter. When the promoter is attached to the clamp, the promoter sequences melt without undergoing strand dissociation. The calculated distribution of melting domains within the promoter differs markedly according to the relative orientation of the clamp and promoter sequences. We find that the behavior of DNA fragments containing the promoter and clamp sequences on denaturing gradient polyacrylamide gels is in close agreement with the theoretical melting calculations. These studies provide the basis for critical evaluation of the parameters for DNA melting calculations, and they establish conditions for determining whether all single base substitutions within the promoter can be separated on denaturing gradient gels. Images PMID:2987873

  1. High resolution detection system of capillary electrophoresis

    NASA Astrophysics Data System (ADS)

    Wang, Jie; Wang, Li Qiang; Shi, Yan; Zheng, Hua; Lu, Zu Kang

    2007-12-01

    The capillary electrophoresis (CE) with laser induced fluorescence detection (LIFD) system was founded according to confocal theory. The 3-D adjustment of the exciting and collecting optical paths was realized. The photomultiplier tube (PMT) is used and the signals are processed by a software designed by ourselves. Under computer control, high voltage is applied to appropriate reservoirs and to inject and separate DNA samples respectively. Two fluorescent dyes Thiazole Orange (TO) and SYBR Green I were contrasted. With both of the dyes, high signals-to-noise images were obtained with the CE-LIFD system. The single-bases can be distinguished from the electrophoretogram and high resolution of DNA sample separation was obtained.

  2. Structure and disorder in basaltic glasses and melts: Insights from high-resolution solid-state NMR study of glasses in diopside-Ca-tschermakite join and diopside-anorthite eutectic composition

    NASA Astrophysics Data System (ADS)

    Park, Sun Young; Lee, Sung Keun

    2012-03-01

    obtained by solution calorimetry. The observed increase in NBO fraction (as also expected from the chemical composition) with increasing XDiopside indicates an obvious decrease in melt viscosity toward a diopside endmember. The partitioning of Ca2+ and Mg2+ and/or unmixing of these cations between NBOs and BOs may result in variations in the activity coefficients of CaO and MgO, thus the compositions of melts.

  3. DARPA high resolution display technologies

    NASA Astrophysics Data System (ADS)

    Slusarczuk, Marko

    1990-11-01

    Much of the information of interest to pilots in flight is display-limited, and is undergoing substantial expansion due to improved sensor output and signal processing; attention is accordingly given to digitally-based instrument display imaging in the present evaluation of high-resolution cockpit display technologies. Also noted are the advantages of digitally transmitted sensor data in cases where the airborne reconnaissance user may be able to analyze telemetered airborne data in real time and respond with requests to the pilot for more detailed information of specific battlefield sites.

  4. High-Resolution Imaging Spectrometer

    NASA Technical Reports Server (NTRS)

    Dozier, Jeff; Goetz, Alexander F. H.

    1990-01-01

    Earth resources observed in greater detail. High-Resolution Imaging Spectrometer, undergoing development for use in NASA's Earth Observing System, measures reflectance of Earth's surface in visible and near-infrared wavelengths. From an orbit around Earth, instrument scans surface of Earth in 200 wavelength bands simultaneously. Produces images enabling identification of minerals in rocks and soils, important algal pigments in oceans and inland waters, changes in spectra associated with biochemistry of plant canopies, compositions of atmospheric aerosols, sizes of grains in snow, and contamination of snow by impurities that absorb visible light.

  5. High Resolution Scanning Reflectarray Antenna

    NASA Technical Reports Server (NTRS)

    Romanofsky, Robert R. (Inventor); Miranda, Felix A. (Inventor)

    2000-01-01

    The present invention provides a High Resolution Scanning Reflectarray Antenna (HRSRA) for the purpose of tracking ground terminals and space craft communication applications. The present invention provides an alternative to using gimbaled parabolic dish antennas and direct radiating phased arrays. When compared to a gimbaled parabolic dish, the HRSRA offers the advantages of vibration free steering without incurring appreciable cost or prime power penalties. In addition, it offers full beam steering at a fraction of the cost of direct radiating arrays and is more efficient.

  6. Detection of orthopoxvirus DNA by real-time PCR and identification of variola virus DNA by melting analysis.

    PubMed

    Nitsche, Andreas; Ellerbrok, Heinz; Pauli, Georg

    2004-03-01

    Although variola virus was eradicated by the World Health Organization vaccination program in the 1970s, the diagnosis of smallpox infection has attracted great interest in the context of a possible deliberate release of variola virus in bioterrorist attacks. Obviously, fast and reliable diagnostic tools are required to detect variola virus and to distinguish it from orthopoxviruses that have identical morphological characteristics, including vaccinia virus. The advent of real-time PCR for the clinical diagnosis of viral infections has facilitated the detection of minute amounts of viral nucleic acids in a fast, safe, and precise manner, including the option to quantify and to genotype the target reliably. In this study a complete set of four hybridization probe-based real-time PCR assays for the specific detection of orthopoxvirus DNA is presented. Melting analysis following PCR enables the identification of variola virus by the PCR product's characteristic melting temperature, permitting the discrimination of variola virus from other orthopoxviruses. In addition, an assay for the specific amplification of variola virus DNA is presented. All assays can be performed simultaneously in the same cycler, and results of a PCR run are obtained in less than 1 h. The application of more than one assay for the same organism significantly contributes to the diagnostic reliability, reducing the risk of false-negative results due to unknown sequence variations. In conclusion, the assays presented will improve the speed and reliability of orthopoxvirus diagnostics and variola virus identification.

  7. High Resolution Thermography In Medicine

    NASA Astrophysics Data System (ADS)

    Clark, R. P.; Goff, M. R.; Culley, J. E.

    1988-10-01

    A high resolution medical thermal imaging system using an 8 element SPRI1E detector is described. Image processing is by an Intellect 100 processor and is controlled by a DEC LSI 11/23 minicomputer. Image storage is with a 170 Mbyte winchester disc together with archival storage on 12 inch diameter optical discs having a capacity of 1 Gbyte per side. The system is currently being evaluated for use in physiology and medicine. Applications outlined include the potential of thermographic screening to identify genetic carriers in X-linked hypohidrotic ectodermal dysplasia (XED), detailed vas-cular perfusion studies in health and disease and the relation-ship between cutaneous blood flow, neurological peripheral function and skin surface temperature.

  8. Enhanced High Resolution RBS System

    NASA Astrophysics Data System (ADS)

    Pollock, Thomas J.; Hass, James A.; Klody, George M.

    2011-06-01

    Improvements in full spectrum resolution with the second NEC high resolution RBS system are summarized. Results for 50 Å TiN/HfO films on Si yielding energy resolution on the order of 1 keV are also presented. Detector enhancements include improved pulse processing electronics, upgraded shielding for the MCP/RAE detector, and reduced noise generated from pumping. Energy resolution measurements on spectra front edge coupled with calculations using 0.4mStr solid angle show that beam energy spread at 400 KeV from the Pelletron® accelerator is less than 100 eV. To improve user throughput, magnet control has been added to the automatic data collection. Depth profiles derived from experimental data are discussed. For the thin films profiled, depth resolutions were on the Angstrom level with the non-linear energy/channel conversions ranging from 100 to 200 eV.

  9. High Resolution Neutral Atom Microscope

    NASA Astrophysics Data System (ADS)

    Bucay, Igal; Castillo-Garza, Rodrigo; Stratis, Georgios; Raizen, Mark

    2015-03-01

    We are developing a high resolution neutral atom microscope based on metastable atom electron spectroscopy (MAES). When a metastable atom of a noble gas is near a solid, a surface electron will tunnel to an empty energy level of the metastable atom, thereby ejecting the excited electron from the atom. The emitted electrons carry information regarding the local topography and electronic, magnetic, and chemical structures of most hard materials. Furthermore, using a chromatic aberration corrected magnetic hexapole lens we expect to attain a spatial resolution below 10 nm. We will use this microscope to investigate how local phenomena can give rise to macroscopic effects in materials that cannot be probed using a scanning tunneling microscope, namely insulating transition metal oxides.

  10. High resolution spectrograph. [for LST

    NASA Technical Reports Server (NTRS)

    Peacock, K.

    1975-01-01

    The high resolution spectrograph (HRS) is designed to be used with the Large Space Telescope (LST) for the study of spectra of point and extended targets in the spectral range 110 to 410 nm. It has spectral resolutions of 1,000; 30,000; and 100,000 and has a field of view as large as 10 arc sec. The spectral range and resolution are selectable using interchangeable optical components and an echelle spectrograph is used to display a cross dispersed spectrum on the photocathode of either of 2 SEC orthicon image tubes. Provisions are included for wavelength calibration, target identification and acquisition and thermal control. The system considerations of the instrument are described.

  11. High-resolution imaging ellipsometer.

    PubMed

    Zhan, Qiwen; Leger, James R

    2002-08-01

    We report on a novel imaging ellipsometer using a high-numerical-aperture (NA) objective lens capable of measuring a two-dimensional ellipsometric signal with high resolution. Two-dimensional ellipsometric imaging is made possible by spatial filtering at the pupil plane of the objective. A Richards-Wolf vectorial diffraction model and geometrical optics model are developed to simulate the system. The thickness profile of patterned polymethyl methacrylate is measured for calibration purposes. Our instrument has a sensitivity of 5 A and provides spatial resolution of approximately 0.5 microm with 632.8-nm illumination. Its capability of measuring refractive-index variations with high spatial resolution is also demonstrated.

  12. A high resolution TDC subsystem

    SciTech Connect

    Geiges, R.; Merle, K. )

    1994-02-01

    A high resolution TDC subsystem was developed at the Institute for Nuclear Physics in Mainz. The TDC chip offers a time resolution of less than 300 ps and a programmable measurement range from 0 to 16 [mu]sec. The time measurement is done with a new, purely digital counting method. The chip can be operated in common start or common stop mode. In common start mode the chip is able to store up to 4 multiple hits per channel. The chip is used to build a transputer controlled subsystem for the measurement of the drift times of a vertical drift chamber. The design of the subsystem will be described and the first results from the tests of the prototype system will be presented.

  13. High resolution tomographic instrument development

    SciTech Connect

    Not Available

    1992-08-01

    Our recent work has concentrated on the development of high-resolution PET instrumentation reflecting in part the growing importance of PET in nuclear medicine imaging. We have developed a number of positron imaging instruments and have the distinction that every instrument has been placed in operation and has had an extensive history of application for basic research and clinical study. The present program is a logical continuation of these earlier successes. PCR-I, a single ring positron tomograph was the first demonstration of analog coding using BGO. It employed 4 mm detectors and is currently being used for a wide range of biological studies. These are of immense importance in guiding the direction for future instruments. In particular, PCR-II, a volume sensitive positron tomograph with 3 mm spatial resolution has benefited greatly from the studies using PCR-I. PCR-II is currently in the final stages of assembly and testing and will shortly be placed in operation for imaging phantoms, animals and ultimately humans. Perhaps the most important finding resulting from our previous study is that resolution and sensitivity must be carefully balanced to achieve a practical high resolution system. PCR-II has been designed to have the detection characteristics required to achieve 3 mm resolution in human brain under practical imaging situations. The development of algorithms by the group headed by Dr. Chesler is based on a long history of prior study including his joint work with Drs. Pelc and Reiderer and Stearns. This body of expertise will be applied to the processing of data from PCR-II when it becomes operational.

  14. High resolution tomographic instrument development

    SciTech Connect

    Not Available

    1992-01-01

    Our recent work has concentrated on the development of high-resolution PET instrumentation reflecting in part the growing importance of PET in nuclear medicine imaging. We have developed a number of positron imaging instruments and have the distinction that every instrument has been placed in operation and has had an extensive history of application for basic research and clinical study. The present program is a logical continuation of these earlier successes. PCR-I, a single ring positron tomograph was the first demonstration of analog coding using BGO. It employed 4 mm detectors and is currently being used for a wide range of biological studies. These are of immense importance in guiding the direction for future instruments. In particular, PCR-II, a volume sensitive positron tomograph with 3 mm spatial resolution has benefited greatly from the studies using PCR-I. PCR-II is currently in the final stages of assembly and testing and will shortly be placed in operation for imaging phantoms, animals and ultimately humans. Perhaps the most important finding resulting from our previous study is that resolution and sensitivity must be carefully balanced to achieve a practical high resolution system. PCR-II has been designed to have the detection characteristics required to achieve 3 mm resolution in human brain under practical imaging situations. The development of algorithms by the group headed by Dr. Chesler is based on a long history of prior study including his joint work with Drs. Pelc and Reiderer and Stearns. This body of expertise will be applied to the processing of data from PCR-II when it becomes operational.

  15. High resolution tomographic instrument development

    NASA Astrophysics Data System (ADS)

    Our recent work has concentrated on the development of high-resolution PET instrumentation reflecting in part the growing importance of PET in nuclear medicine imaging. We have developed a number of positron imaging instruments and have the distinction that every instrument has been placed in operation and has had an extensive history of application for basic research and clinical study. The present program is a logical continuation of these earlier successes. PCR-I, a single ring positron tomograph was the first demonstration of analog coding using BGO. It employed 4 mm detectors and is currently being used for a wide range of biological studies. These are of immense importance in guiding the direction for future instruments. In particular, PCR-II, a volume sensitive positron tomograph with 3 mm spatial resolution has benefitted greatly from the studies using PCR-I. PCR-II is currently in the final stages of assembly and testing and will shortly be placed in operation for imaging phantoms, animals and ultimately humans. Perhaps the most important finding resulting from our previous study is that resolution and sensitivity must be carefully balanced to achieve a practical high resolution system. PCR-II has been designed to have the detection characteristics required to achieve 3 mm resolution in human brain under practical imaging situations. The development of algorithms by the group headed by Dr. Chesler is based on a long history of prior study including his joint work with Drs. Pelc and Reiderer and Stearns. This body of expertise will be applied to the processing of data from PCR-II when it becomes operational.

  16. Mars high-resolution mapping

    NASA Astrophysics Data System (ADS)

    Batson, R. M.; Thomas, P. K.

    1991-06-01

    A series of photomosaics of high-resolution Viking Orbiter images of Mars is being prepared and published to support the Mars 1:500,000 scale geologic mapping program. More than 100 of these photomosaics were made manually, but for the last several years they have all been made digitally. The digital mosaics are published on the Mars Transverse Mercator (MTM) system, and they are also available to the appropriate principal investigators as digital files in the mosaicked digital image model (MDIM) format. The mosaics contain Viking Orbiter images with the highest available resolution: in some areas as high as 10 m/pixel. This resolution, where it exists, will support a 1:100,000 map scale. The full resolution of a mosaic is preserved in a digital file, but conventional lithographic publication of such large-scale inset maps will be done only if required by the geologic map author. When high-resolution images do not fill the neat lines of an MTM quadrangle, the medium-resolution (1/256 degrees/pixel, or 231 m/pixel) MDIM is used. The mosaics are tied by image-matching to the planetwide MDIM, in which random errors as large as 5 km (10 mm at 1:500,000 scale) are common; a few much larger, worst-case errors also occur. Because of the distribution of the errors, many large discrepancies appear along the cutlines between frames with very different resolutions. Furthermore, each block of quadrangles is compiled on its own local control system, and adjacent blocks, compiled later, are unlikely to match. Selection of areas to be mapped is based on geologic mapping proposals reviewed and recommended by the Mars 1:500,000 scale geologic mapping review panel. There is no intention to map the entire planet at this scale.

  17. HRSC: High resolution stereo camera

    USGS Publications Warehouse

    Neukum, G.; Jaumann, R.; Basilevsky, A.T.; Dumke, A.; Van Gasselt, S.; Giese, B.; Hauber, E.; Head, J. W.; Heipke, C.; Hoekzema, N.; Hoffmann, H.; Greeley, R.; Gwinner, K.; Kirk, R.; Markiewicz, W.; McCord, T.B.; Michael, G.; Muller, Jan-Peter; Murray, J.B.; Oberst, J.; Pinet, P.; Pischel, R.; Roatsch, T.; Scholten, F.; Willner, K.

    2009-01-01

    The High Resolution Stereo Camera (HRSC) on Mars Express has delivered a wealth of image data, amounting to over 2.5 TB from the start of the mapping phase in January 2004 to September 2008. In that time, more than a third of Mars was covered at a resolution of 10-20 m/pixel in stereo and colour. After five years in orbit, HRSC is still in excellent shape, and it could continue to operate for many more years. HRSC has proven its ability to close the gap between the low-resolution Viking image data and the high-resolution Mars Orbiter Camera images, leading to a global picture of the geological evolution of Mars that is now much clearer than ever before. Derived highest-resolution terrain model data have closed major gaps and provided an unprecedented insight into the shape of the surface, which is paramount not only for surface analysis and geological interpretation, but also for combination with and analysis of data from other instruments, as well as in planning for future missions. This chapter presents the scientific output from data analysis and highlevel data processing, complemented by a summary of how the experiment is conducted by the HRSC team members working in geoscience, atmospheric science, photogrammetry and spectrophotometry. Many of these contributions have been or will be published in peer-reviewed journals and special issues. They form a cross-section of the scientific output, either by summarising the new geoscientific picture of Mars provided by HRSC or by detailing some of the topics of data analysis concerning photogrammetry, cartography and spectral data analysis.

  18. High resolution time interval counter

    DOEpatents

    Condreva, Kenneth J.

    1994-01-01

    A high resolution counter circuit measures the time interval between the occurrence of an initial and a subsequent electrical pulse to two nanoseconds resolution using an eight megahertz clock. The circuit includes a main counter for receiving electrical pulses and generating a binary word--a measure of the number of eight megahertz clock pulses occurring between the signals. A pair of first and second pulse stretchers receive the signal and generate a pair of output signals whose widths are approximately sixty-four times the time between the receipt of the signals by the respective pulse stretchers and the receipt by the respective pulse stretchers of a second subsequent clock pulse. Output signals are thereafter supplied to a pair of start and stop counters operable to generate a pair of binary output words representative of the measure of the width of the pulses to a resolution of two nanoseconds. Errors associated with the pulse stretchers are corrected by providing calibration data to both stretcher circuits, and recording start and stop counter values. Stretched initial and subsequent signals are combined with autocalibration data and supplied to an arithmetic logic unit to determine the time interval in nanoseconds between the pair of electrical pulses being measured.

  19. High resolution time interval counter

    DOEpatents

    Condreva, K.J.

    1994-07-26

    A high resolution counter circuit measures the time interval between the occurrence of an initial and a subsequent electrical pulse to two nanoseconds resolution using an eight megahertz clock. The circuit includes a main counter for receiving electrical pulses and generating a binary word--a measure of the number of eight megahertz clock pulses occurring between the signals. A pair of first and second pulse stretchers receive the signal and generate a pair of output signals whose widths are approximately sixty-four times the time between the receipt of the signals by the respective pulse stretchers and the receipt by the respective pulse stretchers of a second subsequent clock pulse. Output signals are thereafter supplied to a pair of start and stop counters operable to generate a pair of binary output words representative of the measure of the width of the pulses to a resolution of two nanoseconds. Errors associated with the pulse stretchers are corrected by providing calibration data to both stretcher circuits, and recording start and stop counter values. Stretched initial and subsequent signals are combined with autocalibration data and supplied to an arithmetic logic unit to determine the time interval in nanoseconds between the pair of electrical pulses being measured. 3 figs.

  20. High resolution optoelectronic retinal prosthesis

    NASA Astrophysics Data System (ADS)

    Loudin, Jim; Dinyari, Rostam; Huie, Phil; Butterwick, Alex; Peumans, Peter; Palanker, Daniel

    2009-02-01

    Electronic retinal prostheses seek to restore sight in patients with retinal degeneration by delivering pulsed electric currents to retinal neurons via an array of microelectrodes. Most implants use inductive or optical transmission of information and power to an intraocular receiver, with decoded signals subsequently distributed to retinal electrodes through an intraocular cable. Surgical complexity could be minimized by an "integrated" prosthesis, in which both power and data are delivered directly to the stimulating array without any discrete components or cables. We present here an integrated retinal prosthesis system based on a photodiode array implant. Video frames are processed and imaged onto the retinal implant by a video goggle projection system operating at near-infrared wavelengths (~ 900 nm). Photodiodes convert light into pulsed electric current, with charge injection maximized by specially optimized series photodiode circuits. Prostheses of three different pixel densities (16 pix/mm2, 64 pix/mm2, and 256 pix/mm2) have been designed, simulated, and prototyped. Retinal tissue response to subretinal implants made of various materials has been investigated in RCS rats. The resulting prosthesis can provide sufficient charge injection for high resolution retinal stimulation without the need for implantation of any bulky discrete elements such as coils or tethers. In addition, since every pixel functions independently, pixel arrays may be placed separately in the subretinal space, providing visual stimulation to a larger field of view.

  1. High resolution imaging at Palomar

    NASA Technical Reports Server (NTRS)

    Kulkarni, Shrinivas R.

    1992-01-01

    For the last two years we have embarked on a program of understanding the ultimate limits of ground-based optical imaging. We have designed and fabricated a camera specifically for high resolution imaging. This camera has now been pressed into service at the prime focus of the Hale 5 m telescope. We have concentrated on two techniques: the Non-Redundant Masking (NRM) and Weigelt's Fully Filled Aperture (FFA) method. The former is the optical analog of radio interferometry and the latter is a higher order extension of the Labeyrie autocorrelation method. As in radio Very Long Baseline Interferometry (VLBI), both these techniques essentially measure the closure phase and, hence, true image construction is possible. We have successfully imaged binary stars and asteroids with angular resolution approaching the diffraction limit of the telescope and image quality approaching that of a typical radio VLBI map. In addition, we have carried out analytical and simulation studies to determine the ultimate limits of ground-based optical imaging, the limits of space-based interferometric imaging, and investigated the details of imaging tradeoffs of beam combination in optical interferometers.

  2. Evaluation of Advanced Bionics high resolution mode.

    PubMed

    Buechner, Andreas; Frohne-Buechner, Carolin; Gaertner, Lutz; Lesinski-Schiedat, Anke; Battmer, Rolf-Dieter; Lenarz, Thomas

    2006-07-01

    The objective of this paper is to evaluate the advantages of the Advanced Bionic high resolution mode for speech perception, through a retrospective analysis. Forty-five adult subjects were selected who had a minimum experience of three months' standard mode (mean of 10 months) before switching to high resolution mode. Speech perception was tested in standard mode immediately before fitting with high resolution mode, and again after a maximum of six months high resolution mode usage (mean of two months). A significant improvement was found, between 11 and 17%, depending on the test material. The standard mode preference does not give any indication about the improvement when switching to high resolution. Users who are converted within any study achieve a higher performance improvement than those converted in the clinical routine. This analysis proves the significant benefits of high resolution mode for users, and also indicates the need for guidelines for individual optimization of parameter settings in a high resolution mode program.

  3. High-resolution slug testing.

    PubMed

    Zemansky, G M; McElwee, C D

    2005-01-01

    The hydraulic conductivity (K) variation has important ramifications for ground water flow and the transport of contaminants in ground water. The delineation of the nature of that variation can be critical to complete characterization of a site and the planning of effective and efficient remedial measures. Site-specific features (such as high-conductivity zones) need to be quantified. Our alluvial field site in the Kansas River valley exhibits spatial variability, very high conductivities, and nonlinear behavior for slug tests in the sand and gravel aquifer. High-resolution, multilevel slug tests have been performed in a number of wells that are fully screened. A general nonlinear model based on the Navier-Stokes equation, nonlinear frictional loss, non-Darcian flow, acceleration effects, radius changes in the wellbore, and a Hvorslev model for the aquifer has been used to analyze the data, employing an automated processing system that runs within the Excel spreadsheet program. It is concluded that slug tests can provide the necessary data to identify the nature of both horizontal and vertical K variation in an aquifer and that improved delineation or higher resolution of K structure is possible with shorter test intervals. The gradation into zones of higher conductivity is sharper than seen previously, and the maximum conductivity observed is greater than previously measured. However, data from this project indicate that well development, the presence of fines, and the antecedent history of the well are important interrelated factors in regard to slug-test response and can prevent obtaining consistent results in some cases.

  4. A stitch in time: Efficient computation of genomic DNA melting bubbles

    PubMed Central

    Tøstesen, Eivind

    2008-01-01

    Background It is of biological interest to make genome-wide predictions of the locations of DNA melting bubbles using statistical mechanics models. Computationally, this poses the challenge that a generic search through all combinations of bubble starts and ends is quadratic. Results An efficient algorithm is described, which shows that the time complexity of the task is O(NlogN) rather than quadratic. The algorithm exploits that bubble lengths may be limited, but without a prior assumption of a maximal bubble length. No approximations, such as windowing, have been introduced to reduce the time complexity. More than just finding the bubbles, the algorithm produces a stitch profile, which is a probabilistic graphical model of bubbles and helical regions. The algorithm applies a probability peak finding method based on a hierarchical analysis of the energy barriers in the Poland-Scheraga model. Conclusion Exact and fast computation of genomic stitch profiles is thus feasible. Sequences of several megabases have been computed, only limited by computer memory. Possible applications are the genome-wide comparisons of bubbles with promotors, TSS, viral integration sites, and other melting-related regions. PMID:18637171

  5. High resolution magnetic spectrometer SHARAQ in RIBF

    SciTech Connect

    Shimoura, S.

    2007-05-22

    For a new spectroscopy of nuclei using intense RI beams at RIBF, we started the SHARAQ project where a high-resolution SHARAQ spectrometer is being constructed together with a high-resolution secondary beam line. Physics motivation and the specification of the spectrometer are presented.

  6. High resolution scintillation detector with semiconductor readout

    DOEpatents

    Levin, Craig S.; Hoffman, Edward J.

    2000-01-01

    A novel high resolution scintillation detector array for use in radiation imaging such as high resolution Positron Emission Tomography (PET) which comprises one or more parallelepiped crystals with at least one long surface of each crystal being in intimate contact with a semiconductor photodetector such that photons generated within each crystal by gamma radiation passing therethrough is detected by the photodetector paired therewith.

  7. High Resolution PDF Measurements on Ag Nanoparticles

    SciTech Connect

    Rocha, Tulio C. R.; Martin, Chris; Kycia, Stefan; Zanchet, Daniela

    2009-01-29

    The quantitative analysis of structural defects in Ag nanoparticles was addressed in this work. We performed atomic scale structural characterization by a combination of x-ray diffraction (XRD) using the Pair Distribution Function analysis (PDF) and High Resolution Transmission Electron Microscopy (HRTEM). The XRD measurements were performed using an innovative instrumentation setup to provide high resolution PDF patterns.

  8. Evaluation of post-polymerase chain reaction melting temperature analysis for meat species identification in mixed DNA samples.

    PubMed

    López-Andreo, María; Garrido-Pertierra, Amando; Puyet, Antonio

    2006-10-18

    Real-time uniplex and duplex polymerase chain reaction (PCR) assays with a SYBR Green I post-PCR melting curve analysis were evaluated for the identification and quantification of bovine, porcine, horse, and wallaroo DNA in food products. Quantitative values were derived from threshold-cycle (C(t)) data obtained from serial dilutions of purified DNA. The limits of detection in uniplex reactions were 0.04 pg for porcine and wallaroo DNA and 0.4 pg for cattle and horse DNA. Species specificity of the PCR products was tested by the identification of peaks in DNA melting curves, measured as the decrease of SYBR Green I fluorescence at the dissociation temperature. The peaks could be distinguished above the background even at the lowest amount of template DNA detected by the C(t) method. The system was also tested in duplex reactions, by use of either single-species DNA or DNA admixtures containing different shares of two species. The minimum proportions of each DNA species allowing the resolution of T(m) peaks in the duplex reactions were 5% (cattle or wallaroo) in cattle/wallaroo mixtures, 5% porcine and 1% horse in porcine/horse mixtures, 60% porcine and 1% wallaroo in porcine/wallaroo mixtures, and 1% cattle and 5% horse in cattle/horse mixtures. A loss in the sensitivity of the method was observed for some DNA combinations in the duplex assay. In contrast, the results obtained from SYBR Green I uniplex and duplex reactions with single-species DNA were largely comparable to those obtained previously with species-specific TaqMan probes, showing the suitability of that simpler experimental approach for large-scale analytical applications.

  9. A High Resolution Microprobe Study of EETA79001 Lithology C

    NASA Technical Reports Server (NTRS)

    Schrader, Christian M.; Cohen, B. A.; Donovan, J. J.; Vicenzi, E. P.

    2010-01-01

    Antarctic meteorite EETA79001 has received substantial attention for possibly containing a component of Martian soil in its impact glass (Lithology C) [1]. The composition of Martian soil can illuminate near-surface processes such as impact gardening [2] and hydrothermal and volcanic activity [3,4]. Impact melts in meteorites represent our most direct samples of Martian regolith. We present the initial findings from a high-resolution electron microprobe study of Lithology C from Martian meteorite EETA79001. As this study develops we aim to extract details of a potential soil composition and to examine Martian surface processes using elemental ratios and correlations.

  10. High-resolution color images of Io

    NASA Technical Reports Server (NTRS)

    Mcewen, A. S.; Soderblom, L. A.

    1984-01-01

    Color versions of the highest resolution Voyager images of Io were produced by combining the low resolution color images with the high resolution, clear filter images. High resolution versions of the orange, blue, and violet filter images are produced by: orange = high-res clear * low-res orange / low-res clear blue = high-res clear * low-res blue / low-res clear violet = high-res clear * low-res violet / low-res clear. The spectral responses of the high and low resolution clear filter images cancel, leaving the color, while the spatial frequencies of the two low resolution images cancel, leaving the high resolution.

  11. NOAA's Use of High-Resolution Imagery

    NASA Technical Reports Server (NTRS)

    Hund, Erik

    2007-01-01

    NOAA's use of high-resolution imagery consists of: a) Shoreline mapping and nautical chart revision; b) Coastal land cover mapping; c) Benthic habitat mapping; d) Disaster response; and e) Imagery collection and support for coastal programs.

  12. Real-time detection of DNA hybridization and melting on oligonucleotide arrays by using optical wave guides.

    PubMed

    Stimpson, D I; Hoijer, J V; Hsieh, W T; Jou, C; Gordon, J; Theriault, T; Gamble, R; Baldeschwieler, J D

    1995-07-01

    The challenge of the Human Genome Project is to increase the rate of DNA sequence acquisition by two orders of magnitude to complete sequencing of the human genome by the year 2000. The present work describes a rapid detection method using a two-dimensional optical wave guide that allows measurement of real-time binding or melting of a light-scattering label on a DNA array. A particulate label on the target DNA acts as a light-scattering source when illuminated by the evanescent wave of the wave guide and only the label bound to the surface generates a signal. Imaging/visual examination of the scattered light permits interrogation of the entire array simultaneously. Hybridization specificity is equivalent to that obtained with a conventional system using autoradiography. Wave guide melting curves are consistent with those obtained in the liquid phase and single-base discrimination is facile. Dilution experiments showed an apparent lower limit of detection at 0.4 nM oligonucleotide. This performance is comparable to the best currently known fluorescence-based systems. In addition, wave guide detection allows manipulation of hybridization stringency during detection and thereby reduces DNA chip complexity. It is anticipated that this methodology will provide a powerful tool for diagnostic applications that require rapid cost-effective detection of variations from known sequences.

  13. RAPID DAMAGE ASSESSMENT FROM HIGH RESOLUTION IMAGERY

    SciTech Connect

    Vijayaraj, Veeraraghavan; Bright, Eddie A; Bhaduri, Budhendra L

    2008-01-01

    Disaster impact modeling and analysis uses huge volumes of image data that are produced immediately following a natural or an anthropogenic disaster event. Rapid damage assessment is the key to time critical decision support in disaster management to better utilize available response resources and accelerate recovery and relief efforts. But exploiting huge volumes of high resolution image data for identifying damaged areas with robust consistency in near real time is a challenging task. In this paper, we present an automated image analysis technique to identify areas of structural damage from high resolution optical satellite data using features based on image content.

  14. Evaluation of the Gibbs Free Energy Changes and Melting Temperatures of DNA/DNA Duplexes Using Hybridization Enthalpy Calculated by Molecular Dynamics Simulation.

    PubMed

    Lomzov, Alexander A; Vorobjev, Yury N; Pyshnyi, Dmitrii V

    2015-12-10

    A molecular dynamics simulation approach was applied for the prediction of the thermal stability of oligonucleotide duplexes. It was shown that the enthalpy of the DNA/DNA complex formation could be calculated using this approach. We have studied the influence of various simulation parameters on the secondary structure and the hybridization enthalpy value of Dickerson-Drew dodecamer. The optimal simulation parameters for the most reliable prediction of the enthalpy values were determined. The thermodynamic parameters (enthalpy and entropy changes) of a duplex formation were obtained experimentally for 305 oligonucleotides of various lengths and GC-content. The resulting database was studied with molecular dynamics (MD) simulation using the optimized simulation parameters. Gibbs free energy changes and the melting temperatures were evaluated using the experimental correlation between enthalpy and entropy changes of the duplex formation and the enthalpy values calculated by the MD simulation. The average errors in the predictions of enthalpy, the Gibbs free energy change, and the melting temperature of oligonucleotide complexes were 11%, 10%, and 4.4 °C, respectively. We have shown that the molecular dynamics simulation gives a possibility to calculate the thermal stability of native DNA/DNA complexes a priori with an unexpectedly high accuracy.

  15. A High-Resolution Stopwatch for Cents

    ERIC Educational Resources Information Center

    Gingl, Z.; Kopasz, K.

    2011-01-01

    A very low-cost, easy-to-make stopwatch is presented to support various experiments in mechanics. The high-resolution stopwatch is based on two photodetectors connected directly to the microphone input of a sound card. Dedicated free open-source software has been developed and made available to download. The efficiency is demonstrated by a free…

  16. High-resolution two dimensional advective transport

    USGS Publications Warehouse

    Smith, P.E.; Larock, B.E.

    1989-01-01

    The paper describes a two-dimensional high-resolution scheme for advective transport that is based on a Eulerian-Lagrangian method with a flux limiter. The scheme is applied to the problem of pure-advection of a rotated Gaussian hill and shown to preserve the monotonicity property of the governing conservation law.

  17. Coding and non-coding DNA thermal stability differences in eukaryotes studied by melting simulation, base shuffling and DNA nearest neighbor frequency analysis.

    PubMed

    Long, Dang D; Grosse, Ivo; Marx, Kenneth A

    2004-07-01

    The melting of the coding and non-coding classes of natural DNA sequences was investigated using a program, MELTSIM, which simulates DNA melting based upon an empirically parameterized nearest neighbor thermodynamic model. We calculated T(m) results of 8144 natural sequences from 28 eukaryotic organisms of varying F(GC) (mole fraction of G and C) and of 3775 coding and 3297 non-coding sequences derived from those natural sequences. These data demonstrated that the T(m) vs. F(GC) relationships in coding and non-coding DNAs are both linear but have a statistically significant difference (6.6%) in their slopes. These relationships are significantly different from the T(m) vs. F(GC) relationship embodied in the classical Marmur-Schildkraut-Doty (MSD) equation for the intact long natural sequences. By analyzing the simulation results from various base shufflings of the original DNAs and the average nearest neighbor frequencies of those natural sequences across the F(GC) range, we showed that these differences in the T(m) vs. F(GC) relationships are largely a direct result of systematic F(GC)-dependent biases in nearest neighbor frequencies for those two different DNA classes. Those differences in the T(m) vs. F(GC) relationships and biases in nearest neighbor frequencies also appear between the sequences from multicellular and unicellular organisms in the same coding or non-coding classes, albeit of smaller but significant magnitudes.

  18. Coding and non-coding DNA thermal stability differences in eukaryotes studied by melting simulation, base shuffling and DNA nearest neighbor frequency analysis.

    PubMed

    Long, Dang D; Grosse, Ivo; Marx, Kenneth A

    2004-07-01

    The melting of the coding and non-coding classes of natural DNA sequences was investigated using a program, MELTSIM, which simulates DNA melting based upon an empirically parameterized nearest neighbor thermodynamic model. We calculated T(m) results of 8144 natural sequences from 28 eukaryotic organisms of varying F(GC) (mole fraction of G and C) and of 3775 coding and 3297 non-coding sequences derived from those natural sequences. These data demonstrated that the T(m) vs. F(GC) relationships in coding and non-coding DNAs are both linear but have a statistically significant difference (6.6%) in their slopes. These relationships are significantly different from the T(m) vs. F(GC) relationship embodied in the classical Marmur-Schildkraut-Doty (MSD) equation for the intact long natural sequences. By analyzing the simulation results from various base shufflings of the original DNAs and the average nearest neighbor frequencies of those natural sequences across the F(GC) range, we showed that these differences in the T(m) vs. F(GC) relationships are largely a direct result of systematic F(GC)-dependent biases in nearest neighbor frequencies for those two different DNA classes. Those differences in the T(m) vs. F(GC) relationships and biases in nearest neighbor frequencies also appear between the sequences from multicellular and unicellular organisms in the same coding or non-coding classes, albeit of smaller but significant magnitudes. PMID:15223141

  19. High resolution Arctic snow observations: SnowNet (Invited)

    NASA Astrophysics Data System (ADS)

    Hiemstra, C. A.; Sturm, M.; Gelvin, A. B.; Berezovskaya, S.; Saari, S. P.; Finnegan, D. C.; Liston, G. E.

    2009-12-01

    Snow’s importance has become especially prominent in the terrestrial Arctic, where snow dominates the landscape most of the year and changes in snow arrival, depth, and melt have substantial energy budget and biotic consequences. Yet, the Arctic presents formidable challenges to accurate snow measurements because snow depths can vary greatly over relatively short distances (< 10 m). Snow distribution patterns in windy environments, such as the Arctic, arise from interactions among wind, snow, vegetation, and topography. In this environment, snow is transported easily and is retained in topographic depressions, near taller vegetation, and deposited on the lee sides of hills. Reliable observations of where snow exists in the Arctic landscape can be difficult to obtain, and estimates vary depending on where snow is sampled. Measurements tend to be widely distributed and sparse. In addition, observed changes in Arctic vegetation (e.g., increasing shrubs) and land surfaces (e.g., thermokarst) complicate matters further. In response to this critical shortcoming in Arctic snow measurements, we have developed a prototype observational network (SnowNet) that employs standard meteorological observations and high resolution topographic and vegetation data in concert with a comprehensive spatially-intensive snow measurement program. Our sites at Barrow (started 2007) and Imnavait Creek (started 2008), Alaska, feature frequent site visits and intensive spatial sampling of snow depths and densities and snow-surface topography. Both sites have high resolution (~20 cm) topographic and vegetation data layers generated from remote sensing and ground surveys. Further, we have been incorporating extremely high-resolution (< 10 cm) ground-based LiDAR snow and vegetation datasets that allow us to identify relationships among topography, vegetation, and snow in Arctic environments. In addition, we have collected tens of thousands of manual snow depths across our research sites. This

  20. High resolution 3D nonlinear integrated inversion

    NASA Astrophysics Data System (ADS)

    Li, Yong; Wang, Xuben; Li, Zhirong; Li, Qiong; Li, Zhengwen

    2009-06-01

    The high resolution 3D nonlinear integrated inversion method is based on nonlinear theory. Under layer control, the log data from several wells (or all wells) in the study area and seismic trace data adjacent to the wells are input to a network with multiple inputs and outputs and are integratedly trained to obtain an adaptive weight function of the entire study area. Integrated nonlinear mapping relationships are built and updated by the lateral and vertical geologic variations of the reservoirs. Therefore, the inversion process and its inversion results can be constrained and controlled and a stable seismic inversion section with high resolution with velocity inversion, impedance inversion, and density inversion sections, can be gained. Good geologic effects have been obtained in model computation tests and real data processing, which verified that this method has high precision, good practicality, and can be used for quantitative reservoir analysis.

  1. High-Resolution X-Ray Telescopes

    NASA Technical Reports Server (NTRS)

    ODell, Stephen L.; Brissenden, Roger J.; Davis, William; Elsner, Ronald F.; Elvis, Martin; Freeman, Mark; Gaetz, Terry; Gorenstein, Paul; Gubarev, Mikhail V.

    2010-01-01

    Fundamental needs for future x-ray telescopes: a) Sharp images => excellent angular resolution. b) High throughput => large aperture areas. Generation-X optics technical challenges: a) High resolution => precision mirrors & alignment. b) Large apertures => lots of lightweight mirrors. Innovation needed for technical readiness: a) 4 top-level error terms contribute to image size. b) There are approaches to controlling those errors. Innovation needed for manufacturing readiness. Programmatic issues are comparably challenging.

  2. High resolution schemes for hyperbolic conservation laws

    NASA Technical Reports Server (NTRS)

    Harten, A.

    1983-01-01

    A class of new explicit second order accurate finite difference schemes for the computation of weak solutions of hyperbolic conservation laws is presented. These highly nonlinear schemes are obtained by applying a nonoscillatory first order accurate scheme to an appropriately modified flux function. The so-derived second order accurate schemes achieve high resolution while preserving the robustness of the original nonoscillatory first order accurate scheme. Numerical experiments are presented to demonstrate the performance of these new schemes.

  3. Thermodynamic study of rhodamine 123-calf thymus DNA interaction: determination of calorimetric enthalpy by optical melting study.

    PubMed

    Masum, Abdulla Al; Chakraborty, Maharudra; Pandya, Prateek; Halder, Umesh Chandra; Islam, Md Maidul; Mukhopadhyay, Subrata

    2014-11-20

    In this paper, the interaction of rhodamine123 (R123) with calf thymus DNA has been studied using molecular modeling and other biophysical methods like UV-vis spectroscopy, fluoremetry, optical melting, isothermal titration calorimetry, and circular dichroic studies. Results showed that the binding energy is about -6 to -8 kcal/mol, and the binding process is favored by both negative enthalpy change and positive entropy change. A new method to determine different thermodynamic properties like calorimetric enthalpy and heat capacity change has been introduced in this paper. The obtained data has been crossed-checked by other methods. After dissecting the free-energy contribution, it was observed that the binding was favored by both negative hydrophobic free energy and negative molecular free energy which compensated for the positive free energies due to the conformational change loss of rotational and transitional freedom of the DNA helix.

  4. Thermodynamic study of rhodamine 123-calf thymus DNA interaction: determination of calorimetric enthalpy by optical melting study.

    PubMed

    Masum, Abdulla Al; Chakraborty, Maharudra; Pandya, Prateek; Halder, Umesh Chandra; Islam, Md Maidul; Mukhopadhyay, Subrata

    2014-11-20

    In this paper, the interaction of rhodamine123 (R123) with calf thymus DNA has been studied using molecular modeling and other biophysical methods like UV-vis spectroscopy, fluoremetry, optical melting, isothermal titration calorimetry, and circular dichroic studies. Results showed that the binding energy is about -6 to -8 kcal/mol, and the binding process is favored by both negative enthalpy change and positive entropy change. A new method to determine different thermodynamic properties like calorimetric enthalpy and heat capacity change has been introduced in this paper. The obtained data has been crossed-checked by other methods. After dissecting the free-energy contribution, it was observed that the binding was favored by both negative hydrophobic free energy and negative molecular free energy which compensated for the positive free energies due to the conformational change loss of rotational and transitional freedom of the DNA helix. PMID:25383921

  5. High-Resolution Traction Force Microscopy

    PubMed Central

    Plotnikov, Sergey V.; Sabass, Benedikt; Schwarz, Ulrich S.; Waterman, Clare M.

    2015-01-01

    Cellular forces generated by the actomyosin cytoskeleton and transmitted to the extracellular matrix (ECM) through discrete, integrin-based protein assemblies, that is, focal adhesions, are critical to developmental morphogenesis and tissue homeostasis, as well as disease progression in cancer. However, quantitative mapping of these forces has been difficult since there has been no experimental technique to visualize nanonewton forces at submicrometer spatial resolution. Here, we provide detailed protocols for measuring cellular forces exerted on two-dimensional elastic substrates with a high-resolution traction force microscopy (TFM) method. We describe fabrication of polyacrylamide substrates labeled with multiple colors of fiducial markers, functionalization of the substrates with ECM proteins, setting up the experiment, and imaging procedures. In addition, we provide the theoretical background of traction reconstruction and experimental considerations important to design a high-resolution TFM experiment. We describe the implementation of a new algorithm for processing of images of fiducial markers that are taken below the surface of the substrate, which significantly improves data quality. We demonstrate the application of the algorithm and explain how to choose a regularization parameter for suppression of the measurement error. A brief discussion of different ways to visualize and analyze the results serves to illustrate possible uses of high-resolution TFM in biomedical research. PMID:24974038

  6. High-Resolution US of Rheumatologic Diseases.

    PubMed

    Taljanovic, Mihra S; Melville, David M; Gimber, Lana H; Scalcione, Luke R; Miller, Margaret D; Kwoh, C Kent; Klauser, Andrea S

    2015-01-01

    For the past 15 years, high-resolution ultrasonography (US) is being routinely and increasingly used for initial evaluation and treatment follow-up of rheumatologic diseases. This imaging technique is performed by using high-frequency linear transducers and has proved to be a powerful diagnostic tool in evaluation of articular erosions, simple and complex joint and bursal effusions, tendon sheath effusions, and synovitis, with results comparable to those of magnetic resonance imaging, excluding detection of bone marrow edema. Crystal deposition diseases including gouty arthropathy and calcium pyrophosphate deposition disease (CPPD) have characteristic appearances at US, enabling differentiation between these two diseases and from inflammatory arthropathies. Enthesopathy, which frequently accompanies psoriatic and reactive arthritis, also has a characteristic appearance at high-resolution US, distinguishing these two entities from other inflammatory and metabolic arthropathies. The presence of Doppler signal in examined joints, bursae, and tendon sheaths indicates active synovitis. Microbubble echo contrast agents augment detection of tissue vascularity and may act in the future as a drug delivery vehicle. Frequently, joint, tendon sheath, and bursal fluid aspirations and therapeutic injections are performed under US guidance. The authors describe the high-resolution US technique including gray-scale, color or power Doppler, and contrast agent-enhanced US that is used in evaluation of rheumatologic diseases of the wrist and hand and the ankle and foot in their routine clinical practice. This article demonstrates imaging findings of normal joints, rheumatoid arthritis, gouty arthritis, CPPD, psoriatic and reactive arthritis, and osteoarthritis.

  7. High-resolution mapping of bifurcations in nonlinear biochemical circuits.

    PubMed

    Genot, A J; Baccouche, A; Sieskind, R; Aubert-Kato, N; Bredeche, N; Bartolo, J F; Taly, V; Fujii, T; Rondelez, Y

    2016-08-01

    Analog molecular circuits can exploit the nonlinear nature of biochemical reaction networks to compute low-precision outputs with fewer resources than digital circuits. This analog computation is similar to that employed by gene-regulation networks. Although digital systems have a tractable link between structure and function, the nonlinear and continuous nature of analog circuits yields an intricate functional landscape, which makes their design counter-intuitive, their characterization laborious and their analysis delicate. Here, using droplet-based microfluidics, we map with high resolution and dimensionality the bifurcation diagrams of two synthetic, out-of-equilibrium and nonlinear programs: a bistable DNA switch and a predator-prey DNA oscillator. The diagrams delineate where function is optimal, dynamics bifurcates and models fail. Inverse problem solving on these large-scale data sets indicates interference from enzymatic coupling. Additionally, data mining exposes the presence of rare, stochastically bursting oscillators near deterministic bifurcations.

  8. High-resolution mapping of bifurcations in nonlinear biochemical circuits

    NASA Astrophysics Data System (ADS)

    Genot, A. J.; Baccouche, A.; Sieskind, R.; Aubert-Kato, N.; Bredeche, N.; Bartolo, J. F.; Taly, V.; Fujii, T.; Rondelez, Y.

    2016-08-01

    Analog molecular circuits can exploit the nonlinear nature of biochemical reaction networks to compute low-precision outputs with fewer resources than digital circuits. This analog computation is similar to that employed by gene-regulation networks. Although digital systems have a tractable link between structure and function, the nonlinear and continuous nature of analog circuits yields an intricate functional landscape, which makes their design counter-intuitive, their characterization laborious and their analysis delicate. Here, using droplet-based microfluidics, we map with high resolution and dimensionality the bifurcation diagrams of two synthetic, out-of-equilibrium and nonlinear programs: a bistable DNA switch and a predator–prey DNA oscillator. The diagrams delineate where function is optimal, dynamics bifurcates and models fail. Inverse problem solving on these large-scale data sets indicates interference from enzymatic coupling. Additionally, data mining exposes the presence of rare, stochastically bursting oscillators near deterministic bifurcations.

  9. High-resolution mapping of bifurcations in nonlinear biochemical circuits

    NASA Astrophysics Data System (ADS)

    Genot, A. J.; Baccouche, A.; Sieskind, R.; Aubert-Kato, N.; Bredeche, N.; Bartolo, J. F.; Taly, V.; Fujii, T.; Rondelez, Y.

    2016-08-01

    Analog molecular circuits can exploit the nonlinear nature of biochemical reaction networks to compute low-precision outputs with fewer resources than digital circuits. This analog computation is similar to that employed by gene-regulation networks. Although digital systems have a tractable link between structure and function, the nonlinear and continuous nature of analog circuits yields an intricate functional landscape, which makes their design counter-intuitive, their characterization laborious and their analysis delicate. Here, using droplet-based microfluidics, we map with high resolution and dimensionality the bifurcation diagrams of two synthetic, out-of-equilibrium and nonlinear programs: a bistable DNA switch and a predator-prey DNA oscillator. The diagrams delineate where function is optimal, dynamics bifurcates and models fail. Inverse problem solving on these large-scale data sets indicates interference from enzymatic coupling. Additionally, data mining exposes the presence of rare, stochastically bursting oscillators near deterministic bifurcations.

  10. High-resolution mapping of bifurcations in nonlinear biochemical circuits.

    PubMed

    Genot, A J; Baccouche, A; Sieskind, R; Aubert-Kato, N; Bredeche, N; Bartolo, J F; Taly, V; Fujii, T; Rondelez, Y

    2016-08-01

    Analog molecular circuits can exploit the nonlinear nature of biochemical reaction networks to compute low-precision outputs with fewer resources than digital circuits. This analog computation is similar to that employed by gene-regulation networks. Although digital systems have a tractable link between structure and function, the nonlinear and continuous nature of analog circuits yields an intricate functional landscape, which makes their design counter-intuitive, their characterization laborious and their analysis delicate. Here, using droplet-based microfluidics, we map with high resolution and dimensionality the bifurcation diagrams of two synthetic, out-of-equilibrium and nonlinear programs: a bistable DNA switch and a predator-prey DNA oscillator. The diagrams delineate where function is optimal, dynamics bifurcates and models fail. Inverse problem solving on these large-scale data sets indicates interference from enzymatic coupling. Additionally, data mining exposes the presence of rare, stochastically bursting oscillators near deterministic bifurcations. PMID:27442281

  11. The rolling-circle melting-pot model for porcine circovirus DNA replication

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A stem-loop structure, formed by a pair of inverted repeats during DNA replication, is a conserved feature at the origin of DNA replication (Ori) among plant and animal viruses, bacteriophages and plasmids that replicate their genomes via the rolling-circle replication (RCR) mechanism. Porcine circo...

  12. High-resolution analysis of CpG methylation and in vivo protein-DNA interactions at the alternative Epstein-Barr virus latency promoters Qp and Cp in the nasopharyngeal carcinoma cell line C666-1.

    PubMed

    Bakos, Agnes; Banati, Ferenc; Koroknai, Anita; Takacs, Maria; Salamon, Daniel; Minarovits-Kormuta, Susanna; Schwarzmann, Fritz; Wolf, Hans; Niller, Hans Helmut; Minarovits, Janos

    2007-10-01

    Transcripts for the Epstein-Barr virus (EBV) encoded nuclear antigens (EBNAs) are initiated at alternative promoters (Wp, Cp, for EBNA 1-6 transcripts and Qp, for EBNA 1 transcripts only) located in the BamHI W, C or Q fragment of the viral genome. To understand the host-cell dependent expression of EBNAs in EBV-associated tumors (lymphomas and carcinomas) and in vitro transformed cell lines, it is necessary to analyse the regulatory mechanisms governing the activity of the alternative promoters of EBNA transcripts. Such studies focused mainly on lymphoid cell lines carrying latent EBV genomes, due to the lack of EBV-associated carcinoma cell lines maintaining latent EBV genomes during cultivation in tissue culture. We took advantage of the unique nasopharyngeal carcinoma cell line, C666-1, harboring EBV genomes, and undertook a detailed analysis of CpG methylation patterns and in vivo protein-DNA interactions at the latency promoters Qp and Cp. We found that the active, unmethylated Qp was marked with strong footprints of cellular transcription factors and the viral protein EBNA 1. In contrast, we could not detect binding of relevant transcription factors to the methylated, silent Cp. We concluded that the epigenetic marks at Qp and Cp in C666-1 cells of epithelial origin resemble those of group I Burkitt's lymphoma cell lines.

  13. A high-resolution record of Greenland mass balance

    NASA Astrophysics Data System (ADS)

    McMillan, Malcolm; Leeson, Amber; Shepherd, Andrew; Briggs, Kate; Armitage, Thomas W. K.; Hogg, Anna; Kuipers Munneke, Peter; Broeke, Michiel; Noël, Brice; Berg, Willem Jan; Ligtenberg, Stefan; Horwath, Martin; Groh, Andreas; Muir, Alan; Gilbert, Lin

    2016-07-01

    We map recent Greenland Ice Sheet elevation change at high spatial (5 km) and temporal (monthly) resolution using CryoSat-2 altimetry. After correcting for the impact of changing snowpack properties associated with unprecedented surface melting in 2012, we find good agreement (3 cm/yr bias) with airborne measurements. With the aid of regional climate and firn modeling, we compute high spatial and temporal resolution records of Greenland mass evolution, which correlate (R = 0.96) with monthly satellite gravimetry and reveal glacier dynamic imbalance. During 2011-2014, Greenland mass loss averaged 269 ± 51 Gt/yr. Atmospherically driven losses were widespread, with surface melt variability driving large fluctuations in the annual mass deficit. Terminus regions of five dynamically thinning glaciers, which constitute less than 1% of Greenland's area, contributed more than 12% of the net ice loss. This high-resolution record demonstrates that mass deficits extending over small spatial and temporal scales have made a relatively large contribution to recent ice sheet imbalance.

  14. A Portable, High Resolution, Surface Measurement Device

    NASA Technical Reports Server (NTRS)

    Ihlefeld, Curtis M.; Burns, Bradley M.; Youngquist, Robert C.

    2012-01-01

    A high resolution, portable, surface measurement device has been demonstrated to provide micron-resolution topographical plots. This device was specifically developed to allow in-situ measurements of defects on the Space Shuttle Orbiter windows, but is versatile enough to be used on a wide variety of surfaces. This paper discusses the choice of an optical sensor and then the decisions required to convert a lab bench optical measurement device into an ergonomic portable system. The necessary trade-offs between performance and portability are presented along with a description of the device developed to measure Orbiter window defects.

  15. High resolution SAR applications and instrument design

    NASA Technical Reports Server (NTRS)

    Dionisio, C.; Torre, A.

    1993-01-01

    The Synthetic Aperture Radar (SAR) has viewed, in the last two years, a huge increment of interest from many preset and potential users. The good spatial resolution associated to the all weather capability lead to considering SAR not only a scientific instrument but a tool for verifying and controlling the daily human relationships with the Earth Environment. New missions were identified for SAR as spatial resolution became lower than three meters: disasters, pollution, ships traffic, volcanic eruptions, earthquake effect are only a few of the possible objects which can be effectively detected, controlled and monitored by SAR mounted on satellites. High resolution radar design constraints and dimensioning are discussed.

  16. High resolution millimeter-wave imaging sensor

    NASA Technical Reports Server (NTRS)

    Wilson, W. J.; Howard, R. J.; Parks, G. S.

    1985-01-01

    A scanning 3-mm radiometer is described that has been built for use on a small aircraft to produce real time high resolution images of the ground when atmospheric conditions such as smoke, dust, and clouds make IR and visual sensors unusable. The sensor can be used for a variety of remote sensing applications such as measurements of snow cover and snow water equivalent, precipitation mapping, vegetation type and extent, surface moisture and temperature, and surface thermal inertia. The advantages of millimeter waves for cloud penetration and the ability to observe different physical phenomena make this system an attractive supplement to visible and IR remote sensing systems.

  17. High resolution extremity CT for biomechanics modeling

    SciTech Connect

    Ashby, A.E.; Brand, H.; Hollerbach, K.; Logan, C.M.; Martz, H.E.

    1995-09-23

    With the advent of ever more powerful computing and finite element analysis (FEA) capabilities, the bone and joint geometry detail available from either commercial surface definitions or from medical CT scans is inadequate. For dynamic FEA modeling of joints, precise articular contours are necessary to get appropriate contact definition. In this project, a fresh cadaver extremity was suspended in parafin in a lucite cylinder and then scanned with an industrial CT system to generate a high resolution data set for use in biomechanics modeling.

  18. A High Resolution Scale-of-four

    DOE R&D Accomplishments Database

    Fitch, V.

    1949-08-25

    A high resolution scale-of-four has been developed to be used in conjunction with the nuclear particle detection devices in applications where the counting rate is unusually high. Specifically, it is intended to precede the commercially available medium resolution scaling circuits and so decrease the resolving time of the counting system. The circuit will function reliably on continuously recurring pulses separated by less than 0.1 microseconds. It will resolve two pulses (occurring at a moderate repetition rate) which are spaced at 0.04 microseconds. A five-volt input signal is sufficient to actuate the device.

  19. High Resolution Image From Viking Lander 1

    NASA Technical Reports Server (NTRS)

    1976-01-01

    Viking 1 took this high-resolution picture today, its third day on Mars. Distance from the camera to the nearfield (bottom) is about 4 meters (13 feet); to the horizon, about 3 kilometers (1.8 miles). The photo shows numerous angular blocks ranging in size from a few centimeters to several meters. The surface between the blocks is composed of fine-grained material. Accumulation of some fine-grained material behind blocks indicates wind deposition of dust and sand downwind of obstacles. The large block on the horizon is about 4 meters (13 feet) wide. Distance across the horizon is about 34 meters (110 feet).

  20. High Resolution Spectroscopy with Submillimeter-Wave

    NASA Astrophysics Data System (ADS)

    Kumar, Vinay; Dave, Hemant

    2003-03-01

    In order to explain the characteristic features of planetary atmosphere, detection and precise measurements of environmentally important gases such as CO, CIO, No becomes necessary. Since most of the polyatomic molecules have (ro-vibrational) transitions in submillimeter region 100 μ-1000μ), probing in this wavelength region is vital. The specific rotational and vibrational states are the result of interactions between different atoms in the molecule. Since each molecule has a unique arrangement of atoms, it has an exclusive submillimeter signature. We are developing a portable heterodyne receiver system at Physical Research Laboratory, Ahmedabad to perform high-resolution spectroscopy in this wavelength region.

  1. High-Resolution Scintimammography: A Pilot Study

    SciTech Connect

    Rachel F. Brem; Joelle M. Schoonjans; Douglas A. Kieper; Stan Majewski; Steven Goodman; Cahid Civelek

    2002-07-01

    This study evaluated a novel high-resolution breast-specific gamma camera (HRBGC) for the detection of suggestive breast lesions. Methods: Fifty patients (with 58 breast lesions) for whom a scintimammogram was clinically indicated were prospectively evaluated with a general-purpose gamma camera and a novel HRBGC prototype. The results of conventional and high-resolution nuclear studies were prospectively classified as negative (normal or benign) or positive (suggestive or malignant) by 2 radiologists who were unaware of the mammographic and histologic results. All of the included lesions were confirmed by pathology. Results: There were 30 benign and 28 malignant lesions. The sensitivity for detection of breast cancer was 64.3% (18/28) with the conventional camera and 78.6% (22/28) with the HRBGC. The specificity with both systems was 93.3% (28/30). For the 18 nonpalpable lesions, sensitivity was 55.5% (10/18) and 72.2% (13/18) with the general-purpose camera and the HRBGC, respectively. For lesions 1 cm, 7 of 15 were detected with the general-purpose camera and 10 of 15 with the HRBGC. Four lesions (median size, 8.5 mm) were detected only with the HRBGC and were missed by the conventional camera. Conclusion: Evaluation of indeterminate breast lesions with an HRBGC results in improved sensitivity for the detection of cancer, with greater improvement shown for nonpalpable and 1-cm lesions.

  2. High Resolution Camera for Mapping Titan Surface

    NASA Technical Reports Server (NTRS)

    Reinhardt, Bianca

    2011-01-01

    Titan, Saturn's largest moon, has a dense atmosphere and is the only object besides Earth to have stable liquids at its surface. The Cassini/Huygens mission has revealed the extraordinary breadth of geological processes shaping its surface. Further study requires high resolution imaging of the surface, which is restrained by light absorption by methane and scattering from aerosols. The Visual and Infrared Mapping Spectrometer (VIMS) onboard the Cassini spacecraft has demonstrated that Titan's surface can be observed within several windows in the near infrared, allowing us to process several regions in order to create a geological map and to determine the morphology. Specular reflections monitored on the lakes of the North Pole show little scattering at 5 microns, which, combined with the present study of Titan's northern pole area, refutes the paradigm that only radar can achieve high resolution mapping of the surface. The present data allowed us to monitor the evolution of lakes, to identify additional lakes at the Northern Pole, to examine Titan's hypothesis of non-synchronous rotation and to analyze the albedo of the North Pole surface. Future missions to Titan could carry a camera with 5 micron detectors and a carbon fiber radiator for weight reduction.

  3. High Resolution Laser Spectroscopy of Rhenium Carbide

    NASA Astrophysics Data System (ADS)

    Adam, Allan G.; Hall, Ryan M.; Linton, Colan; Tokaryk, Dennis

    2014-06-01

    The first spectroscopic study of rhenium carbide, ReC, has been performed using both low and high resolution techniques to collect rotationally resolved electronic spectra from 420 to 500nm. Laser-induced fluorescence (LIF), and dispersed fluorescence (DF) techniques were employed. ReC was formed in our laser ablation molecular jet apparatus by ablating a rhenium target rod in the presence of 1% methane in helium. The low resolution spectrum identified four bands of an electronic system belonging to ReC, three of which have been studied so far. Extensive hyperfine structure composed of six hyperfine components was observed in the high resolution spectrum, as well as a clear distinction between the 187ReC and 185ReC isotopologues. The data seems consistent with a ^4Π - ^4Σ- transition, as was predicted before experimentation. Dispersed fluorescence spectra allowed us to determine the ground state vibrational frequency (ωe"=994.4 ± 0.3 wn), and to identify a low-lying electronically excited state at Te"=1118.4 ± 0.4 wn with a vibrational frequency of ωe"=984 ± 2 wn. Personal communication, F. Grein, University of New Brunswick

  4. Common high-resolution MMW scene generator

    NASA Astrophysics Data System (ADS)

    Saylor, Annie V.; McPherson, Dwight A.; Satterfield, H. DeWayne; Sholes, William J.; Mobley, Scott B.

    2001-08-01

    The development of a modularized millimeter wave (MMW) target and background high resolution scene generator is reported. The scene generator's underlying algorithms are applicable to both digital and real-time hardware-in-the-loop (HWIL) simulations. The scene generator will be configurable for a variety of MMW and multi-mode sensors employing state of the art signal processing techniques. At present, digital simulations for MMW and multi-mode sensor development and testing are custom-designed by the seeker vendor and are verified, validated, and operated by both the vendor and government in simulation-based acquisition. A typical competition may involve several vendors, each requiring high resolution target and background models for proper exercise of seeker algorithms. There is a need and desire by both the government and sensor vendors to eliminate costly re-design and re-development of digital simulations. Additional efficiencies are realized by assuring commonality between digital and HWIL simulation MMW scene generators, eliminating duplication of verification and validation efforts.

  5. High-Resolution PET Detector. Final report

    SciTech Connect

    Karp, Joel

    2014-03-26

    The objective of this project was to develop an understanding of the limits of performance for a high resolution PET detector using an approach based on continuous scintillation crystals rather than pixelated crystals. The overall goal was to design a high-resolution detector, which requires both high spatial resolution and high sensitivity for 511 keV gammas. Continuous scintillation detectors (Anger cameras) have been used extensively for both single-photon and PET scanners, however, these instruments were based on NaI(Tl) scintillators using relatively large, individual photo-multipliers. In this project we investigated the potential of this type of detector technology to achieve higher spatial resolution through the use of improved scintillator materials and photo-sensors, and modification of the detector surface to optimize the light response function.We achieved an average spatial resolution of 3-mm for a 25-mm thick, LYSO continuous detector using a maximum likelihood position algorithm and shallow slots cut into the entrance surface.

  6. Comparative Very-High-Resolution VUV Spectroscopy

    NASA Astrophysics Data System (ADS)

    Lewis, B. R.; Gibson, S. T.; Baldwin, K. G. H.; Dooley, P. M.; Waring, K.

    Despite their importance to the photochemistry of the terrestrial atmosphere, and many experimental studies, previous characterization of the Schumann-Runge (SR) bands of O2, B3 Σ u- <- X3 Σ_g^- (v, 0) (1750-2050 Å) has been limited by poor experimental resolution. In addition, our understanding of the SR spectrum is incomplete, many rovibrational transitions in the perturbed region of the spectrum [B(v > 15)] remaining unassigned. We review new very-high-resolution measurements of the O2 photoabsorption cross section in the SR bands. Tunable, narrow-bandwidth background vacuum-ultraviolet (VUV) radiation for the measurements ( 7 × 105 resolving power) was generated by the two-photon-resonant difference-frequency four-wave mixing in Xe of excimer-pumped dye-laser radiation. With the aid of these cross-section measurements, rovibrational and line-shape analyses have led to new insights into the molecular structure and predissociation dynamics of O2. The current VUV laser-spectroscopic measurements are shown to compare favourably with results from two other very-high-resolution experimental techniques, namely laser-induced fluorescence spectroscopy and VUV Fourier-transform spectroscopy, the latter performed using a synchrotron source.

  7. Limiting liability via high resolution image processing

    SciTech Connect

    Greenwade, L.E.; Overlin, T.K.

    1996-12-31

    The utilization of high resolution image processing allows forensic analysts and visualization scientists to assist detectives by enhancing field photographs, and by providing the tools and training to increase the quality and usability of field photos. Through the use of digitized photographs and computerized enhancement software, field evidence can be obtained and processed as `evidence ready`, even in poor lighting and shadowed conditions or darkened rooms. These images, which are most often unusable when taken with standard camera equipment, can be shot in the worst of photographic condition and be processed as usable evidence. Visualization scientists have taken the use of digital photographic image processing and moved the process of crime scene photos into the technology age. The use of high resolution technology will assist law enforcement in making better use of crime scene photography and positive identification of prints. Valuable court room and investigation time can be saved and better served by this accurate, performance based process. Inconclusive evidence does not lead to convictions. Enhancement of the photographic capability helps solve one major problem with crime scene photos, that if taken with standard equipment and without the benefit of enhancement software would be inconclusive, thus allowing guilty parties to be set free due to lack of evidence.

  8. High Resolution Spectroscopy to Support Atmospheric Measurements

    NASA Technical Reports Server (NTRS)

    Venkataraman, Malathy Devi

    2003-01-01

    Spectroscopic parameters (such as line position, intensity, broadening and shifting coefficients and their temperature dependences, line mixing coefficients etc.) for various molecular species of atmospheric interest are determined. In order to achieve these results, infrared spectra of several molecular bands are obtained using high-resolution recording instruments such as tunable diode laser spectrometer and Fourier transform spectrometers. Using sophisticated analysis routines (Multispectrum nonlinear least squares technique) these high-resolution infrared spectra are processed to determine the various spectral line parameters that are cited above. Spectra were taken using the McMath-Pierce Fourier transform spectrometer (FTS) at the National Solar Observatory on Kitt Peak, Arizona as well as the Bruker FTS at the Pacific Northwest National Laboratory (PNNL) at Richland, Washington. Most of the spectra are acquired not only at room temperature, but also at several different cold temperatures. This procedure is necessary to study the variation of the spectral line parameters as a function of temperature in order to simulate the Earth's and other planetary atmospheric environments. Depending upon the strength or weakness of the various bands recorded and analyzed, the length(s) of the absorption cells in which the gas samples under study are kept varied from a few centimeters up to several meters and the sample temperatures varied from approximately +30 C to -63 C. Research on several infrared bands of various molecular species and their isotopomers are undertaken. Those studies are briefly described.

  9. Observation of HIV-1 Nucleocapsid Protein induced TAR DNA melting at the single molecule level

    NASA Astrophysics Data System (ADS)

    Cosa, Gonzalo; Harbron, Elizabeth; O'Connor, Donald; Musier-Forsyth, Karin; Barbara, Paul

    2003-03-01

    Reverse transcription of the HIV-1 RNA genome involves several nucleic acid rearrangement steps, and the HIV-1 nucleocapsid protein (NC) plays a key role in this process. NC is a nucleic acid chaperone protein, which facilitates the formation of the most stable nucleic acid structures. Single molecule fluorescence resonance energy transfer (SM-FRET) measurements enable us to observe the NC-induced conformational fluctuations of a transactivation response region (TAR) DNA hairpin, which is part of the initial product of reverse transcription known as minus-strand strong-stop DNA. SM-FRET studies show that the majority of conformational fluctuations of the fluorescently-labeled TAR DNA hairpin in the presence of NC occur in <100 ms. A single molecule explores a wide range of confomations unpon NC binding, with fluctuations encompassing as many as 40 bases in both arms of the hairpin. No conformational fluctuations are observed with the dye-labeled TAR DNA hairpin in the absence of NC or when a labeled TAR DNA hairpin variant lacking bulges and internal loops is analyzed in the presence of NC. This study represents the first real-time observation of NC-mediated nucleic acid conformational fluctuations, revealing new insights into NC's nucleic acid chaperone activity.

  10. Effect of surface binding on heterogeneous DNA melting equilibria: a Monte Carlo simulation study.

    PubMed

    Allen, John H; Schoch, Emily T; Stubbs, John M

    2011-02-24

    Deoxyribonucleic acid (DNA) microarrays are constructed with a surface-immobilized single-stranded probe sequence that hydrogen bonds with its complementary target strand from solution and is subsequently detected, making their hybridization equilibrium of central importance. Unexpectedly, the effect of surface immobilization is that if the sequences of probe and target are exchanged, the hybridization equilibrium shifts. Here, configurational-bias Monte Carlo simulations using a coarse-grained model for DNA were carried out for an undecamer double helix both in solution and bound to a surface to determine dissociation equilibria. Four possible surface binding orientations were independently investigated. Analysis shows that the effect of surface binding is to destabilize hydrogen-bonding interactions of bases proximal to the binding site and enhance those of distal bases due to the double helix lying flat on the surface. Results have implications for predicting surface-bound DNA hybridization equilibria.

  11. Clementine High Resolution Camera Mosaicking Project

    NASA Technical Reports Server (NTRS)

    1998-01-01

    This report constitutes the final report for NASA Contract NASW-5054. This project processed Clementine I high resolution images of the Moon, mosaicked these images together, and created a 22-disk set of compact disk read-only memory (CD-ROM) volumes. The mosaics were produced through semi-automated registration and calibration of the high resolution (HiRes) camera's data against the geometrically and photometrically controlled Ultraviolet/Visible (UV/Vis) Basemap Mosaic produced by the US Geological Survey (USGS). The HiRes mosaics were compiled from non-uniformity corrected, 750 nanometer ("D") filter high resolution nadir-looking observations. The images were spatially warped using the sinusoidal equal-area projection at a scale of 20 m/pixel for sub-polar mosaics (below 80 deg. latitude) and using the stereographic projection at a scale of 30 m/pixel for polar mosaics. Only images with emission angles less than approximately 50 were used. Images from non-mapping cross-track slews, which tended to have large SPICE errors, were generally omitted. The locations of the resulting image population were found to be offset from the UV/Vis basemap by up to 13 km (0.4 deg.). Geometric control was taken from the 100 m/pixel global and 150 m/pixel polar USGS Clementine Basemap Mosaics compiled from the 750 nm Ultraviolet/Visible Clementine imaging system. Radiometric calibration was achieved by removing the image nonuniformity dominated by the HiRes system's light intensifier. Also provided are offset and scale factors, achieved by a fit of the HiRes data to the corresponding photometrically calibrated UV/Vis basemap, that approximately transform the 8-bit HiRes data to photometric units. The sub-polar mosaics are divided into tiles that cover approximately 1.75 deg. of latitude and span the longitude range of the mosaicked frames. Images from a given orbit are map projected using the orbit's nominal central latitude. Polar mosaics are tiled into squares 2250 pixels on a

  12. High Resolution Powder Diffraction and Structure Determination

    SciTech Connect

    Cox, D. E.

    1999-04-23

    It is clear that high-resolution synchrotrons X-ray powder diffraction is a very powerful and convenient tool for material characterization and structure determination. Most investigations to date have been carried out under ambient conditions and have focused on structure solution and refinement. The application of high-resolution techniques to increasingly complex structures will certainly represent an important part of future studies, and it has been seen how ab initio solution of structures with perhaps 100 atoms in the asymmetric unit is within the realms of possibility. However, the ease with which temperature-dependence measurements can be made combined with improvements in the technology of position-sensitive detectors will undoubtedly stimulate precise in situ structural studies of phase transitions and related phenomena. One challenge in this area will be to develop high-resolution techniques for ultra-high pressure investigations in diamond anvil cells. This will require highly focused beams and very precise collimation in front of the cell down to dimensions of 50 {micro}m or less. Anomalous scattering offers many interesting possibilities as well. As a means of enhancing scattering contrast it has applications not only to the determination of cation distribution in mixed systems such as the superconducting oxides discussed in Section 9.5.3, but also to the location of specific cations in partially occupied sites, such as the extra-framework positions in zeolites, for example. Another possible application is to provide phasing information for ab initio structure solution. Finally, the precise determination of f as a function of energy through an absorption edge can provide useful information about cation oxidation states, particularly in conjunction with XANES data. In contrast to many experiments at a synchrotron facility, powder diffraction is a relatively simple and user-friendly technique, and most of the procedures and software for data analysis

  13. Clementine High Resolution Camera Mosaicking Project

    NASA Astrophysics Data System (ADS)

    1998-10-01

    This report constitutes the final report for NASA Contract NASW-5054. This project processed Clementine I high resolution images of the Moon, mosaicked these images together, and created a 22-disk set of compact disk read-only memory (CD-ROM) volumes. The mosaics were produced through semi-automated registration and calibration of the high resolution (HiRes) camera's data against the geometrically and photometrically controlled Ultraviolet/Visible (UV/Vis) Basemap Mosaic produced by the US Geological Survey (USGS). The HiRes mosaics were compiled from non-uniformity corrected, 750 nanometer ("D") filter high resolution nadir-looking observations. The images were spatially warped using the sinusoidal equal-area projection at a scale of 20 m/pixel for sub-polar mosaics (below 80 deg. latitude) and using the stereographic projection at a scale of 30 m/pixel for polar mosaics. Only images with emission angles less than approximately 50 were used. Images from non-mapping cross-track slews, which tended to have large SPICE errors, were generally omitted. The locations of the resulting image population were found to be offset from the UV/Vis basemap by up to 13 km (0.4 deg.). Geometric control was taken from the 100 m/pixel global and 150 m/pixel polar USGS Clementine Basemap Mosaics compiled from the 750 nm Ultraviolet/Visible Clementine imaging system. Radiometric calibration was achieved by removing the image nonuniformity dominated by the HiRes system's light intensifier. Also provided are offset and scale factors, achieved by a fit of the HiRes data to the corresponding photometrically calibrated UV/Vis basemap, that approximately transform the 8-bit HiRes data to photometric units. The sub-polar mosaics are divided into tiles that cover approximately 1.75 deg. of latitude and span the longitude range of the mosaicked frames. Images from a given orbit are map projected using the orbit's nominal central latitude. Polar mosaics are tiled into squares 2250 pixels on a

  14. High Resolution, High Frame Rate Video Technology

    NASA Technical Reports Server (NTRS)

    1990-01-01

    Papers and working group summaries presented at the High Resolution, High Frame Rate Video (HHV) Workshop are compiled. HHV system is intended for future use on the Space Shuttle and Space Station Freedom. The Workshop was held for the dual purpose of: (1) allowing potential scientific users to assess the utility of the proposed system for monitoring microgravity science experiments; and (2) letting technical experts from industry recommend improvements to the proposed near-term HHV system. The following topics are covered: (1) State of the art in the video system performance; (2) Development plan for the HHV system; (3) Advanced technology for image gathering, coding, and processing; (4) Data compression applied to HHV; (5) Data transmission networks; and (6) Results of the users' requirements survey conducted by NASA.

  15. High-Resolution Broadband Spectral Interferometry

    SciTech Connect

    Erskine, D J; Edelstein, J

    2002-08-09

    We demonstrate solar spectra from a novel interferometric method for compact broadband high-resolution spectroscopy. The spectral interferometer (SI) is a hybrid instrument that uses a spectrometer to externally disperse the output of a fixed-delay interferometer. It also has been called an externally dispersed interferometer (EDI). The interferometer can be used with linear spectrometers for imaging spectroscopy or with echelle spectrometers for very broad-band coverage. EDI's heterodyning technique enhances the spectrometer's response to high spectral-density features, increasing the effective resolution by factors of several while retaining its bandwidth. The method is extremely robust to instrumental insults such as focal spot size or displacement. The EDI uses no moving parts, such as purely interferometric FTS spectrometers, and can cover a much wider simultaneous bandpass than other internally dispersed interferometers (e.g. HHS or SHS).

  16. High resolution wavefront measurement of aspheric optics

    NASA Astrophysics Data System (ADS)

    Erichsen, I.; Krey, S.; Heinisch, J.; Ruprecht, A.; Dumitrescu, E.

    2008-08-01

    With the recently emerged large volume production of miniature aspheric lenses for a wide range of applications, a new fast fully automatic high resolution wavefront measurement instrument has been developed. The Shack-Hartmann based system with reproducibility better than 0.05 waves is able to measure highly aspheric optics and allows for real time comparison with design data. Integrated advanced analysis tools such as calculation of Zernike coefficients, 2D-Modulation Transfer Function (MTF), Point Spread Function (PSF), Strehl-Ratio and the measurement of effective focal length (EFL) as well as flange focal length (FFL) allow for the direct verification of lens properties and can be used in a development as well as in a production environment.

  17. Improved methods for high resolution electron microscopy

    SciTech Connect

    Taylor, J.R.

    1987-04-01

    Existing methods of making support films for high resolution transmission electron microscopy are investigated and novel methods are developed. Existing methods of fabricating fenestrated, metal reinforced specimen supports (microgrids) are evaluated for their potential to reduce beam induced movement of monolamellar crystals of C/sub 44/H/sub 90/ paraffin supported on thin carbon films. Improved methods of producing hydrophobic carbon films by vacuum evaporation, and improved methods of depositing well ordered monolamellar paraffin crystals on carbon films are developed. A novel technique for vacuum evaporation of metals is described which is used to reinforce microgrids. A technique is also developed to bond thin carbon films to microgrids with a polymer bonding agent. Unique biochemical methods are described to accomplish site specific covalent modification of membrane proteins. Protocols are given which covalently convert the carboxy terminus of papain cleaved bacteriorhodopsin to a free thiol. 53 refs., 19 figs., 1 tab.

  18. High-resolution climate simulation using CAM

    NASA Astrophysics Data System (ADS)

    Bacmeister, J.; Neale, R. B.; Hannay, C.; Lauritzen, P. H.; Wehner, M. F.

    2012-12-01

    Thanks to the development of highly scalable dynamical cores that can exploit massively parallel computer architectures, we expect that global climate models in the next decade will run routinely at horizontal resolutions of 25 km or finer. Early results at these resolutions show clear improvements in simulating climatologically and societally-important mesoscale meteorology such as tropical cyclones. Improvements in regional circulations likely associated with topography are also obtained. Nevertheless many long-standing biases in climate simulations, e.g., the "double ITCZ" bias in precipitation, remain remarkably insensitive to increased resolution. This talk will present high-resolution global simulations using the community atmosphere model. Sensitivity of tropical cyclone climatology and precipitation statistics to model physics suites will be shown

  19. Improved methods for high resolution electron microscopy

    NASA Astrophysics Data System (ADS)

    Taylor, J. R.

    1987-04-01

    Existing methods of making support films for high resolution transmission electron microscopy are investigated and novel methods are developed. Existing methods of fabricating fenestrated, metal reinforced specimen supports (microgrids) are evaluated for their potential to reduce beam induced movement of monolamellar crystals of C44H90 paraffin supported on thin carbon films. Improved methods of producing hydrophobic carbon films by vacuum evaporation, and improved methods of depositing well ordered monolamellar paraffin crystals on carbon films are developed. A novel technique for vacuum evaporation of metals is described which is used to reinforce microgrids. A technique is also developed to bond thin carbon films to microgrids with a polymer bonding agent. Unique biochemical methods are described to accomplish site specific covalent modification of membrane proteins. Protocols are given which covalently convert the carboxy terminus of papain cleaved bacteriorhodopsin to a free thiol.

  20. Ultra-high resolution computed tomography imaging

    DOEpatents

    Paulus, Michael J.; Sari-Sarraf, Hamed; Tobin, Jr., Kenneth William; Gleason, Shaun S.; Thomas, Jr., Clarence E.

    2002-01-01

    A method for ultra-high resolution computed tomography imaging, comprising the steps of: focusing a high energy particle beam, for example x-rays or gamma-rays, onto a target object; acquiring a 2-dimensional projection data set representative of the target object; generating a corrected projection data set by applying a deconvolution algorithm, having an experimentally determined a transfer function, to the 2-dimensional data set; storing the corrected projection data set; incrementally rotating the target object through an angle of approximately 180.degree., and after each the incremental rotation, repeating the radiating, acquiring, generating and storing steps; and, after the rotating step, applying a cone-beam algorithm, for example a modified tomographic reconstruction algorithm, to the corrected projection data sets to generate a 3-dimensional image. The size of the spot focus of the beam is reduced to not greater than approximately 1 micron, and even to not greater than approximately 0.5 microns.

  1. Low noise and high resolution microchannel plate

    NASA Astrophysics Data System (ADS)

    Liu, Shulin; Pan, Jingsheng; Deng, Guangxu; Su, Detan; Xu, Zhiqing; Zhang, Yanyun

    2008-02-01

    To improve the Figure of Merit (FOM) and reduce the Equivalent Background Input (EBI) and Fixed-Pattern-Noise (FPN) in image intensifier, NVT (North Night Vision Technology Co., Ltd) has been researching and developing a low noise and high resolution Micro Channel Plate (MCP). The density of dark current of this new MCP is less than 0.5PA/cm2 (when MCP voltage at 1000V). The FPN and scintillation noise are reduced remarkably. Channel diameter is 6 μm and open area ratio is 60%~70%. The vacuum bakeout temperature could be as high as 500°C. This new kind of MCP will be extensively used in the supper generation and the third generation image intensifiers.

  2. High resolution analysis of satellite gradiometry

    NASA Technical Reports Server (NTRS)

    Colombo, O. L.

    1989-01-01

    Satellite gravity gradiometry is a technique now under development which, by the middle of the next decade, may be used for the high resolution charting from space of the gravity field of the earth and, afterwards, of other planets. Some data analysis schemes are reviewed for getting detailed gravity maps from gradiometry on both a global and a local basis. It also presents estimates of the likely accuracies of such maps, in terms of normalized spherical harmonics expansions, both using gradiometry alone and in combination with data from a Global Positioning System (GPS) receiver carried on the same spacecraft. It compares these accuracies with those of current and future maps obtained from other data (conventional tracking, satellite-satellite tracking, etc.), and also with the spectra of various signals of geophysical interest.

  3. High-resolution MRI: in vivo histology?

    PubMed Central

    Bridge, Holly; Clare, Stuart

    2005-01-01

    For centuries scientists have been fascinated with the question of how the brain works. Investigators have looked at both where different functions are localized and how the anatomical microstructure varies across the brain surface. Here we discuss how advances in magnetic resonance imaging (MRI) have allowed in vivo visualization of the fine structure of the brain that was previously only visible in post-mortem brains. We present data showing the correspondence between definitions of the primary visual cortex defined anatomically using very high-resolution MRI and functionally using functional MRI. We consider how this technology can be applied to allow the investigation of brains that differ from normal, and what this ever-evolving technology may be able to reveal about in vivo brain structure in the next few years. PMID:16553313

  4. A high resolution ultraviolet Shuttle glow spectrograph

    NASA Technical Reports Server (NTRS)

    Carruthers, George R.

    1993-01-01

    The High Resolution Shuttle Glow Spectrograph-B (HRSGS-B) is a small payload being developed by the Naval Research Laboratory. It is intended for study of shuttle surface glow in the 180-400 nm near- and middle-ultraviolet wavelength range, with a spectral resolution of 0.2 nm. It will search for, among other possible features, the band systems of excited NO which result from surface-catalyzed combination of N and O. It may also detect O2 Hertzberg bands and N2 Vegard-Kaplan bands resulting from surface recombination. This wavelength range also includes possible N2+ and OH emissions. The HRSGS-B will be housed in a Get Away Special canister, mounted in the shuttle orbiter payload bay, and will observe the glow on the tail of the orbiter.

  5. High-resolution reconstruction for terahertz imaging.

    PubMed

    Xu, Li-Min; Fan, Wen-Hui; Liu, Jia

    2014-11-20

    We present a high-resolution (HR) reconstruction model and algorithms for terahertz imaging, taking advantage of super-resolution methodology and algorithms. The algorithms used include projection onto a convex sets approach, iterative backprojection approach, Lucy-Richardson iteration, and 2D wavelet decomposition reconstruction. Using the first two HR reconstruction methods, we successfully obtain HR terahertz images with improved definition and lower noise from four low-resolution (LR) 22×24 terahertz images taken from our homemade THz-TDS system at the same experimental conditions with 1.0 mm pixel. Using the last two HR reconstruction methods, we transform one relatively LR terahertz image to a HR terahertz image with decreased noise. This indicates potential application of HR reconstruction methods in terahertz imaging with pulsed and continuous wave terahertz sources.

  6. HIRIS - The High Resolution Imaging Spectrometer

    NASA Technical Reports Server (NTRS)

    Dozier, Jeff

    1988-01-01

    The High-Resolution Imaging Spectrometer (HIRIS) is a JPL facility instrument designed for NASA's Earth Observing System (Eos).It will have 10-nm wide spectral bands from 0.4-2.5 microns at 30 m spatial resolution over a 30 km swath. The spectral resolution allows identification of many minerals in rocks and soils, important algal pigments in oceans and inland waters, spectral changes associated with plant canopy biochemistry, composition of atmospheric aerosols, and grain size of snow and its contamination by absorbing impurities. The bands wil have 12-bit quantization over a dynamic range suitable for bright targets, such as snow. For targets of low brightness, such as water bodies, image-motion compensation will allow gains up to a factor of eight to increase signal-to-noise ratios. In the 824-km orbit altitude proposed for Eos, the crosstrack pointing capability will allow 4-5 views during a 16-day revisit cycle.

  7. Constructing a WISE High Resolution Galaxy Atlas

    NASA Astrophysics Data System (ADS)

    Jarrett, T. H.; Masci, F.; Tsai, C. W.; Petty, S.; Cluver, M.; Assef, Roberto J.; Benford, D.; Blain, A.; Bridge, C.; Donoso, E.; Eisenhardt, P.; Fowler, J.; Koribalski, B.; Lake, S.; Neill, James D.; Seibert, M.; Sheth, K.; Stanford, S.; Wright, E.

    2012-08-01

    After eight months of continuous observations, the Wide-field Infrared Survey Explorer (WISE) mapped the entire sky at 3.4 μm, 4.6 μm, 12 μm, and 22 μm. We have begun a dedicated WISE High Resolution Galaxy Atlas project to fully characterize large, nearby galaxies and produce a legacy image atlas and source catalog. Here we summarize the deconvolution techniques used to significantly improve the spatial resolution of WISE imaging, specifically designed to study the internal anatomy of nearby galaxies. As a case study, we present results for the galaxy NGC 1566, comparing the WISE enhanced-resolution image processing to that of Spitzer, Galaxy Evolution Explorer, and ground-based imaging. This is the first paper in a two-part series; results for a larger sample of nearby galaxies are presented in the second paper.

  8. Computer synthesis of high resolution electron micrographs

    NASA Technical Reports Server (NTRS)

    Nathan, R.

    1976-01-01

    Specimen damage, spherical aberration, low contrast and noisy sensors combine to prevent direct atomic viewing in a conventional electron microscope. The paper describes two methods for obtaining ultra-high resolution in biological specimens under the electron microscope. The first method assumes the physical limits of the electron objective lens and uses a series of dark field images of biological crystals to obtain direct information on the phases of the Fourier diffraction maxima; this information is used in an appropriate computer to synthesize a large aperture lens for a 1-A resolution. The second method assumes there is sufficient amplitude scatter from images recorded in focus which can be utilized with a sensitive densitometer and computer contrast stretching to yield fine structure image details. Cancer virus characterization is discussed as an illustrative example. Numerous photographs supplement the text.

  9. High-resolution adaptive spiking sonar.

    PubMed

    Alvarez, Fernando J; Kuc, Roman

    2009-05-01

    A new sonar system based on the conventional 6500 ranging module is presented that generates a sequence of spikes whose temporal density is related to the strength of the received echo. This system notably improves the resolution of a previous system by shortening the discharge cycle of the integrator included in the module. The operation is controlled by a PIC18F452 device, which can adapt the duration of the discharge to changing features of the echo, providing the system with a novel adaptive behavior. The performance of the new sensor is characterized and compared with that of the previous system by performing rotational scans of simple objects with different reflecting strengths. Some applications are suggested that exploit the high resolution and adaptability of this sensor.

  10. Constructing a WISE High Resolution Galaxy Atlas

    NASA Technical Reports Server (NTRS)

    Jarrett, T. H.; Masci, F.; Tsai, C. W.; Petty, S.; Cluver, M.; Assef, Roberto J.; Benford, D.; Blain, A.; Bridge, C.; Donoso, E.; Eisenhardt, P.; Fowler, J.; Koribalski, B.; Lake, S.; Neill, James D.; Seibert, M.; Stanford, S.; Wright, E.

    2012-01-01

    After eight months of continuous observations, the Wide-field Infrared Survey Explorer (WISE) mapped the entire sky at 3.4 micron, 4.6 micron, 12 micron, and 22 micron. We have begun a dedicated WISE High Resolution Galaxy Atlas project to fully characterize large, nearby galaxies and produce a legacy image atlas and source catalog. Here we summarize the deconvolution techniques used to significantly improve the spatial resolution of WISE imaging, specifically designed to study the internal anatomy of nearby galaxies. As a case study, we present results for the galaxy NGC 1566, comparing the WISE enhanced-resolution image processing to that of Spitzer, Galaxy Evolution Explorer, and ground-based imaging. This is the first paper in a two-part series; results for a larger sample of nearby galaxies are presented in the second paper.

  11. Limits of simulation based high resolution EBSD.

    PubMed

    Alkorta, Jon

    2013-08-01

    High resolution electron backscattered diffraction (HREBSD) is a novel technique for a relative determination of both orientation and stress state in crystals through digital image correlation techniques. Recent works have tried to use simulated EBSD patterns as reference patterns to achieve the absolute orientation and stress state of crystals. However, a precise calibration of the pattern centre location is needed to avoid the occurrence of phantom stresses. A careful analysis of the projective transformation involved in the formation of EBSD patterns has permitted to understand these phantom stresses. This geometrical analysis has been confirmed by numerical simulations. The results indicate that certain combinations of crystal strain states and sample locations (pattern centre locations) lead to virtually identical EBSD patterns. This ambiguity makes the problem of solving the absolute stress state of a crystal unfeasible in a single-detector configuration. PMID:23676453

  12. High-Resolution Anamorphic SPECT Imaging

    PubMed Central

    Durko, Heather L.; Barrett, Harrison H.; Furenlid, Lars R.

    2015-01-01

    We have developed a gamma-ray imaging system that combines a high-resolution silicon detector with two sets of movable, half-keel-edged copper-tungsten blades configured as crossed slits. These apertures can be positioned independently between the object and detector, producing an anamorphic image in which the axial and transaxial magnifications are not constrained to be equal. The detector is a 60 mm × 60 mm, one-millimeter-thick, one-megapixel silicon double-sided strip detector with a strip pitch of 59 μm. The flexible nature of this system allows the application of adaptive imaging techniques. We present system details; calibration, acquisition, and reconstruction methods; and imaging results. PMID:26160983

  13. High resolution solar X-ray studies

    NASA Technical Reports Server (NTRS)

    Blake, R. L.

    1974-01-01

    Two high resolution solar X-ray payloads and their launches on Aerobee rockets with pointing system are described. The payloads included 5 to 25A X-ray spectrometers, multiaperture X-ray cameras, and command box attitude control inflight by means of a television image radioed to ground. Spatial resolution ranged from five arc minutes to ten arc seconds and spectral resolution ranged from 500 to 3000. Several laboratory tasks were completed in order to achieve the desired resolution. These included (1) development of techniques to align grid collimators, (2) studies of the spectrometric properties of crystals, (3) measurements of the absorption coefficients of various materials used in X-ray spectrometers, (4) evaluation of the performance of multiaperture cameras, and (5) development of facilities.

  14. A simple, high efficiency, high resolution spectropolarimeter

    NASA Astrophysics Data System (ADS)

    Barden, Samuel C.

    2012-09-01

    A simple concept is described that uses volume phase holographic gratings as polarizing dispersers for a high efficiency, high resolution spectropolarimeter. Although the idea has previously been mentioned in the literature as possible, such a concept has not been explored in detail. Performance analysis is presented for a VPHG spectropolarimeter concept that could be utilized for both solar and night-time astronomy. Instrumental peak efficiency can approach 100% with spectral dispersions permitting R~200,000 spectral resolution with diffraction limited telescopes. The instrument has 3-channels: two dispersed image planes with orthogonal polarization and an undispersed image plane. The concept has a range of versatility where it could be configured (with appropriate half-wave plates) for slit-fed spectroscopy or without slits for snapshot/hyperspectral/tomographic spectroscopic imaging. Multiplex gratings could also be used for the simultaneous recording of two separate spectral bands or multiple instruments could be daisy chained with beam splitters for further spectral coverage.

  15. High Resolution Spectroscopy to Support Atmospheric Measurements

    NASA Technical Reports Server (NTRS)

    Venkataraman, Malathy Devi

    2006-01-01

    The major research activities performed during the cooperative agreement enhanced our spectroscopic knowledge of molecules of atmospheric interest such as H2O (water vapor), O3 (ozone), HCN (hydrogen cyanide), CH4 (methane), NO2 (nitrogen dioxide) and CO (carbon monoxide). The data required for the analyses were obtained from two different Fourier Transform Spectrometers (FTS); one of which is located at the National Solar Observatory (NSO) on Kitt Peak, Arizona and the other instrument is located at the Pacific Northwest National Laboratories (PNNL) at Richland, Washington. The data were analyzed using a modified multispectrum nonlinear least squares fitting algorithm developed by Dr. D. Chris Benner of the College of William and Mary. The results from these studies made significant improvements in the line positons and intensities for these molecules. The measurements of pressure broadening and pressure induced line shift coefficients and the temperature dependence of pressure broadening and pressure induced shift coefficients for hundreds of infrared transitions of HCN, CO3 CH4 and H2O were also performed during this period. Results from these studies have been used for retrievals of stratospheric gas concentration profiles from data collected by several Upper Atmospheric Research Satellite (UARS) infrared instruments as well as in the analysis of high resolution atmospheric spectra such as those acquired by space-based, ground-based, and various balloon- and aircraft-borne experiments. Our results made significant contributions in several updates of the HITRAN (HIgh resolution TRANsmission) spectral line parameters database. This database enjoys worldwide recognition in research involving diversified scientific fields. The research conducted during the period 2003-2006 has resulted in publications given in this paper. In addition to Journal publications, several oral and poster presentations were given at various Scientific conferences within the United States

  16. High resolution multimodal clinical ophthalmic imaging system.

    PubMed

    Mujat, Mircea; Ferguson, R Daniel; Patel, Ankit H; Iftimia, Nicusor; Lue, Niyom; Hammer, Daniel X

    2010-05-24

    We developed a multimodal adaptive optics (AO) retinal imager which is the first to combine high performance AO-corrected scanning laser ophthalmoscopy (SLO) and swept source Fourier domain optical coherence tomography (SSOCT) imaging modes in a single compact clinical prototype platform. Such systems are becoming ever more essential to vision research and are expected to prove their clinical value for diagnosis of retinal diseases, including glaucoma, diabetic retinopathy (DR), age-related macular degeneration (AMD), and retinitis pigmentosa. The SSOCT channel operates at a wavelength of 1 microm for increased penetration and visualization of the choriocapillaris and choroid, sites of major disease activity for DR and wet AMD. This AO system is designed for use in clinical populations; a dual deformable mirror (DM) configuration allows simultaneous low- and high-order aberration correction over a large range of refractions and ocular media quality. The system also includes a wide field (33 deg.) line scanning ophthalmoscope (LSO) for initial screening, target identification, and global orientation, an integrated retinal tracker (RT) to stabilize the SLO, OCT, and LSO imaging fields in the presence of lateral eye motion, and a high-resolution LCD-based fixation target for presentation of visual cues. The system was tested in human subjects without retinal disease for performance optimization and validation. We were able to resolve and quantify cone photoreceptors across the macula to within approximately 0.5 deg (approximately 100-150 microm) of the fovea, image and delineate ten retinal layers, and penetrate to resolve features deep into the choroid. The prototype presented here is the first of a new class of powerful flexible imaging platforms that will provide clinicians and researchers with high-resolution, high performance adaptive optics imaging to help guide therapies, develop new drugs, and improve patient outcomes.

  17. Ecological applications of high resolution spectrometry

    NASA Technical Reports Server (NTRS)

    Lawrence, William T.

    1989-01-01

    Future directions of NASA's space program plans include a significant effort at studying the Earth as a system of interrelated ecosystems. As part of NASA's Earth Observing System (Eos) Program a series of space platforms will be launched and operated to study the Earth with a variety of active and passive instruments. Several of the Eos instruments will be capable of imaging the planet's surface reflectance on a large number of very narrow portions of the solar spectrum. After the development of appropriate algorithms, this reflectance information will be used to determine key parameters about the structure and function of terrestrial and aquatic ecosystems and the pattern and processes of those systems across large areas of the globe. Algorithm development applicable to terrestrial systems will permit the inference of ecological processes from high resolution spectrometry data, similar to that to be forthcoming from the Eos mission. The first summer was spent working with tropical soils and relating their reflectance characteristics to particle size, iron content, and color. This summer the emphasis is on vegetation and work was begun with the Forest Ecosystems Dynamics Project in the Earth Resources Branch where both optical and radar characteristics of a mixed conifer/hardwood forest in Maine are being studied for use in a ecological modeling effort. A major series of aircraft overflights will take place throughout the summer. Laboratory and field spectrometers are used to measure the spectral reflectance of a hierarchy of vegetation from individual leaves to whole canopies for eventual modeling of their nutrient content using reflectance data. Key leaf/canopy parameters are being approximated including chlorophyll, nitrogen, phosphorus, water content, and leaf specific weight using high resolution spectrometry alone. Measurements are made of carbon exchange across the landscape for input to a spatial modeling effort to gauge production within the forest. A

  18. Pyramidal fractal dimension for high resolution images.

    PubMed

    Mayrhofer-Reinhartshuber, Michael; Ahammer, Helmut

    2016-07-01

    Fractal analysis (FA) should be able to yield reliable and fast results for high-resolution digital images to be applicable in fields that require immediate outcomes. Triggered by an efficient implementation of FA for binary images, we present three new approaches for fractal dimension (D) estimation of images that utilize image pyramids, namely, the pyramid triangular prism, the pyramid gradient, and the pyramid differences method (PTPM, PGM, PDM). We evaluated the performance of the three new and five standard techniques when applied to images with sizes up to 8192 × 8192 pixels. By using artificial fractal images created by three different generator models as ground truth, we determined the scale ranges with minimum deviations between estimation and theory. All pyramidal methods (PM) resulted in reasonable D values for images of all generator models. Especially, for images with sizes ≥1024×1024 pixels, the PMs are superior to the investigated standard approaches in terms of accuracy and computation time. A measure for the possibility to differentiate images with different intrinsic D values did show not only that the PMs are well suited for all investigated image sizes, and preferable to standard methods especially for larger images, but also that results of standard D estimation techniques are strongly influenced by the image size. Fastest results were obtained with the PDM and PGM, followed by the PTPM. In terms of absolute D values best performing standard methods were magnitudes slower than the PMs. Concluding, the new PMs yield high quality results in short computation times and are therefore eligible methods for fast FA of high-resolution images.

  19. Pyramidal fractal dimension for high resolution images

    NASA Astrophysics Data System (ADS)

    Mayrhofer-Reinhartshuber, Michael; Ahammer, Helmut

    2016-07-01

    Fractal analysis (FA) should be able to yield reliable and fast results for high-resolution digital images to be applicable in fields that require immediate outcomes. Triggered by an efficient implementation of FA for binary images, we present three new approaches for fractal dimension (D) estimation of images that utilize image pyramids, namely, the pyramid triangular prism, the pyramid gradient, and the pyramid differences method (PTPM, PGM, PDM). We evaluated the performance of the three new and five standard techniques when applied to images with sizes up to 8192 × 8192 pixels. By using artificial fractal images created by three different generator models as ground truth, we determined the scale ranges with minimum deviations between estimation and theory. All pyramidal methods (PM) resulted in reasonable D values for images of all generator models. Especially, for images with sizes ≥1024 ×1024 pixels, the PMs are superior to the investigated standard approaches in terms of accuracy and computation time. A measure for the possibility to differentiate images with different intrinsic D values did show not only that the PMs are well suited for all investigated image sizes, and preferable to standard methods especially for larger images, but also that results of standard D estimation techniques are strongly influenced by the image size. Fastest results were obtained with the PDM and PGM, followed by the PTPM. In terms of absolute D values best performing standard methods were magnitudes slower than the PMs. Concluding, the new PMs yield high quality results in short computation times and are therefore eligible methods for fast FA of high-resolution images.

  20. Pyramidal fractal dimension for high resolution images.

    PubMed

    Mayrhofer-Reinhartshuber, Michael; Ahammer, Helmut

    2016-07-01

    Fractal analysis (FA) should be able to yield reliable and fast results for high-resolution digital images to be applicable in fields that require immediate outcomes. Triggered by an efficient implementation of FA for binary images, we present three new approaches for fractal dimension (D) estimation of images that utilize image pyramids, namely, the pyramid triangular prism, the pyramid gradient, and the pyramid differences method (PTPM, PGM, PDM). We evaluated the performance of the three new and five standard techniques when applied to images with sizes up to 8192 × 8192 pixels. By using artificial fractal images created by three different generator models as ground truth, we determined the scale ranges with minimum deviations between estimation and theory. All pyramidal methods (PM) resulted in reasonable D values for images of all generator models. Especially, for images with sizes ≥1024×1024 pixels, the PMs are superior to the investigated standard approaches in terms of accuracy and computation time. A measure for the possibility to differentiate images with different intrinsic D values did show not only that the PMs are well suited for all investigated image sizes, and preferable to standard methods especially for larger images, but also that results of standard D estimation techniques are strongly influenced by the image size. Fastest results were obtained with the PDM and PGM, followed by the PTPM. In terms of absolute D values best performing standard methods were magnitudes slower than the PMs. Concluding, the new PMs yield high quality results in short computation times and are therefore eligible methods for fast FA of high-resolution images. PMID:27475069

  1. High-resolution mapping of protein sequence-function relationships.

    PubMed

    Fowler, Douglas M; Araya, Carlos L; Fleishman, Sarel J; Kellogg, Elizabeth H; Stephany, Jason J; Baker, David; Fields, Stanley

    2010-09-01

    We present a large-scale approach to investigate the functional consequences of sequence variation in a protein. The approach entails the display of hundreds of thousands of protein variants, moderate selection for activity and high-throughput DNA sequencing to quantify the performance of each variant. Using this strategy, we tracked the performance of >600,000 variants of a human WW domain after three and six rounds of selection by phage display for binding to its peptide ligand. Binding properties of these variants defined a high-resolution map of mutational preference across the WW domain; each position had unique features that could not be captured by a few representative mutations. Our approach could be applied to many in vitro or in vivo protein assays, providing a general means for understanding how protein function relates to sequence.

  2. Rapid identification and classification of bacteria by 16S rDNA restriction fragment melting curve analyses (RFMCA).

    PubMed

    Rudi, Knut; Kleiberg, Gro H; Heiberg, Ragnhild; Rosnes, Jan T

    2007-08-01

    The aim of this work was to evaluate restriction fragment melting curve analyses (RFMCA) as a novel approach for rapid classification of bacteria during food production. RFMCA was evaluated for bacteria isolated from sous vide food products, and raw materials used for sous vide production. We identified four major bacterial groups in the material analysed (cluster I-Streptococcus, cluster II-Carnobacterium/Bacillus, cluster III-Staphylococcus and cluster IV-Actinomycetales). The accuracy of RFMCA was evaluated by comparison with 16S rDNA sequencing. The strains satisfying the RFMCA quality filtering criteria (73%, n=57), with both 16S rDNA sequence information and RFMCA data (n=45) gave identical group assignments with the two methods. RFMCA enabled rapid and accurate classification of bacteria that is database compatible. Potential application of RFMCA in the food or pharmaceutical industry will include development of classification models for the bacteria expected in a given product, and then to build an RFMCA database as a part of the product quality control. PMID:17367680

  3. High Resolution Continuous Flow Analysis System for Polar Ice Cores

    NASA Astrophysics Data System (ADS)

    Dallmayr, Remi; Azuma, Kumiko; Yamada, Hironobu; Kjær, Helle Astrid; Vallelonga, Paul; Azuma, Nobuhiko; Takata, Morimasa

    2014-05-01

    35.636 kyr b2k 7), respectively. The results show the conductivity measured upstream and downstream of the debubbler. We will calculate the depth resolution of our system and compare it with earlier studies. 1) Bigler at al, "Optimization of High-Resolution Continuous Flow Analysis For Transient Climate Signals in Ice Cores". Environ. Sci. Technol. 2011, 45, 4483-4489 2) Kaufmann et al, "An Improved Continuous Flow Analysis System for High Resolution Field Measurements on Ice Cores". Environmental Environ. Sci. Technol. 2008, 42, 8044-8050 3) Gkinis, V., T. J. Popp, S. J. Johnsen and T, Blunier, 2010: A continuous stream flash evaporator for the calibration of an IR cavity ring down spectrometer for the isotopic analysis of water. Isotopes in Environmental and Health Studies, 46(4), 463-475. 4) McConnell et al, "Continuous ice-core chemical analyses using inductively coupled plasma mass spectrometry. Environ. Sci. Technol. 2002, 36, 7-11 5) Rhodes et al, "Continuous methane measurements from a late Holocene Greenland ice core : Atmospheric and in-situ signals" Earth and Planetary Science Letters. 2013, 368, 9-19 6) Breton et al, "Quantifying Signal Dispersion in a Hybrid Ice Core Melting System". Environ. Sci. Technol. 2012, 46, 11922-11928 7) Rasmussen et al, " A first chronology for the NEEM ice core". Climate of the Past. 2013, 9, 2967--3013

  4. Crusta: Visualizing High-resolution Global Data

    NASA Astrophysics Data System (ADS)

    Bernardin, T. S.; Kreylos, O.; Bowles, C. J.; Cowgill, E.; Hamann, B.; Kellogg, L. H.

    2009-12-01

    Virtual globes have become indispensable tools for visualizing, understanding and presenting data from Earth and other planetary bodies. The scientific community has invested much effort into exploiting existing globes to their fullest potential by refining and adapting their capabilities to better satisfy specific needs. For example, Google Earth provides users with the ability to view hillshade images derived from airborne LiDAR data such as the 2007 Northern California GeoEarthScope data. However, because most available globes were not designed with the specific needs of geoscientists in mind, shortcomings are becoming increasingly evident in geoscience applications such as terrain visualization. In particular, earth scientists struggle to visualize digital elevation models with both high spatial resolution (0.5 - 1 square meters per sample) and large extent (>2000 square kilometers), such as those obtained with airborne LiDAR. To address the specific earth science need of real-time terrain visualization of LiDAR data, we are developing Crusta as part of a close collaboration involving earth and computer scientists. Crusta is a new virtual globe that differs from widely used globes by both providing accurate global data representation and the ability to easily visualize custom topographic and image data. As a result, Crusta enables real-time, interactive visualization of high resolution digital elevation data spanning thousands of square kilometers, such as the complete 2007 Northern California GeoEarthScope airborne LiDAR data set. To implement an accurate data representation and avoid distortion of the display at the poles, where other projections have singularities, Crusta represents the globe as a thirty-sided polyhedron. Each side of this polyhedron can be subdivided to an arbitrarily fine grid on the surface of the globe, which allows Crusta to accommodate input data of arbitrary resolution ranging from global (e.g., Blue Marble) to local (e.g., a tripod

  5. Superconducting High Resolution Fast-Neutron Spectrometers

    SciTech Connect

    Hau, Ionel Dragos

    2006-01-01

    Superconducting high resolution fast-neutron calorimetric spectrometers based on 6LiF and TiB{sub 2} absorbers have been developed. These novel cryogenic spectrometers measure the temperature rise produced in exothermal (n, α) reactions with fast neutrons in 6Li and 10B-loaded materials with heat capacity C operating at temperatures T close to 0.1 K. Temperature variations on the order of 0.5 mK are measured with a Mo/Cu thin film multilayer operated in the transition region between its superconducting and its normal state. The advantage of calorimetry for high resolution spectroscopy is due to the small phonon excitation energies kBT on the order of μeV that serve as signal carriers, resulting in an energy resolution ΔE ~ (kBT2C)1/2, which can be well below 10 keV. An energy resolution of 5.5 keV has been obtained with a Mo/Cu superconducting sensor and a TiB2 absorber using thermal neutrons from a 252Cf neutron source. This resolution is sufficient to observe the effect of recoil nuclei broadening in neutron spectra, which has been related to the lifetime of the first excited state in 7Li. Fast-neutron spectra obtained with a 6Li-enriched LiF absorber show an energy resolution of 16 keV FWHM, and a response in agreement with the 6Li(n, α)3H reaction cross section and Monte Carlo simulations for energies up to several MeV. The energy resolution of order of a few keV makes this novel instrument applicable to fast-neutron transmission spectroscopy based on the unique elemental signature provided by the neutron absorption and scattering resonances. The optimization of the energy resolution based on analytical and numerical models of the detector response is discussed in the context of these applications.

  6. A melting pot of multicontinental mtDNA lineages in admixed Venezuelans.

    PubMed

    Gómez-Carballa, Alberto; Ignacio-Veiga, Ana; Alvarez-Iglesias, Vanesa; Pastoriza-Mourelle, Ana; Ruíz, Yarimar; Pineda, Lennie; Carracedo, Angel; Salas, Antonio

    2012-01-01

    The arrival of Europeans in Colonial and post-Colonial times coupled with the forced introduction of sub-Saharan Africans have dramatically changed the genetic background of Venezuela. The main aim of the present study was to evaluate, through the study of mitochondrial DNA (mtDNA) variation, the extent of admixture and the characterization of the most likely continental ancestral sources of present-day urban Venezuelans. We analyzed two admixed populations that have experienced different demographic histories, namely, Caracas (n = 131) and Pueblo Llano (n = 219). The native American component of admixed Venezuelans accounted for 80% (46% haplogroup [hg] A2, 7% hg B2, 21% hg C1, and 6% hg D1) of all mtDNAs; while the sub-Saharan and European contributions made up ∼10% each, indicating that Trans-Atlantic immigrants have only partially erased the native American nature of Venezuelans. A Bayesian-based model allowed the different contributions of European countries to admixed Venezuelans to be disentangled (Spain: ∼38.4%, Portugal: ∼35.5%, Italy: ∼27.0%), in good agreement with the documented history. Seventeen entire mtDNA genomes were sequenced, which allowed five new native American branches to be discovered. B2j and B2k, are supported by two different haplotypes and control region data, and their coalescence ages are 3.9 k.y. (95% C.I. 0-7.8) and 2.6 k.y. (95% C.I. 0.1-5.2), respectively. The other clades were exclusively observed in Pueblo Llano and they show the fingerprint of strong recent genetic drift coupled with severe historical consanguinity episodes that might explain the high prevalence of certain Mendelian and complex multi-factorial diseases in this region. PMID:22120584

  7. High resolution scanning electron microscopy of plasmodesmata.

    PubMed

    Brecknock, Sarah; Dibbayawan, Teresa P; Vesk, Maret; Vesk, Peter A; Faulkner, Christine; Barton, Deborah A; Overall, Robyn L

    2011-10-01

    Symplastic transport occurs between neighbouring plant cells through functionally and structurally dynamic channels called plasmodesmata (PD). Relatively little is known about the composition of PD or the mechanisms that facilitate molecular transport into neighbouring cells. While transmission electron microscopy (TEM) provides 2-dimensional information about the structural components of PD, 3-dimensional information is difficult to extract from ultrathin sections. This study has exploited high-resolution scanning electron microscopy (HRSEM) to reveal the 3-dimensional morphology of PD in the cell walls of algae, ferns and higher plants. Varied patterns of PD were observed in the walls, ranging from uniformly distributed individual PD to discrete clusters. Occasionally the thick walls of the giant alga Chara were fractured, revealing the surface morphology of PD within. External structures such as spokes, spirals and mesh were observed surrounding the PD. Enzymatic digestions of cell wall components indicate that cellulose or pectin either compose or stabilise the extracellular spokes. Occasionally, the PD were fractured open and desmotubule-like structures and other particles were observed in their central regions. Our observations add weight to the argument that Chara PD contain desmotubules and are morphologically similar to higher plant PD.

  8. High-resolution imaging using endoscopic holography

    NASA Astrophysics Data System (ADS)

    Bjelkhagen, Hans I.

    1990-08-01

    Endoscopic holography or endoholography combines the features of endoscopy and holography. The purpose of endoholographic imaging is to provide the physician with a unique means of extending diagnosis by providing a life-like record of tissue. Endoholographic recording will provide means for microscopic examination of tissue and in some cases may obviate the need to excise specimens for biopsy. In this method holograms which have the unique properties of three-dimensionality large focal depth and high resolution are made with a newly designed endoscope. The endoscope uses a single-mode optical fiber for illumination and single-beam reflection holograms are recorded in close contact with the tissue at the distal end of the endoscope. The holograms are viewed under a microscope. By using the proper combinations of dyes for staining specific tissue types with various wavelengths of laser illumination increased contrast on the cellular level can be obtained. Using dyes such as rose bengal in combination with the 514. 5 nm line of an argon ion laser and trypan blue or methylene blue with the 647. 1 nm line of a krypton ion laser holograms of the stained colon of a dog showed the architecture of the colon''s columnar epithelial cells. It is hoped through chronological study using this method in-vivo an increased understanding of the etiology and pathology of diseases such as Crohn''s diseases colitis proctitis and several different forms of cancer will help to their control. 1.

  9. Holographic high-resolution endoscopic image recording

    NASA Astrophysics Data System (ADS)

    Bjelkhagen, Hans I.

    1991-03-01

    Endoscopic holography or endoholography combines the features of endoscopy and holography. The purpose of endoholographic imaging is to provide the physician with a unique means of extending diagnosis by providing a life-like record of tissue. Endoholographic recording will provide means for microscopic examination of tissue and in some cases may obviate the need to excise specimens for biopsy. In this method holograms which have the unique properties of three-dimensionality large focal depth and high resolution are made with a newly designed endoscope. The endoscope uses a single-mode optical fiber for illumination and single-beam reflection holograms are recorded in close contact with the tissue at the distal end of the endoscope. The holograms are viewed under a microscope. By using the proper combinations of dyes for staining specific tissue types with various wavelengths of laser illumination increased contrast on the cellular level can be obtained. Using dyes such as rose bengal in combination with the 514. 5 nm line of an argon ion laser and trypan blue or methylene blue with the 647. 1 nm line of a krypton ion laser holograms of the stained colon of a dog showed the architecture of the colon''s columnar epithelial cells. It is hoped through chronological study using this method in-vivo an increased understanding of the etiology and pathology of diseases such as Crohn''s diseases colitis proctitis and several different forms of cancer will help

  10. High-resolution light microscopy of nanoforms

    NASA Astrophysics Data System (ADS)

    Vodyanoy, Vitaly; Pustovyy, Oleg; Vainrub, Arnold

    2007-09-01

    We developed a high resolution light imaging system. Diffraction gratings with 100 nm width lines as well as less than 100 nm size features of different-shaped objects are clearly visible on a calibrated microscope test slide (Vainrub et al., Optics Letters, 2006, 31, 2855). The two-point resolution increase results from a known narrowing of the central diffraction peak for the annular aperture. Better visibility and advanced contrast of the smallest features in the image are due to enhancement of high spatial frequencies in the optical transfer function. The imaging system is portable, low energy, and battery operated. It has been adapted to use in both transmitting and reflecting light. It is particularly applicable for motile nanoform systems where structure and functions can be depicted in real time. We have isolated micrometer and submicrometer particles, termed proteons, from human and animal blood. Proteons form by reversible seeded aggregation of proteins around proteon nucleating centers (PNCs). PNCs are comprised of 1-2nm metallic nanoclusters containing 40-300 atoms. Proteons are capable of spontaneous assembling into higher nanoform systems assuming structure of complicated topology. The arrangement of complex proteon system mimics the structure of a small biological cell. It has structures that imitate membrane and nucleolus or nuclei. Some of these nanoforms are motile. They interact and divide. Complex nanoform systems can spontaneously reduce to simple proteons. The physical properties of these nanoforms could shed some light on the properties of early life forms or forms at extreme conditions.

  11. High-resolution CCD imaging alternatives

    NASA Astrophysics Data System (ADS)

    Brown, D. L.; Acker, D. E.

    1992-08-01

    High resolution CCD color cameras have recently stimulated the interest of a large number of potential end-users for a wide range of practical applications. Real-time High Definition Television (HDTV) systems are now being used or considered for use in applications ranging from entertainment program origination through digital image storage to medical and scientific research. HDTV generation of electronic images offers significant cost and time-saving advantages over the use of film in such applications. Further in still image systems electronic image capture is faster and more efficient than conventional image scanners. The CCD still camera can capture 3-dimensional objects into the computing environment directly without having to shoot a picture on film develop it and then scan the image into a computer. 2. EXTENDING CCD TECHNOLOGY BEYOND BROADCAST Most standard production CCD sensor chips are made for broadcast-compatible systems. One popular CCD and the basis for this discussion offers arrays of roughly 750 x 580 picture elements (pixels) or a total array of approximately 435 pixels (see Fig. 1). FOR. A has developed a technique to increase the number of available pixels for a given image compared to that produced by the standard CCD itself. Using an inter-lined CCD with an overall spatial structure several times larger than the photo-sensitive sensor areas each of the CCD sensors is shifted in two dimensions in order to fill in spatial gaps between adjacent sensors.

  12. High resolution animated scenes from stills.

    PubMed

    Lin, Zhouchen; Wang, Lifeng; Wang, Yunbo; Kang, Sing Bing; Fang, Tian

    2007-01-01

    Current techniques for generating animated scenes involve either videos (whose resolution is limited) or a single image (which requires a significant amount of user interaction). In this paper, we describe a system that allows the user to quickly and easily produce a compelling-looking animation from a small collection of high resolution stills. Our system has two unique features. First, it applies an automatic partial temporal order recovery algorithm to the stills in order to approximate the original scene dynamics. The output sequence is subsequently extracted using a second-order Markov Chain model. Second, a region with large motion variation can be automatically decomposed into semiautonomous regions such that their temporal orderings are softly constrained. This is to ensure motion smoothness throughout the original region. The final animation is obtained by frame interpolation and feathering. Our system also provides a simple-to-use interface to help the user to fine-tune the motion of the animated scene. Using our system, an animated scene can be generated in minutes. We show results for a variety of scenes. PMID:17356221

  13. High-resolution microwave images of Saturn

    NASA Technical Reports Server (NTRS)

    Grossman, A. W.; Muhleman, D. O.; Berge, G. L.

    1989-01-01

    An analysis of high-resolution microwave images of Saturn and Saturn's individual rings is presented. Radio interferometric observations of Saturn taken at the Very Large Array in New Mexico at wavelengths of 2 and 6 centimeters reveal interesting new features in both the atmosphere and rings. The resulting maps show an increase in brightness temperature of about 3 K from equator to pole at both wavelengths, while the 6-centimeter map shows a bright band at northern midlatitudes. The data are consistent with a radiative transfer model of the atmosphere that constrains the well-mixed, fully saturated, NH3 mixing ratio to be 0.00012 in a region just below the NH3 clouds, while the observed bright band indicates a 25 percent relative decrease of NH3 in northern midlatitudes. Brightness temperatures for the classical rings are presented. Ring brightness shows a variation with azimuth and is linearly polarized at an average value of about 5 percent. The variations in ring polarization suggest that at least 20 percent of the ring brightness is the result of a single scattering process.

  14. Laser wavelength comparison by high resolution interferometry.

    PubMed

    Layer, H P; Deslattes, R D; Schweitzer, W G

    1976-03-01

    High resolution interferometry has been used to determine the wavelength ratio between two molecularly stabilized He-Ne lasers, one locked to a methane absorption at 3.39 microm and the other locked to the k peak of (129)I(2) at 633 nm. An optical beat frequency technique gave fractional orders while a microwave sideband method yielded the integer parts. Conventional (third derivative) peak seeking servoes stabilized both laser and cavity lengths. Reproducibility of the electronic control system and optics was a few parts in 10(12), while systematic errors associated with curvature of the cavity mirrors limited the accuracy of the wavelength ratio measurement to 2 parts in 10(10). The measured wavelength ratio of the methane stabilized He-Ne laser at 3.39 microm [P(7) line, nu(3) band] to the (129)I(2) (k peak) stabilized He-Ne laser at 633 nm was 5.359 049 260 6 (0.000 2 ppm). This ratio agrees with that calculated from the (lower accuracy) results of earlier wavelength measurements made relative to the (86)Kr standard. Its higher accuracy thus permits a provisional extension of the frequency scale based on the cesium oscillator into the visible spectrum.

  15. High-resolution microwave images of saturn.

    PubMed

    Grossman, A W; Muhleman, D O; Berge, G L

    1989-09-15

    An analysis of high-resolution microwave images of Saturn and Saturn's individual rings is presented. Radio interferometric observations of Saturn taken at the Very Large Array in New Mexico at wavelengths of 2 and 6 centimeters reveal interesting new features in both the atmosphere and rings. The resulting maps show an increase in brightness temperature of about 3 K from equator to pole at both wavelengths, while the 6-centimeter map shows a bright band at northern mid-latitudes. The data are consistent with a radiative transfer model of the atmosphere that constrains the well-mixed, fully saturated, NH(3) mixing ratio to be 1.2 x 10(-4) in a region just below the NH(3) clouds, while the observed bright band indicates a 25 percent relative decrease of NH(3) in northern mid-latitudes. Brightness temperatures for the classical rings are presented. Ring brightness shows a variation with azimuth and is linearly polarized at an average value of about 5 percent. The variations in ring polarization suggest that at least 20 percent of the ring brightness is the result of a single scattering process.

  16. Potential High Resolution Dosimeters For MRT

    NASA Astrophysics Data System (ADS)

    Bräuer-Krisch, E.; Rosenfeld, A.; Lerch, M.; Petasecca, M.; Akselrod, M.; Sykora, J.; Bartz, J.; Ptaszkiewicz, M.; Olko, P.; Berg, A.; Wieland, M.; Doran, S.; Brochard, T.; Kamlowski, A.; Cellere, G.; Paccagnella, A.; Siegbahn, E. A.; Prezado, Y.; Martinez-Rovira, I.; Bravin, A.; Dusseau, L.; Berkvens, P.

    2010-07-01

    Microbeam Radiation Therapy (MRT) uses highly collimated, quasi-parallel arrays of X-ray microbeams of 50-600 keV, produced by 2nd and 3rd generation synchrotron sources, such as the National Synchrotron Light Source (NSLS) in the U.S., and the European Synchrotron Radiation Facility (ESRF) in France, respectively. High dose rates are necessary to deliver therapeutic doses in microscopic volumes, to avoid spreading of the microbeams by cardiosynchronous movement of the tissues. A small beam divergence and a filtered white beam spectrum in the energy range between 30 and 250 keV results in the advantage of steep dose gradients with a sharper penumbra than that produced in conventional radiotherapy. MRT research over the past 20 years has allowed a vast number of results from preclinical trials on different animal models, including mice, rats, piglets and rabbits. Microbeams in the range between 10 and 100 micron width show an unprecedented sparing of normal radiosensitive tissues as well as preferential damage to malignant tumor tissues. Typically, MRT uses arrays of narrow (˜25-100 micron-wide) microplanar beams separated by wider (100-400 microns centre-to-centre, c-t-c) microplanar spaces. We note that thicker microbeams of 0.1-0.68 mm used by investigators at the NSLS are still called microbeams, although some invesigators in the community prefer to call them minibeams. This report, however, limits it discussion to 25-100 μm microbeams. Peak entrance doses of several hundreds of Gy are surprisingly well tolerated by normal tissues. High resolution dosimetry has been developed over the last two decades, but typical dose ranges are adapted to dose delivery in conventional Radiation Therapy (RT). Spatial resolution in the sub-millimetric range has been achieved, which is currently required for quality assurance measurements in Gamma-knife RT. Most typical commercially available detectors are not suitable for MRT applications at a dose rate of 16000 Gy/s, micron

  17. Large Scale, High Resolution, Mantle Dynamics Modeling

    NASA Astrophysics Data System (ADS)

    Geenen, T.; Berg, A. V.; Spakman, W.

    2007-12-01

    To model the geodynamic evolution of plate convergence, subduction and collision and to allow for a connection to various types of observational data, geophysical, geodetical and geological, we developed a 4D (space-time) numerical mantle convection code. The model is based on a spherical 3D Eulerian fem model, with quadratic elements, on top of which we constructed a 3D Lagrangian particle in cell(PIC) method. We use the PIC method to transport material properties and to incorporate a viscoelastic rheology. Since capturing small scale processes associated with localization phenomena require a high resolution, we spend a considerable effort on implementing solvers suitable to solve for models with over 100 million degrees of freedom. We implemented Additive Schwartz type ILU based methods in combination with a Krylov solver, GMRES. However we found that for problems with over 500 thousend degrees of freedom the convergence of the solver degraded severely. This observation is known from the literature [Saad, 2003] and results from the local character of the ILU preconditioner resulting in a poor approximation of the inverse of A for large A. The size of A for which ILU is no longer usable depends on the condition of A and on the amount of fill in allowed for the ILU preconditioner. We found that for our problems with over 5×105 degrees of freedom convergence became to slow to solve the system within an acceptable amount of walltime, one minute, even when allowing for considerable amount of fill in. We also implemented MUMPS and found good scaling results for problems up to 107 degrees of freedom for up to 32 CPU¡¯s. For problems with over 100 million degrees of freedom we implemented Algebraic Multigrid type methods (AMG) from the ML library [Sala, 2006]. Since multigrid methods are most effective for single parameter problems, we rebuild our model to use the SIMPLE method in the Stokes solver [Patankar, 1980]. We present scaling results from these solvers for 3D

  18. Potential High Resolution Dosimeters For MRT

    SciTech Connect

    Braeuer-Krisch, E.; Brochard, T.; Prezado, Y.; Bravin, A.; Berkvens, P.; Rosenfeld, A.; Lerch, M.; Petasecca, M.; Akselrod, M.; Sykora, J.; Bartz, J.; Ptaszkiewicz, M.; Olko, P.; Berg, A.; Wieland, M.; Doran, S.; Kamlowski, A.; Cellere, G.

    2010-07-23

    Microbeam Radiation Therapy (MRT) uses highly collimated, quasi-parallel arrays of X-ray microbeams of 50-600 keV, produced by 2nd and 3rd generation synchrotron sources, such as the National Synchrotron Light Source (NSLS) in the U.S., and the European Synchrotron Radiation Facility (ESRF) in France, respectively. High dose rates are necessary to deliver therapeutic doses in microscopic volumes, to avoid spreading of the microbeams by cardiosynchronous movement of the tissues. A small beam divergence and a filtered white beam spectrum in the energy range between 30 and 250 keV results in the advantage of steep dose gradients with a sharper penumbra than that produced in conventional radiotherapy. MRT research over the past 20 years has allowed a vast number of results from preclinical trials on different animal models, including mice, rats, piglets and rabbits. Microbeams in the range between 10 and 100 micron width show an unprecedented sparing of normal radiosensitive tissues as well as preferential damage to malignant tumor tissues. Typically, MRT uses arrays of narrow ({approx}25-100 micron-wide) microplanar beams separated by wider (100-400 microns centre-to-centre, c-t-c) microplanar spaces. We note that thicker microbeams of 0.1-0.68 mm used by investigators at the NSLS are still called microbeams, although some invesigators in the community prefer to call them minibeams. This report, however, limits it discussion to 25-100 {mu}m microbeams. Peak entrance doses of several hundreds of Gy are surprisingly well tolerated by normal tissues. High resolution dosimetry has been developed over the last two decades, but typical dose ranges are adapted to dose delivery in conventional Radiation Therapy (RT). Spatial resolution in the sub-millimetric range has been achieved, which is currently required for quality assurance measurements in Gamma-knife RT. Most typical commercially available detectors are not suitable for MRT applications at a dose rate of 16000 Gy

  19. High-Resolution Radar Imagery of Mars

    NASA Astrophysics Data System (ADS)

    Harmon, John K.; Nolan, M. C.

    2009-09-01

    We present high-resolution radar images of Mars obtained during the 2005 and 2007 oppositions. The images were constructed from long-code delay-Doppler observations made with the Arecibo S-band (13-cm) radar. The average image resolution of 3 km represented a better than order-of-magnitude improvement over pre-upgrade Arecibo imagery of the planet. Images of depolarized reflectivity (an indicator primarily of wavelength-scale surface roughness) show the same bright volcanic flow features seen in earlier imagery, but with much finer detail. A new image of the Elysium region shows fine detail in the radar-bright channels of Athabasca Vallis, Marte Vallis, and Grjota Vallis. The new images of Tharsis and Olympus Mons also show a complex array of radar-bright and radar-dark features. Southern Amazonis exhibits some of the most complex and puzzling radar-bright structure on the planet. Another curiosity is the Chryse/Xanthe/Channels region, where we find some radar-bright features in or adjacent to fluvial chaos structures. Chryse/Xanthe is also the only region of Mars showing radar-bright craters (which are rare on Mars but common on the Moon and Mercury). We also obtained the first delay-Doppler image showing the enhanced backscatter from the residual south polar ice cap. In addition to the depolarized imagery, we were able to make the first delay-Doppler images of the circular polarization ratio (an important diagnostic for surface roughness texture). We find that vast areas of the radar-bright volcanic regions have polarization ratios close to unity. Such high ratios are rare for terrestrial lava flows and only seen for extremely blocky surfaces giving high levels of multiple scattering.

  20. Toward high-resolution optoelectronic retinal prosthesis

    NASA Astrophysics Data System (ADS)

    Palanker, Daniel; Huie, Philip; Vankov, Alexander; Asher, Alon; Baccus, Steven

    2005-04-01

    It has been already demonstrated that electrical stimulation of retina can produce visual percepts in blind patients suffering from macular degeneration and retinitis pigmentosa. Current retinal implants provide very low resolution (just a few electrodes), while several thousand pixels are required for functional restoration of sight. We present a design of the optoelectronic retinal prosthetic system that can activate a retinal stimulating array with pixel density up to 2,500 pix/mm2 (geometrically corresponding to a visual acuity of 20/80), and allows for natural eye scanning rather than scanning with a head-mounted camera. The system operates similarly to "virtual reality" imaging devices used in military and medical applications. An image from a video camera is projected by a goggle-mounted infrared LED-LCD display onto the retina, activating an array of powered photodiodes in the retinal implant. Such a system provides a broad field of vision by allowing for natural eye scanning. The goggles are transparent to visible light, thus allowing for simultaneous utilization of remaining natural vision along with prosthetic stimulation. Optical control of the implant allows for simple adjustment of image processing algorithms and for learning. A major prerequisite for high resolution stimulation is the proximity of neural cells to the stimulation sites. This can be achieved with sub-retinal implants constructed in a manner that directs migration of retinal cells to target areas. Two basic implant geometries are described: perforated membranes and protruding electrode arrays. Possibility of the tactile neural stimulation is also examined.

  1. Decadal prediction with a high resolution model

    NASA Astrophysics Data System (ADS)

    Monerie, Paul-Arthur; Valcke, Sophie; Terray, Laurent; Moine, Marie-Pierre

    2016-04-01

    The ability of a high resolution coupled atmosphere-ocean general circulation model (with a horizontal resolution of the quarter degree in the ocean and of about 50 km in the atmosphere) to predict the annual means of temperature, precipitation, sea-ice volume and extent is assessed. Reasonable skill in predicting sea surface temperatures and surface air temperature is obtained, especially over the North Atlantic, the tropical Atlantic and the Indian Ocean. The skill in predicting precipitations is weaker and not significant. The Sea Ice Extent and volume are also reasonably predicted in winter (March) and summer (September). It is however argued that the skill is mainly due to the atmosphere feeding in well-mixed GHGs. The mid-90's subpolar gyre warming is assessed. The model simulates a warming of the North Atlantic Ocean, associated with an increase of the meridional heat transport, a strengthening of the North Atlantic current and a deepening of the mixed layer over the Labrador Sea. The atmosphere plays a role in the warming through a modulation of the North Atlantic Oscillation and a shrinking of the subpolar gyre. At the 3-8 years lead-time, a negative anomaly of pressure, located south of the subpolar gyre is associated with the wind speed decrease over the subpolar gyre. It prevents oceanic heat-loss and favors the northward move, from the subtropical to the subpolar gyre, of anomalously warm and salty water, leading to its warming. We finally argued that the subpolar gyre warming is triggered by the ocean dynamic but the atmosphere can contributes to its sustaining. This work is realised in the framework of the EU FP7 SPECS Project.

  2. High-resolution phylogenetic microbial community profiling

    SciTech Connect

    Singer, Esther; Coleman-Derr, Devin; Bowman, Brett; Schwientek, Patrick; Clum, Alicia; Copeland, Alex; Ciobanu, Doina; Cheng, Jan-Fang; Gies, Esther; Hallam, Steve; Tringe, Susannah; Woyke, Tanja

    2014-03-17

    The representation of bacterial and archaeal genome sequences is strongly biased towards cultivated organisms, which belong to merely four phylogenetic groups. Functional information and inter-phylum level relationships are still largely underexplored for candidate phyla, which are often referred to as microbial dark matter. Furthermore, a large portion of the 16S rRNA gene records in the GenBank database are labeled as environmental samples and unclassified, which is in part due to low read accuracy, potential chimeric sequences produced during PCR amplifications and the low resolution of short amplicons. In order to improve the phylogenetic classification of novel species and advance our knowledge of the ecosystem function of uncultivated microorganisms, high-throughput full length 16S rRNA gene sequencing methodologies with reduced biases are needed. We evaluated the performance of PacBio single-molecule real-time (SMRT) sequencing in high-resolution phylogenetic microbial community profiling. For this purpose, we compared PacBio and Illumina metagenomic shotgun and 16S rRNA gene sequencing of a mock community as well as of an environmental sample from Sakinaw Lake, British Columbia. Sakinaw Lake is known to contain a large age of microbial species from candidate phyla. Sequencing results show that community structure based on PacBio shotgun and 16S rRNA gene sequences is highly similar in both the mock and the environmental communities. Resolution power and community representation accuracy from SMRT sequencing data appeared to be independent of GC content of microbial genomes and was higher when compared to Illumina-based metagenome shotgun and 16S rRNA gene (iTag) sequences, e.g. full-length sequencing resolved all 23 OTUs in the mock community, while iTags did not resolve closely related species. SMRT sequencing hence offers various potential benefits when characterizing uncharted microbial communities.

  3. High Resolution Global View of Io

    NASA Technical Reports Server (NTRS)

    1997-01-01

    Io, the most volcanic body in the solar system is seen in the highest resolution obtained to date by NASA's Galileo spacecraft. The smallest features that can be discerned are 2.5 kilometers in size. There are rugged mountains several kilometers high, layered materials forming plateaus, and many irregular depressions called volcanic calderas. Several of the dark, flow-like features correspond to hot spots, and may be active lava flows. There are no landforms resembling impact craters, as the volcanism covers the surface with new deposits much more rapidly than the flux of comets and asteroids can create large impact craters. The picture is centered on the side of Io that always faces away from Jupiter; north is to the top.

    Color images acquired on September 7, 1996 have been merged with higher resolution images acquired on November 6, 1996 by the Solid State Imaging (CCD) system aboard NASA's Galileo spacecraft. The color is composed of data taken, at a range of 487,000 kilometers, in the near-infrared, green, and violet filters and has been enhanced to emphasize the extraordinary variations in color and brightness that characterize Io's face. The high resolution images were obtained at ranges which varied from 245,719 kilometers to 403,100 kilometers.

    Launched in October 1989, Galileo entered orbit around Jupiter on December 7, 1995. The spacecraft's mission is to conduct detailed studies of the giant planet, its largest moons and the Jovian magnetic environment. The Jet Propulsion Laboratory, Pasadena, CA manages the mission for NASA's Office of Space Science, Washington, DC.

    This image and other images and data received from Galileo are posted on the World Wide Web, on the Galileo mission home page at URL http://galileo.jpl.nasa.gov. Background information and educational context for the images can be found at URL http://www.jpl.nasa.gov/galileo/sepo

  4. High Resolution Airborne Shallow Water Mapping

    NASA Astrophysics Data System (ADS)

    Steinbacher, F.; Pfennigbauer, M.; Aufleger, M.; Ullrich, A.

    2012-07-01

    In order to meet the requirements of the European Water Framework Directive (EU-WFD), authorities face the problem of repeatedly performing area-wide surveying of all kinds of inland waters. Especially for mid-sized or small rivers this is a considerable challenge imposing insurmountable logistical efforts and costs. It is therefore investigated if large-scale surveying of a river system on an operational basis is feasible by employing airborne hydrographic laser scanning. In cooperation with the Bavarian Water Authority (WWA Weilheim) a pilot project was initiated by the Unit of Hydraulic Engineering at the University of Innsbruck and RIEGL Laser Measurement Systems exploiting the possibilities of a new LIDAR measurement system with high spatial resolution and high measurement rate to capture about 70 km of riverbed and foreland for the river Loisach in Bavaria/Germany and the estuary and parts of the shoreline (about 40km in length) of lake Ammersee. The entire area surveyed was referenced to classic terrestrial cross-section surveys with the aim to derive products for the monitoring and managing needs of the inland water bodies forced by the EU-WFD. The survey was performed in July 2011 by helicopter and airplane and took 3 days in total. In addition, high resolution areal images were taken to provide an optical reference, offering a wide range of possibilities on further research, monitoring, and managing responsibilities. The operating altitude was about 500 m to maintain eye-safety, even for the aided eye, the airspeed was about 55 kts for the helicopter and 75 kts for the aircraft. The helicopter was used in the alpine regions while the fixed wing aircraft was used in the plains and the urban area, using appropriate scan rates to receive evenly distributed point clouds. The resulting point density ranged from 10 to 25 points per square meter. By carefully selecting days with optimum water quality, satisfactory penetration down to the river bed was achieved

  5. High Resolution Velocity Structure in Eastern Turkey

    SciTech Connect

    Pasyanos, M; Gok, R; Zor, E; Walter, W

    2004-09-03

    We investigate the crustal and upper mantle structure of eastern Turkey where the Anatolian, Arabian and Eurasian Plates meet and form a complex tectonic structure. The Bitlis suture is a continental collision zone between the Anatolian plateau and the Arabian plate. Broadband data available through the Eastern Turkey Seismic Experiment (ETSE) provided a unique opportunity for studying the high resolution velocity structure. Zor et al. found an average 46 km thick crust in Anatolian plateau using six-layered grid search inversion of the ETSE receiver functions. Receiver functions are sensitive to the velocity contrast of interfaces and the relative travel time of converted and reverberated waves between those interfaces. The interpretation of receiver function alone with many-layered parameterization may result in an apparent depth-velocity tradeoff. In order to improve previous velocity model, we employed the joint inversion method with many layered parameterization of Julia et al. (2000) to the ETSE receiver functions. In this technique, the receiver function and surface-wave observations are combined into a single algebraic equation and each data set is weighted by an estimate of the uncertainty in the observations. We consider azimuthal changes of receiver functions and have stacked them into different groups. We calculated the receiver functions using iterative time-domain deconvolution technique and surface wave group velocity dispersion curves between 10-100 sec. We are making surface wave dispersion measurements at the ETSE stations and have incorporated them into a regional group velocity model. Preliminary results indicate a strong trend in the long period group velocity in the northeast. This indicates slow upper mantle velocities in the region consistent with Pn, Sn and receiver function results. We started with both the 1-D model that is obtained with the 12 tones dam explosion shot data recorded by ETSE network and the existing receiver function

  6. The HFIP High Resolution Hurricane Forecast Test

    NASA Astrophysics Data System (ADS)

    Nance, L. B.; Bernardet, L.; Bao, S.; Brown, B.; Carson, L.; Fowler, T.; Halley Gotway, J.; Harrop, C.; Szoke, E.; Tollerud, E. I.; Wolff, J.; Yuan, H.

    2010-12-01

    Tropical cyclones are a serious concern for the nation, causing significant risk to life, property and economic vitality. The National Oceanic and Atmospheric Administration (NOAA) National Weather Service has a mission of issuing tropical cyclone forecasts and warnings, aimed at protecting life and property and enhancing the national economy. In the last 10 years, the errors in hurricane track forecasts have been reduced by about 50% through improved model guidance, enhanced observations, and forecaster expertise. However, little progress has been made during this period toward reducing forecasted intensity errors. To address this shortcoming, NOAA established the Hurricane Forecast Improvement Project (HFIP) in 2007. HFIP is a 10-year plan to improve one to five day tropical cyclone forecasts, with a focus on rapid intensity change. Recent research suggests that prediction models with grid spacing less than 1 km in the inner core of the hurricane may provide a substantial improvement in intensity forecasts. The 2008-09 staging of the High Resolution Hurricane (HRH) Test focused on quantifying the impact of increased horizontal resolution in numerical models on hurricane intensity forecasts. The primary goal of this test was an evaluation of the effect of increasing horizontal resolution within a given model across a variety of storms with different intensity, location and structure. The test focused on 69 retrospectives cases from the 2005 and 2007 hurricane seasons. Six modeling groups participated in the HRH test utilizing a variety of models, including three configurations of the Weather Research and Forecasting (WRF) model, the operational GFDL model, the Navy’s tropical cyclone model, and a model developed at the University of Wisconsin-Madison (UWM). The Development Testbed Center (DTC) was tasked with providing objective verification statistics for a variety of metrics. This presentation provides an overview of the HRH Test and a summary of the standard

  7. High resolution atomic force microscopy of double-stranded RNA

    NASA Astrophysics Data System (ADS)

    Ares, Pablo; Fuentes-Perez, Maria Eugenia; Herrero-Galán, Elías; Valpuesta, José M.; Gil, Adriana; Gomez-Herrero, Julio; Moreno-Herrero, Fernando

    2016-06-01

    Double-stranded (ds) RNA mediates the suppression of specific gene expression, it is the genetic material of a number of viruses, and a key activator of the innate immune response against viral infections. The ever increasing list of roles played by dsRNA in the cell and its potential biotechnological applications over the last decade has raised an interest for the characterization of its mechanical properties and structure, and that includes approaches using Atomic Force Microscopy (AFM) and other single-molecule techniques. Recent reports have resolved the structure of dsDNA with AFM at unprecedented resolution. However, an equivalent study with dsRNA is still lacking. Here, we have visualized the double helix of dsRNA under near-physiological conditions and at sufficient resolution to resolve the A-form sub-helical pitch periodicity. We have employed different high-sensitive force-detection methods and obtained images with similar spatial resolution. Therefore, we show here that the limiting factors for high-resolution AFM imaging of soft materials in liquid medium are, rather than the imaging mode, the force between the tip and the sample and the sharpness of the tip apex.Double-stranded (ds) RNA mediates the suppression of specific gene expression, it is the genetic material of a number of viruses, and a key activator of the innate immune response against viral infections. The ever increasing list of roles played by dsRNA in the cell and its potential biotechnological applications over the last decade has raised an interest for the characterization of its mechanical properties and structure, and that includes approaches using Atomic Force Microscopy (AFM) and other single-molecule techniques. Recent reports have resolved the structure of dsDNA with AFM at unprecedented resolution. However, an equivalent study with dsRNA is still lacking. Here, we have visualized the double helix of dsRNA under near-physiological conditions and at sufficient resolution to

  8. The implementation of sea ice model on a regional high-resolution scale

    NASA Astrophysics Data System (ADS)

    Prasad, Siva; Zakharov, Igor; Bobby, Pradeep; McGuire, Peter

    2015-09-01

    The availability of high-resolution atmospheric/ocean forecast models, satellite data and access to high-performance computing clusters have provided capability to build high-resolution models for regional ice condition simulation. The paper describes the implementation of the Los Alamos sea ice model (CICE) on a regional scale at high resolution. The advantage of the model is its ability to include oceanographic parameters (e.g., currents) to provide accurate results. The sea ice simulation was performed over Baffin Bay and the Labrador Sea to retrieve important parameters such as ice concentration, thickness, ridging, and drift. Two different forcing models, one with low resolution and another with a high resolution, were used for the estimation of sensitivity of model results. Sea ice behavior over 7 years was simulated to analyze ice formation, melting, and conditions in the region. Validation was based on comparing model results with remote sensing data. The simulated ice concentration correlated well with Advanced Microwave Scanning Radiometer for EOS (AMSR-E) and Ocean and Sea Ice Satellite Application Facility (OSI-SAF) data. Visual comparison of ice thickness trends estimated from the Soil Moisture and Ocean Salinity satellite (SMOS) agreed with the simulation for year 2010-2011.

  9. High Resolution Global Electrical Conductivity Variations in the Earth's Mantle

    NASA Astrophysics Data System (ADS)

    Kelbert, A.; Sun, J.; Egbert, G. D.

    2013-12-01

    Electrical conductivity of the Earth's mantle is a valuable constraint on the water content and melting processes. In Kelbert et al. (2009), we obtained the first global inverse model of electrical conductivity in the mantle capable of providing constraints on the lateral variations in mantle water content. However, in doing so we had to compromise on the problem complexity by using the historically very primitive ionospheric and magnetospheric source assumptions. In particular, possible model contamination by the auroral current systems had greatly restricted our use of available data. We have now addressed this problem by inverting for the external sources along with the electrical conductivity variations. In this study, we still focus primarily on long period data that are dominated by quasi-zonal source fields. The improved understanding of the ionospheric sources allows us to invert the magnetic fields directly, without a correction for the source and/or the use of transfer functions. It allows us to extend the period range of available data to 1.2 days - 102 days, achieving better sensitivity to the upper mantle and transition zone structures. Finally, once the source effects in the data are accounted for, a much larger subset of observatories may be used in the electrical conductivity inversion. Here, we use full magnetic fields at 207 geomagnetic observatories, which include mid-latitude, equatorial and high latitude data. Observatory hourly means from the years 1958-2010 are employed. The improved quality and spatial distribution of the data set, as well as the high resolution modeling and inversion using degree and order 40 spherical harmonics mapped to a 2x2 degree lateral grid, all contribute to the much improved resolution of our models, representing a conceptual step forward in global electromagnetic sounding. We present a fully three-dimensional, global electrical conductivity model of the Earth's mantle as inferred from ground geomagnetic

  10. High-resolution ground-based spectroscopy: where and how ?

    NASA Astrophysics Data System (ADS)

    Pallavicini, R.

    2002-07-01

    An overview is presented of high-resolution optical spectrographs in operation or under development at large telescopes, with emphasis on those facilities best suited for the study of late-type stars and stellar surface inhomogeneities. Plans for the development of new high-resolution spectroscopic instruments are discussed with emphasis on the ICE spectrograph for the PEPSI spectropolarimeter at the LBT.

  11. High resolution airborne geophysics at hazardous waste disposal sites

    SciTech Connect

    Beard, L.P.; Nyquist, J.E.; Doll, W.E.; Chong Foo, M.; Gamey, T.J.

    1995-06-01

    In 1994, a high resolution helicopter geophysical survey was conducted over portions of the Oak Ridge Reservation, Tennessee. The 1800 line kilometer survey included multi-frequency electromagnetic and magnetic sensors. The areas covered by the high resolution portion of the survey were selected on the basis of their importance to the environmental restoration effort and on data obtained from the reconnaissance phase of the airborne survey in which electromagnetic, magnetic, and radiometric data were collected over the entire Oak Ridge Reservation in 1992--1993. The high resolution phase had lower sensor heights, more and higher EM frequencies, and tighter line spacings than did the reconnaissance survey. When flying over exceptionally clear areas, the high resolution bird came within a few meters of the ground surface. Unfortunately, even sparse trees and power or phone lines could prevent the bird from being towed safely at low altitude, and over such areas it was more usual for it to be flown at about the same altitude as the bird in the reconnaissance survey, about 30m. Even so, the magnetometers used in the high resolution phase were 20m closer to the ground than in the reconnaissance phase because they were mounted on the tail of the bird rather than on the tow cable above the bird. The EM frequencies used in the high resolution survey ranged from 7400Hz to 67000Hz. Only the horizontal coplanar loop configuration was used in the high resolution flyovers.

  12. High resolution integral holography using Fourier ptychographic approach.

    PubMed

    Li, Zhaohui; Zhang, Jianqi; Wang, Xiaorui; Liu, Delian

    2014-12-29

    An innovative approach is proposed for calculating high resolution computer generated integral holograms by using the Fourier Ptychographic (FP) algorithm. The approach initializes a high resolution complex hologram with a random guess, and then stitches together low resolution multi-view images, synthesized from the elemental images captured by integral imaging (II), to recover the high resolution hologram through an iterative retrieval with FP constrains. This paper begins with an analysis of the principle of hologram synthesis from multi-projections, followed by an accurate determination of the constrains required in the Fourier ptychographic integral-holography (FPIH). Next, the procedure of the approach is described in detail. Finally, optical reconstructions are performed and the results are demonstrated. Theoretical analysis and experiments show that our proposed approach can reconstruct 3D scenes with high resolution.

  13. Update on High-Resolution Geodetically Controlled LROC Polar Mosaics

    NASA Astrophysics Data System (ADS)

    Archinal, B.; Lee, E.; Weller, L.; Richie, J.; Edmundson, K.; Laura, J.; Robinson, M.; Speyerer, E.; Boyd, A.; Bowman-Cisneros, E.; Wagner, R.; Nefian, A.

    2015-10-01

    We describe progress on high-resolution (1 m/pixel) geodetically controlled LROC mosaics of the lunar poles, which can be used for locating illumination resources (for solar power or cold traps) or landing site and surface operations planning.

  14. High Resolution CryoFESEM of Microbial Surfaces

    NASA Astrophysics Data System (ADS)

    Erlandsen, Stanley; Lei, Ming; Martin-Lacave, Ines; Dunny, Gary; Wells, Carol

    2003-08-01

    The outer surfaces of three microorganisms, Giardia lamblia, Enterococcus faecalis, and Proteus mirabilis, were investigated by cryo-immobilization followed by sublimation of extracellular ice and cryocoating with either Pt alone or Pt plus carbon. Cryocoated samples were examined at [minus sign]125°C in either an in-lens field emission SEM or a below-the-lens field emission SEM. Cryocoating with Pt alone was sufficient for low magnification observation, but attempts to do high-resolution imaging resulted in radiolysis and cracking of the specimen surface. Double coating with Pt and carbon, in combination with high resolution backscatter electron detectors, enabled high-resolution imaging of the glycocalyx of bacteria, revealing a sponge-like network over the surface. High resolution examination of bacterial flagella also revealed a periodic substructure. Common artifacts included radiolysis leading to “cracking” of the surface, and insufficient deposition of Pt resulting in the absence of detectable surface topography.

  15. High resolution difference schemes for compressible gas dynamics

    SciTech Connect

    Woodward, P.; Colella, P.

    1980-07-30

    The advantages and disadvantages of four new high-resolution difference schemes, namely the von Neumann-Richtmyer, Godunovs, MUSCL and Glimms, for mathematically representing physical conditions in compressible gas flows are compared. (LCL)

  16. Methodology of high-resolution photography for mural condition database

    NASA Astrophysics Data System (ADS)

    Higuchi, R.; Suzuki, T.; Shibata, M.; Taniguchi, Y.

    2015-08-01

    Digital documentation is one of the most useful techniques to record the condition of cultural heritage. Recently, high-resolution images become increasingly useful because it is possible to show general views of mural paintings and also detailed mural conditions in a single image. As mural paintings are damaged by environmental stresses, it is necessary to record the details of painting condition on high-resolution base maps. Unfortunately, the cost of high-resolution photography and the difficulty of operating its instruments and software have commonly been an impediment for researchers and conservators. However, the recent development of graphic software makes its operation simpler and less expensive. In this paper, we suggest a new approach to make digital heritage inventories without special instruments, based on our recent our research project in Üzümlü church in Cappadocia, Turkey. This method enables us to achieve a high-resolution image database with low costs, short time, and limited human resources.

  17. AVHRR/1-FM Advanced Very High Resolution Radiometer

    NASA Technical Reports Server (NTRS)

    1979-01-01

    The advanced very high resolution radiometer is discussed. The program covers design, construction, and test of a breadboard model, engineering model, protoflight model, mechanical/structural model, and a life test model. Special bench test and calibration equipment was developed for use on the program. The flight model program objectives were to fabricate, assemble and test four of the advanced very high resolution radiometers along with a bench cooler and collimator.

  18. Volumetric expiratory high-resolution CT of the lung.

    PubMed

    Nishino, Mizuki; Hatabu, Hiroto

    2004-11-01

    We developed a volumetric expiratory high-resolution CT (HRCT) protocol that provides combined inspiratory and expiratory volumetric imaging of the lung without increasing radiation exposure, and conducted a preliminary feasibility assessment of this protocol to evaluate diffuse lung disease with small airway abnormalities. The volumetric expiratory high-resolution CT increased the detectability of the conducting airway to the areas of air trapping (P<0.0001), and added significant information about extent and distribution of air trapping (P<0.0001).

  19. Development of a Melting Curve-Based Allele-Specific PCR of Apolipoprotein E (APOE) Genotyping Method for Genomic DNA, Guthrie Blood Spot, and Whole Blood.

    PubMed

    Chen, Chia-Hsiang

    2016-01-01

    Genetic polymorphisms of apolipoprotein E (APOE) are associated with various health conditions and diseases, such as Alzheimer's disease, cardiovascular diseases, type 2 diabetes, etc. Hence, genotyping of APOE has broad applications in biomedical research and clinical settings, particularly in the era of precision medicine. The study aimed to develop a convenient and accurate method with flexible throughput to genotype the APOE polymorphisms. A melting curve-based allele-specific PCR method was developed to genotype two single nucleotide polymorphisms (SNPs) of APOE, i.e. rs429358 at codon 112 and rs7412 at codon 158. These two SNPs determine the genotype of APOE2, E3, and E4. PCR-based Sanger sequencing was used as the reference method for APOE genotyping. A 100% concordance rate was obtained in 300 subjects between the melting curve-based allele-specific PCR method and the Sanger sequencing method. This method was applied to a genetic association analysis of APOE and schizophrenia consisting of 711 patients with schizophrenia and 665 control subjects from Taiwan. However, no significant differences in the allele and genotype frequencies were detected between these two groups. Further experiments showed that DNA dissolved from blood collected on Guthrie filter paper and total blood cell lysate without DNA extraction can be used in the melting curve-based allele-specific PCR method. Thus, we suggest that this is a fast, accurate and robust APOE genotyping method with a flexible throughput and suitable for DNA template from different preparations. This convenient method shall meet the different needs of various research and clinical laboratories. PMID:27078154

  20. Palindrome regeneration by template strand-switching mechanism at the origin of DNA replication of porcine circovirus via the rolling-circle melting-pot replication model.

    PubMed

    Cheung, Andrew K

    2004-09-01

    Palindromic sequences (inverted repeats) flanking the origin of DNA replication with the potential of forming single-stranded stem-loop cruciform structures have been reported to be essential for replication of the circular genomes of many prokaryotic and eukaryotic systems. In this study, mutant genomes of porcine circovirus with deletions in the origin-flanking palindrome and incapable of forming any cruciform structures invariably yielded progeny viruses containing longer and more stable palindromes. These results suggest that origin-flanking palindromes are essential for termination but not for initiation of DNA replication. Detection of template strand switching in the middle of an inverted repeat strand among the progeny viruses demonstrated that both the minus genome and a corresponding palindromic strand served as templates simultaneously during DNA biosynthesis and supports the recently proposed rolling-circle "melting-pot" replication model. The genome configuration presented by this model, a four-stranded tertiary structure, provides insights into the mechanisms of DNA replication, inverted repeat correction (or conversion), and illegitimate recombination of any circular DNA molecule with an origin-flanking palindrome.

  1. NASA/American Cancer Society High-Resolution Flow Cytometry Project-I

    NASA Technical Reports Server (NTRS)

    Thomas, R. A.; Krishan, A.; Robinson, D. M.; Sams, C.; Costa, F.

    2001-01-01

    BACKGROUND: The NASA/American Cancer Society (ACS) flow cytometer can simultaneously analyze the electronic nuclear volume (ENV) and DNA content of cells. This study describes the schematics, resolution, reproducibility, and sensitivity of biological standards analyzed on this unit. METHODS: Calibrated beads and biological standards (lymphocytes, trout erythrocytes [TRBC], calf thymocytes, and tumor cells) were analyzed for ENV versus DNA content. Parallel data (forward scatter versus DNA) from a conventional flow cytometer were obtained. RESULTS: ENV linearity studies yielded an R value of 0.999. TRBC had a coefficient of variation (CV) of 1.18 +/- 0.13. DNA indexes as low as 1.02 were detectable. DNA content of lymphocytes from 42 females was 1.9% greater than that for 60 males, with a noninstrumental variability in total DNA content of 0.5%. The ENV/DNA ratio was constant in 15 normal human tissue samples, but differed in the four animal species tested. The ENV/DNA ratio for a hypodiploid breast carcinoma was 2.3 times greater than that for normal breast tissue. CONCLUSIONS: The high-resolution ENV versus DNA analyses are highly reliable, sensitive, and can be used for the detection of near-diploid tumor cells that are difficult to identify with conventional cytometers. ENV/DNA ratio may be a useful parameter for detection of aneuploid populations.

  2. Cloud-Based Tools to Support High-Resolution Modeling (Invited)

    NASA Astrophysics Data System (ADS)

    Jones, N.; Nelson, J.; Swain, N.; Christensen, S.

    2013-12-01

    The majority of watershed models developed to support decision-making by water management agencies are simple, lumped-parameter models. Maturity in research codes and advances in the computational power from multi-core processors on desktop machines, commercial cloud-computing resources, and supercomputers with thousands of cores have created new opportunities for employing more accurate, high-resolution distributed models for routine use in decision support. The barriers for using such models on a more routine basis include massive amounts of spatial data that must be processed for each new scenario and lack of efficient visualization tools. In this presentation we will review a current NSF-funded project called CI-WATER that is intended to overcome many of these roadblocks associated with high-resolution modeling. We are developing a suite of tools that will make it possible to deploy customized web-based apps for running custom scenarios for high-resolution models with minimal effort. These tools are based on a software stack that includes 52 North, MapServer, PostGIS, HT Condor, CKAN, and Python. This open source stack provides a simple scripting environment for quickly configuring new custom applications for running high-resolution models as geoprocessing workflows. The HT Condor component facilitates simple access to local distributed computers or commercial cloud resources when necessary for stochastic simulations. The CKAN framework provides a powerful suite of tools for hosting such workflows in a web-based environment that includes visualization tools and storage of model simulations in a database to archival, querying, and sharing of model results. Prototype applications including land use change, snow melt, and burned area analysis will be presented. This material is based upon work supported by the National Science Foundation under Grant No. 1135482

  3. Genotyping of velvet antlers for identification of country of origin using mitochondrial DNA and fluorescence melting curve analysis with locked nucleic acid probes.

    PubMed

    Ahn, Jeong Jin; Kim, Youngjoo; Hong, Ji Young; Kim, Gi Won; Hwang, Seung Yong

    2016-07-01

    Velvet antlers are used medicinally in Asia and possess various therapeutic effects. Prices are set according to the country of origin, which is unidentifiable to the naked eye, and therefore counterfeiting is prevalent. Additionally, antlers of the Canadian elk, which can generate chronic wasting disease, are prevalently smuggled and distributed in the market. Thus, a method for identifying the country of origin of velvet antlers was developed, using polymorphisms in mitochondrial DNA, fluorescence melting curve analysis and analysis of locked nucleic acids (LNA). This combined method is capable of identifying five genotypes of velvet antlers in a single experiment using two probes. It also has advantages in multiplexing, simplicity and efficiency in genotyping, when compared to real-time PCR or microarrays. The developed method can be used to improve identification rates in the velvet antler market and, by extension, research based on polymorphisms in DNA sequences.

  4. An ultraviolet melting study of the stability of the DNA double helix in the NaDNA-bipyridyl-(ethylenediamine)platintum(II) complex.

    PubMed

    Szabó, S; Lee, S A

    2008-08-01

    Complexes of NaDNA with the bipyridyl-(ethylenediamine)platintum(II) (abbreviated [(bipy)Pt(en)]2+) molecular ion have been studied in solution via ultraviolet absorption experiments at 260 nm between 50 and 90 degrees C. These measurements, performed as a function of the molar ratio of the [(bipy)Pt(en)]2+ complex to DNA base pairs, show that the stability of the DNA double helix is increased by the formation of the DNA.[(bipy)Pt(en)]2+ complex: at a molar ratio of 0.33, the temperature at which the DNA double helix separates into two single strands is increased by about 15 degrees C.

  5. Design and implementation of spaceborne high resolution infrared touch screen

    NASA Astrophysics Data System (ADS)

    Li, Tai-guo; Li, Wen-xin; Dong, Yi-peng; Ma, Wen; Xia, Jia-gao

    2015-10-01

    For the consideration of the special application environment of the electronic products used in aerospace and to further more improve the human-computer interaction of the manned aerospace area. The research is based on the design and implementation way of the high resolution spaceborne infrared touch screen on the basis of FPGA and DSP frame structure. Beside the introduction of the whole structure for the high resolution spaceborne infrared touch screen system, this essay also gives the detail information about design of hardware for the high resolution spaceborne infrared touch screen system, FPGA design, GUI design and DSP algorithm design based on Lagrange interpolation. What is more, the easy makes a comprehensive research of the reliability design for the high resolution spaceborne infrared touch screen for the special purpose of it. Besides, the system test is done after installation of spaceborne infrared touch screen. The test result shows that the system is simple and reliable enough, which has a stable running environment and high resolution, which certainly can meet the special requirement of the manned aerospace instrument products.

  6. EDITORIAL: High-resolution noncontact atomic force microscopy High-resolution noncontact atomic force microscopy

    NASA Astrophysics Data System (ADS)

    Pérez, Rubén; García, Ricardo; Schwarz, Udo

    2009-06-01

    original papers authored by many of the leading groups in the field with the goal of providing a well-balanced overview on the state-of-the-art in this rapidly evolving field. These papers, many of which are based on notable presentations given during the Madrid conference, feature highlights such as (1) the development of sophisticated force spectroscopy procedures that are able to map the complete 3D tip-sample force field on different surfaces; (2) the considerable resolution improvement of Kelvin probe force microscopy (reaching, in some cases, the atomic scale), which is accompanied by a thorough, quantitative understanding of the contrast observed; (3) the perfecting of atomic resolution imaging on insulating substrates, which helps reshape our microscopic understanding of surface properties and chemical activity of these surfaces; (4) the description of instrumental and methodological developments that pave the way to the atomic-scale characterization of magnetic and electronic properties of nanostructures, and last but not least (5) the extension of dynamic imaging modes to high-resolution operation in liquids, ultimately achieving atomic resolution. The latter developments are already having a significant impact in the highly competitive field of biological imaging under physiological conditions. This special issue of Nanotechnology would not have been possible without the highly professional support from Nina Couzin, Amy Harvey, Alex Wotherspoon and the entire Nanotechnology team at IOP Publishing. We are thankful for their help in pushing this project forward. We also thank the authors who have contributed their excellent original articles to this issue, the referees whose comments have helped make the issue an accurate portrait of this rapidly moving field, and the entire NC-AFM community that continues to drive NC-AFM to new horizons.

  7. High-resolution Urban Image Classification Using Extended Features

    SciTech Connect

    Vatsavai, Raju

    2011-01-01

    High-resolution image classification poses several challenges because the typical object size is much larger than the pixel resolution. Any given pixel (spectral features at that location) by itself is not a good indicator of the object it belongs to without looking at the broader spatial footprint. Therefore most modern machine learning approaches that are based on per-pixel spectral features are not very effective in high- resolution urban image classification. One way to overcome this problem is to extract features that exploit spatial contextual information. In this study, we evaluated several features in- cluding edge density, texture, and morphology. Several machine learning schemes were tested on the features extracted from a very high-resolution remote sensing image and results were presented.

  8. High resolution single particle refinement in EMAN2.1.

    PubMed

    Bell, James M; Chen, Muyuan; Baldwin, Philip R; Ludtke, Steven J

    2016-05-01

    EMAN2.1 is a complete image processing suite for quantitative analysis of grayscale images, with a primary focus on transmission electron microscopy, with complete workflows for performing high resolution single particle reconstruction, 2-D and 3-D heterogeneity analysis, random conical tilt reconstruction and subtomogram averaging, among other tasks. In this manuscript we provide the first detailed description of the high resolution single particle analysis pipeline and the philosophy behind its approach to the reconstruction problem. High resolution refinement is a fully automated process, and involves an advanced set of heuristics to select optimal algorithms for each specific refinement task. A gold standard FSC is produced automatically as part of refinement, providing a robust resolution estimate for the final map, and this is used to optimally filter the final CTF phase and amplitude corrected structure. Additional methods are in-place to reduce model bias during refinement, and to permit cross-validation using other computational methods.

  9. High resolution single particle refinement in EMAN2.1.

    PubMed

    Bell, James M; Chen, Muyuan; Baldwin, Philip R; Ludtke, Steven J

    2016-05-01

    EMAN2.1 is a complete image processing suite for quantitative analysis of grayscale images, with a primary focus on transmission electron microscopy, with complete workflows for performing high resolution single particle reconstruction, 2-D and 3-D heterogeneity analysis, random conical tilt reconstruction and subtomogram averaging, among other tasks. In this manuscript we provide the first detailed description of the high resolution single particle analysis pipeline and the philosophy behind its approach to the reconstruction problem. High resolution refinement is a fully automated process, and involves an advanced set of heuristics to select optimal algorithms for each specific refinement task. A gold standard FSC is produced automatically as part of refinement, providing a robust resolution estimate for the final map, and this is used to optimally filter the final CTF phase and amplitude corrected structure. Additional methods are in-place to reduce model bias during refinement, and to permit cross-validation using other computational methods. PMID:26931650

  10. Single sensor processing to obtain high resolution color component signals

    NASA Technical Reports Server (NTRS)

    Glenn, William E. (Inventor)

    2010-01-01

    A method for generating color video signals representative of color images of a scene includes the following steps: focusing light from the scene on an electronic image sensor via a filter having a tri-color filter pattern; producing, from outputs of the sensor, first and second relatively low resolution luminance signals; producing, from outputs of the sensor, a relatively high resolution luminance signal; producing, from a ratio of the relatively high resolution luminance signal to the first relatively low resolution luminance signal, a high band luminance component signal; producing, from outputs of the sensor, relatively low resolution color component signals; and combining each of the relatively low resolution color component signals with the high band luminance component signal to obtain relatively high resolution color component signals.

  11. High resolution melting (HRM) analysis in sugar beet: identification of SNP markers associated to Fusarium resistance

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Fusarium spp. cause severe damage in many agricultural crops including sugar beet. Sugar beet needs to be protected from these soil borne pathogens to guarantee an optimal sugar yield in the field. The genetic control is the key to overcoming this disease. Identification of single nucleotide polymor...

  12. Temperature-induced melting of double-stranded DNA in the absence and presence of covalently bonded antitumour drugs: insight from molecular dynamics simulations

    PubMed Central

    Bueren-Calabuig, Juan A.; Giraudon, Christophe; Galmarini, Carlos M.; Egly, Jean Marc; Gago, Federico

    2011-01-01

    The difference in melting temperature of a double-stranded (ds) DNA molecule in the absence and presence of bound ligands can provide experimental information about the stabilization brought about by ligand binding. By simulating the dynamic behaviour of a duplex of sequence 5′-d(TAATAACGGATTATT)·5′-d(AATAATCCGTTATTA) in 0.1 M NaCl aqueous solution at 400 K, we have characterized in atomic detail its complete thermal denaturation profile in <200 ns. A striking asymmetry was observed on both sides of the central CGG triplet and the strand separation process was shown to be strongly affected by bonding in the minor groove of the prototypical interstrand crosslinker mitomycin C or the monofunctional tetrahydroisoquinolines trabectedin (Yondelis®), Zalypsis® and PM01183®. Progressive helix unzipping was clearly interspersed with some reannealing events, which were most noticeable in the oligonucleotides containing the monoadducts, which maintained an average of 6 bp in the central region at the end of the simulations. These significant differences attest to the demonstrated ability of these drugs to stabilize dsDNA, stall replication and transcription forks, and recruit DNA repair proteins. This stabilization, quantified here in terms of undisrupted base pairs, supports the view that these monoadducts can functionally mimic a DNA interstrand crosslink. PMID:21727089

  13. Modified Noise Power Ratio testing of high resolution digitizers

    SciTech Connect

    McDonald, T.S.

    1994-05-01

    A broadband, full signal range, side-by-side (tandem) test method for estimating the internal noise performance of high resolution digitizers is described and illustrated. The technique involves a re-definition of the traditional Noise Power Ratio (NPR) test, a change that not only makes this test applicable to higher resolution systems than was previously practical, but also enhances its value and flexibility. Since coherence analysis is the basis of this new definition, and since the application of coherence procedures to high resolution data poses several problems, this report discusses these problems and their resolution.

  14. High-resolution seismic studies applied to injected geothermal fluids

    SciTech Connect

    Smith, A.T.; Kasameyer, P.

    1985-01-01

    The application of high-resolution microseismicity studies to the problem of monitoring injected fluids is one component of the Geothermal Injection Monitoring Project at LLNL. The evaluation of microseismicity includes the development of field techniques, and the acquisition and processing of events during the initial development of a geothermal field. To achieve a specific detection threshold and location precision, design criteria are presented for seismic networks. An analysis of a small swarm near Mammoth Lakes, California, demonstrates these relationships and the usefulness of high-resolution seismic studies. A small network is currently monitoring the Mammoth-Pacific geothermal power plant at Casa Diablo as it begins production.

  15. Theoretical Problems in High Resolution Solar Physics, 2

    NASA Technical Reports Server (NTRS)

    Athay, G. (Editor); Spicer, D. S. (Editor)

    1987-01-01

    The Science Working Group for the High Resolution Solar Observatory (HRSO) laid plans beginning in 1984 for a series of workshops designed to stimulate a broadbased input from the scientific community to the HRSO mission. These workshops have the dual objectives of encouraging an early start on the difficult theoretical problems in radiative transfer, magnetohydrodynamics, and plasma physics that will be posed by the HRSO data, and maintaining current discussions of results in high resolution solar studies. This workshop was the second in the series. The workshop format presented invited review papers during the formal sessions and contributed poster papers for discussions during open periods. Both are presented.

  16. High Resolution EUV & FUV Spectroscopy of DA White Dwarfs

    NASA Astrophysics Data System (ADS)

    Barstow, M. A.; Good, S. A.; Bannister, N. P.; Burleigh, M. R.; Holberg, J. B.; Bruhweiler, F. C.; Napiwotzki, R.; Cruddace, R. G.; Kowalski, M. P.

    We report on recent results from a high-resolution spectroscopic survey of hot DA white dwarfs, based on IUE, FUSE and HST observations. For the first time, we address the measurement of element abundances in a completely objective manner with a spectroscopic model fitting technique, which allows us to consider formally the limits that can be placed on abundances in stars where no heavy elements are detected. We also include our latest analysis of the high resolution EUV spectrum of G191-B2B recorded by J-PEX.

  17. Wide swath and high resolution optical imaging satellite of Japan

    NASA Astrophysics Data System (ADS)

    Katayama, Haruyoshi; Kato, Eri; Imai, Hiroko; Sagisaka, Masakazu

    2016-05-01

    The "Advanced optical satellite" (tentative name) is a follow-on mission from ALOS. Mission objectives of the advanced optical satellite is to build upon the existing advanced techniques for global land observation using optical sensors, as well as to promote data utilization for social needs. Wide swath and high resolution optical imager onboard the advanced optical satellite will extend the capabilities of earlier ALOS missions. The optical imager will be able to collect high-resolution (< 1 m) and wide-swath (70 km) images with high geo-location accuracy. This paper introduces a conceptual design of the advanced optical satellite.

  18. Microbeam X-Ray Standing Wave and High Resolution Diffraction

    SciTech Connect

    Kazimirov, A.; Bilderback, D.H.; Huang, R.; Sirenko, A.

    2004-05-12

    Post-focusing collimating optics are introduced as a tool to condition X-ray microbeams for the use in high-resolution X-ray diffraction and scattering techniques. As an example, a one-bounce imaging capillary and miniature Si(004) channel-cut crystal were used to produce a microbeam with 10 {mu}m size and an ultimate angular resolution of 2.5 arc sec. This beam was used to measure the strain in semiconductor microstructures by using X-ray high resolution diffraction and standing wave techniques to {delta}d/d < 5x10-4.

  19. On the application and extension of Harten's high resolution scheme

    NASA Technical Reports Server (NTRS)

    Yee, H. C.; Warming, R. F.; Harten, A.

    1982-01-01

    Extensions of a second order high resolution explicit method for the numerical computation of weak solutions of one dimensonal hyperbolic conservation laws are discussed. The main objectives were (1) to examine the shock resoluton of Harten's method for a two dimensional shock reflection problem, (2) to study the use of a high resolution scheme as a post-processor to an approximate steady state solution, and (3) to construct an implicit in the delta-form using Harten's scheme for the explicit operator and a simplified iteration matrix for the implicit operator.

  20. High resolution BPMS with integrated gain correction system

    SciTech Connect

    Wendt, M.; Briegel, C.; Eddy, N.; Fellenz, B.; Gianfelice, E.; Prieto, P.; Rechenmacher, R.; Voy, D.; Terunuma, N.; Urakawa, J.; /KEK, Tsukuba

    2009-08-01

    High resolution beam position monitors (BPM) are an essential tool to achieve and reproduce a low vertical beam emittance at the KEK Accelerator Test Facility (ATF) damping ring. The ATF damping ring (DR) BPMs are currently upgraded with new high resolution read-out electronics. Based on analog and digital down-conversion techniques, the upgrade includes an automatic gain calibration system to correct for slow drift effects and ensure high reproducible beam position readings. The concept and its technical realization, as well as preliminary results of beam studies are presented.

  1. High-resolution low-dose scanning transmission electron microscopy

    PubMed Central

    Buban, James P.; Ramasse, Quentin; Gipson, Bryant; Browning, Nigel D.; Stahlberg, Henning

    2010-01-01

    During the past two decades instrumentation in scanning transmission electron microscopy (STEM) has pushed toward higher intensity electron probes to increase the signal-to-noise ratio of recorded images. While this is suitable for robust specimens, biological specimens require a much reduced electron dose for high-resolution imaging. We describe here protocols for low-dose STEM image recording with a conventional field-emission gun STEM, while maintaining the high-resolution capability of the instrument. Our findings show that a combination of reduced pixel dwell time and reduced gun current can achieve radiation doses comparable to low-dose TEM. PMID:19915208

  2. High-resolution low-dose scanning transmission electron microscopy.

    PubMed

    Buban, James P; Ramasse, Quentin; Gipson, Bryant; Browning, Nigel D; Stahlberg, Henning

    2010-01-01

    During the past two decades instrumentation in scanning transmission electron microscopy (STEM) has pushed toward higher intensity electron probes to increase the signal-to-noise ratio of recorded images. While this is suitable for robust specimens, biological specimens require a much reduced electron dose for high-resolution imaging. We describe here protocols for low-dose STEM image recording with a conventional field-emission gun STEM, while maintaining the high-resolution capability of the instrument. Our findings show that a combination of reduced pixel dwell time and reduced gun current can achieve radiation doses comparable to low-dose TEM.

  3. Human enamel structure studied by high resolution electron microscopy

    SciTech Connect

    Wen, S.L. )

    1989-01-01

    Human enamel structural features are characterized by high resolution electron microscopy. The human enamel consists of polycrystals with a structure similar to Ca10(PO4)6(OH)2. This article describes the structural features of human enamel crystal at atomic and nanometer level. Besides the structural description, a great number of high resolution images are included. Research into the carious process in human enamel is very important for human beings. This article firstly describes the initiation of caries in enamel crystal at atomic and unit-cell level and secondly describes the further steps of caries with structural and chemical demineralization. The demineralization in fact, is the origin of caries in human enamel. The remineralization of carious areas in human enamel has drawn more and more attention as its potential application is realized. This process has been revealed by high resolution electron microscopy in detail in this article. On the other hand, the radiation effects on the structure of human enamel are also characterized by high resolution electron microscopy. In order to reveal this phenomenon clearly, a great number of electron micrographs have been shown, and a physical mechanism is proposed. 26 references.

  4. Reproducible high-resolution multispectral image acquisition in dermatology

    NASA Astrophysics Data System (ADS)

    Duliu, Alexandru; Gardiazabal, José; Lasser, Tobias; Navab, Nassir

    2015-07-01

    Multispectral image acquisitions are increasingly popular in dermatology, due to their improved spectral resolution which enables better tissue discrimination. Most applications however focus on restricted regions of interest, imaging only small lesions. In this work we present and discuss an imaging framework for high-resolution multispectral imaging on large regions of interest.

  5. High-resolution imaging of upper limb neuropathies.

    PubMed

    Howe, Benjamin Matthew; Spinner, Robert J; Felmlee, Joel P; Amrami, Kimberly K

    2015-04-01

    MRI of the peripheral nerves continues to grow technologically and in clinical use. This article reviews the technological aspects and basic interpretation of high-resolution MR imaging of the upper extremity nerves. These techniques work with 1.5-, or preferably 3-T, scanners regardless of vendors. The article also includes selected pitfalls in the interpretation of upper extremity nerve MRI.

  6. Persistence Diagrams of High-Resolution Temporal Rainfall

    NASA Astrophysics Data System (ADS)

    Fernández Méndez, F.; Carsteanu, A. A.

    2015-12-01

    This study applies Topological Data Analysis (TDA), by generating persistence diagrams to uncover patterns in the data of high-resolution temporal rainfall intensities from Iowa City (IIHR, U of Iowa). Persistence diagrams are a way to identify essential cycles in state-space representations of the data.

  7. Application of Classification Models to Pharyngeal High-Resolution Manometry

    ERIC Educational Resources Information Center

    Mielens, Jason D.; Hoffman, Matthew R.; Ciucci, Michelle R.; McCulloch, Timothy M.; Jiang, Jack J.

    2012-01-01

    Purpose: The authors present 3 methods of performing pattern recognition on spatiotemporal plots produced by pharyngeal high-resolution manometry (HRM). Method: Classification models, including the artificial neural networks (ANNs) multilayer perceptron (MLP) and learning vector quantization (LVQ), as well as support vector machines (SVM), were…

  8. High-Resolution Land Use and Land Cover Mapping

    USGS Publications Warehouse

    ,

    1999-01-01

    As the Nation?s population grows, quantifying, monitoring, and managing land use becomes increasingly important. The U.S. Geological Survey (USGS) has a long heritage of leadership and innovation in land use and land cover (LULC) mapping that has been the model both nationally and internationally for over 20 years. At present, the USGS is producing high-resolution LULC data for several watershed and urban areas within the United States. This high-resolution LULC mapping is part of an ongoing USGS Land Cover Characterization Program (LCCP). The four components of the LCCP are global (1:2,000,000-scale), national (1:100,000-scale), urban (1:24,000-scale), and special projects (various scales and time periods). Within the urban and special project components, the USGS Rocky Mountain Mapping Center (RMMC) is collecting historical as well as contemporary high-resolution LULC data. RMMC?s high-resolution LULC mapping builds on the heritage and success of previous USGS LULC programs and provides LULC information to meet user requirements.

  9. High-Resolution Nuclear Magnetic Resonance of Solids.

    ERIC Educational Resources Information Center

    Maciel, Gary E.

    1984-01-01

    Examines recent developments in techniques for obtaining high-resolution nuclear magnetic resonance (NMR) spectra on solid samples, discussing the kinds of applications for which these techniques are well suited. Also discusses the characteristics of NMR of solids and generating magnetization for NMR in solids. (JN)

  10. High resolution bone mineral densitometry with a gamma camera

    NASA Technical Reports Server (NTRS)

    Leblanc, A.; Evans, H.; Jhingran, S.; Johnson, P.

    1983-01-01

    A technique by which the regional distribution of bone mineral can be determined in bone samples from small animals is described. The technique employs an Anger camera interfaced to a medical computer. High resolution imaging is possible by producing magnified images of the bone samples. Regional densitometry of femurs from oophorectomised and bone mineral loss.

  11. Workshop on high-resolution, large-acceptance spectrometers

    SciTech Connect

    Zeidman, B.

    1981-01-01

    The purpose of the Workshop on High-Resolution, Large-Acceptance Spectrometers was to provide a means for exchange of information among those actively engaged in the design and construction of these new spectrometers. Thirty-seven papers were prepared for the data base.

  12. High-resolution TFT-LCD for spatial light modulator

    NASA Astrophysics Data System (ADS)

    Lee, JaeWon; Kim, Yong-Hae; Byun, Chun-Won; Pi, Jae-Eun; Oh, Himchan; Kim, GiHeon; Lee, Myung-Lae; Chu, Hye-Yong; Hwang, Chi-Sun

    2014-06-01

    SLM with very fine pixel pitch is needed for the holographic display system. Among various kinds of SLMs, commercially available high resolution LCoS has been widely used as a spatial light modulator. But the size of commercially available LCoS SLM is limited because the manufacturing technology of LCoS is based on the semiconductor process developed on small size Si wafer. Recently very high resolution flat panel display panel (~500ppi) was developed as a "retina display". Until now, the pixel pitch of flat panel display is several times larger than the pixel pitch of LCoS. But considering the possibility of shrink down the pixel pitch with advanced lithographic tools, the application of flat panel display will make it possible to build a SLM with high spatial bandwidth product. We simulated High resolution TFT-LCD panel on glass substrate using oxide semiconductor TFT with pixel pitch of 20um. And we considered phase modulation behavior of LC(ECB) mode. The TFT-LCD panel is reflective type with 4-metal structure with organic planarization layers. The technical challenge for high resolution large area SLM will be discussed with very fine pixel.

  13. Plant respirometer enables high resolution of oxygen consumption rates

    NASA Technical Reports Server (NTRS)

    Foster, D. L.

    1966-01-01

    Plant respirometer permits high resolution of relatively small changes in the rate of oxygen consumed by plant organisms undergoing oxidative metabolism in a nonphotosynthetic state. The two stage supply and monitoring system operates by a differential pressure transducer and provides a calibrated output by digital or analog signals.

  14. A DVD Spectroscope: A Simple, High-Resolution Classroom Spectroscope

    ERIC Educational Resources Information Center

    Wakabayashi, Fumitaka; Hamada, Kiyohito

    2006-01-01

    Digital versatile disks (DVDs) have successfully made up an inexpensive but high-resolution spectroscope suitable for classroom experiments that can easily be made with common material and gives clear and fine spectra of various light sources and colored material. The observed spectra can be photographed with a digital camera, and such images can…

  15. High Resolution Digital Imaging of Paintings: The Vasari Project.

    ERIC Educational Resources Information Center

    Martinez, Kirk

    1991-01-01

    Describes VASARI (the Visual Art System for Archiving and Retrieval of Images), a project funded by the European Community to show the feasibility of high resolution colormetric imaging directly from paintings. The hardware and software used in the system are explained, storage on optical disks is described, and initial results are reported. (five…

  16. HIGH RESOLUTION RESISTIVITY LEAK DETECTION DATA PROCESSING & EVALUATION MEHTODS & REQUIREMENTS

    SciTech Connect

    SCHOFIELD JS

    2007-10-04

    This document has two purposes: {sm_bullet} Describe how data generated by High Resolution REsistivity (HRR) leak detection (LD) systems deployed during single-shell tank (SST) waste retrieval operations are processed and evaluated. {sm_bullet} Provide the basic review requirements for HRR data when Hrr is deployed as a leak detection method during SST waste retrievals.

  17. Large-field high-resolution mosaic movies

    NASA Astrophysics Data System (ADS)

    Hammerschlag, Robert H.; Sliepen, Guus; Bettonvil, Felix C. M.; Jägers, Aswin P. L.; Sütterlin, Peter; Lin, Yong; Martin, Sara F.; Panasenco, Olga; Romashets, Eugene P.

    2013-08-01

    Movies with fields-of-view larger than normal, for high-resolution telescopes, will give a better understanding of processes on the Sun such as filament and active region developments and their possible interactions. New active regions can serve as an igniter of the eruption of a nearby filament. A method to create a large field-of-view is to join several fields-of-view into a mosaic. Fields are imaged quickly, one after another, using fast telescope-pointing. Such a pointing cycle has been automated at the Dutch open telescope (DOT), a high-resolution solar telescope located on the Canary Island La Palma. The number and positions of the subfields are calculated automatically and represented by an array of bright points in the guider image which indicates the subfield centers inside the drawn rectangle of the total field on the computer screen with the whole-sun image. Automatic production of flats is also programmed. For the first time, mosaic movies were programmed from stored information on automated telescope motions. The mosaic movies show larger regions of the solar disk in high resolution and fill a gap between available whole-sun images with limited spatial resolution of synoptic telescopes including space instruments and small-field high-cadence movies of high-resolution solar telescopes.

  18. Evacuee Compliance Behavior Analysis using High Resolution Demographic Information

    SciTech Connect

    Lu, Wei; Han, Lee; Liu, Cheng; Tuttle, Mark A; Bhaduri, Budhendra L

    2014-01-01

    The purpose of this study is to examine whether evacuee compliance behavior with route assignments from different resolutions of demographic data would impact the evacuation performance. Most existing evacuation strategies assume that travelers will follow evacuation instructions, while in reality a certain percent of evacuees do not comply with prescribed instructions. In this paper, a comparison study of evacuation assignment based on Traffic Analysis Zones (TAZ) and high resolution LandScan USA Population Cells (LPC) were conducted for the detailed road network representing Alexandria, Virginia. A revised platform for evacuation modeling built on high resolution demographic data and activity-based microscopic traffic simulation is proposed. The results indicate that evacuee compliance behavior affects evacuation efficiency with traditional TAZ assignment, but it does not significantly compromise the efficiency with high resolution LPC assignment. The TAZ assignment also underestimates the real travel time during evacuation, especially for high compliance simulations. This suggests that conventional evacuation studies based on TAZ assignment might not be effective at providing efficient guidance to evacuees. From the high resolution data perspective, traveler compliance behavior is an important factor but it does not impact the system performance significantly. The highlight of evacuee compliance behavior analysis should be emphasized on individual evacuee level route/shelter assignments, rather than the whole system performance.

  19. High resolution data base for use with MAP

    SciTech Connect

    Tapley, W.C.; Harris, D.B.

    1987-05-05

    A high resolution cartographic data base of thw World is available from the CIA. We obtained this data, extracted portions of the data, and produced cartographic files of varying resolutions. The resulting data files are of the proper format for use with MAP (2), our in-house cartographic plotting program.

  20. High-resolution axial MR imaging of tibial stress injuries

    PubMed Central

    2012-01-01

    Purpose To evaluate the relative involvement of tibial stress injuries using high-resolution axial MR imaging and the correlation with MR and radiographic images. Methods A total of 33 patients with exercise-induced tibial pain were evaluated. All patients underwent radiograph and high-resolution axial MR imaging. Radiographs were taken at initial presentation and 4 weeks later. High-resolution MR axial images were obtained using a microscopy surface coil with 60 × 60 mm field of view on a 1.5T MR unit. All images were evaluated for abnormal signals of the periosteum, cortex and bone marrow. Results Nineteen patients showed no periosteal reaction at initial and follow-up radiographs. MR imaging showed abnormal signals in the periosteal tissue and partially abnormal signals in the bone marrow. In 7 patients, periosteal reaction was not seen at initial radiograph, but was detected at follow-up radiograph. MR imaging showed abnormal signals in the periosteal tissue and entire bone marrow. Abnormal signals in the cortex were found in 6 patients. The remaining 7 showed periosteal reactions at initial radiograph. MR imaging showed abnormal signals in the periosteal tissue in 6 patients. Abnormal signals were seen in the partial and entire bone marrow in 4 and 3 patients, respectively. Conclusions Bone marrow abnormalities in high-resolution axial MR imaging were related to periosteal reactions at follow-up radiograph. Bone marrow abnormalities might predict later periosteal reactions, suggesting shin splints or stress fractures. High-resolution axial MR imaging is useful in early discrimination of tibial stress injuries. PMID:22574840

  1. In-Situ High-Resolution Transmission Electron Microscopy Investigation of Overheating of Cu Nanoparticles

    PubMed Central

    Chen, Chunlin; Hu, Ziyu; Li, Yanfen; Liu, Limin; Mori, Hirotaro; Wang, Zhangchang

    2016-01-01

    Synthesizing and functionalizing metal nanoparticles supported on substrates is currently the subject of intensive study owing to their outstanding catalytic performances for heterogeneous catalysis. Revealing the fundamental effect of the substrates on metal nanoparticles represents a key step in clarifying mechanisms of stability and catalytic properties of these heterogeneous systems. However, direct identification of these effects still poses a significant challenge due to the complicacy of interactions between substrates and nanoparticles and also for the technical difficulty, restraining our understanding of these heterogeneous systems. Here, we combine in situ high-resolution transmission electron microscopy with molecular dynamics simulations to investigate Cu nanoparticles supported on graphite and Cu2O substrates, and demonstrate that melting behavior and thermal stability of Cu nanoparticles can be markedly influenced by substrates. The graphite-supported Cu nanoparticles do not melt during annealing at 1073 K until they vanish completely, i.e. only the sublimation occurs, while the Cu2O-supported Cu nanoparticles suffer melting during annealing at 973 K. Such selective superheating of the Cu nanoparticles can be attributed to the adsorption of a thin carbon layer on the surface of the Cu nanoparticles, which helps guide further stability enhancement of functional nanoparticles for realistic applications. PMID:26785839

  2. In-Situ High-Resolution Transmission Electron Microscopy Investigation of Overheating of Cu Nanoparticles

    NASA Astrophysics Data System (ADS)

    Chen, Chunlin; Hu, Ziyu; Li, Yanfen; Liu, Limin; Mori, Hirotaro; Wang, Zhangchang

    2016-01-01

    Synthesizing and functionalizing metal nanoparticles supported on substrates is currently the subject of intensive study owing to their outstanding catalytic performances for heterogeneous catalysis. Revealing the fundamental effect of the substrates on metal nanoparticles represents a key step in clarifying mechanisms of stability and catalytic properties of these heterogeneous systems. However, direct identification of these effects still poses a significant challenge due to the complicacy of interactions between substrates and nanoparticles and also for the technical difficulty, restraining our understanding of these heterogeneous systems. Here, we combine in situ high-resolution transmission electron microscopy with molecular dynamics simulations to investigate Cu nanoparticles supported on graphite and Cu2O substrates, and demonstrate that melting behavior and thermal stability of Cu nanoparticles can be markedly influenced by substrates. The graphite-supported Cu nanoparticles do not melt during annealing at 1073 K until they vanish completely, i.e. only the sublimation occurs, while the Cu2O-supported Cu nanoparticles suffer melting during annealing at 973 K. Such selective superheating of the Cu nanoparticles can be attributed to the adsorption of a thin carbon layer on the surface of the Cu nanoparticles, which helps guide further stability enhancement of functional nanoparticles for realistic applications.

  3. In-Situ High-Resolution Transmission Electron Microscopy Investigation of Overheating of Cu Nanoparticles.

    PubMed

    Chen, Chunlin; Hu, Ziyu; Li, Yanfen; Liu, Limin; Mori, Hirotaro; Wang, Zhangchang

    2016-01-01

    Synthesizing and functionalizing metal nanoparticles supported on substrates is currently the subject of intensive study owing to their outstanding catalytic performances for heterogeneous catalysis. Revealing the fundamental effect of the substrates on metal nanoparticles represents a key step in clarifying mechanisms of stability and catalytic properties of these heterogeneous systems. However, direct identification of these effects still poses a significant challenge due to the complicacy of interactions between substrates and nanoparticles and also for the technical difficulty, restraining our understanding of these heterogeneous systems. Here, we combine in situ high-resolution transmission electron microscopy with molecular dynamics simulations to investigate Cu nanoparticles supported on graphite and Cu2O substrates, and demonstrate that melting behavior and thermal stability of Cu nanoparticles can be markedly influenced by substrates. The graphite-supported Cu nanoparticles do not melt during annealing at 1073 K until they vanish completely, i.e. only the sublimation occurs, while the Cu2O-supported Cu nanoparticles suffer melting during annealing at 973 K. Such selective superheating of the Cu nanoparticles can be attributed to the adsorption of a thin carbon layer on the surface of the Cu nanoparticles, which helps guide further stability enhancement of functional nanoparticles for realistic applications. PMID:26785839

  4. Quadruplex MAPH: improvement of throughput in high-resolution copy number screening

    PubMed Central

    Tyson, Jess; Majerus, Tamsin MO; Walker, Susan; Armour, John AL

    2009-01-01

    Background Copy number variation (CNV) in the human genome is recognised as a widespread and important source of human genetic variation. Now the challenge is to screen for these CNVs at high resolution in a reliable, accurate and cost-effective way. Results Multiplex Amplifiable Probe Hybridisation (MAPH) is a sensitive, high-resolution technology appropriate for screening for CNVs in a defined region, for a targeted population. We have developed MAPH to a highly multiplexed format ("QuadMAPH") that allows the user a four-fold increase in the number of loci tested simultaneously. We have used this method to analyse a genomic region of 210 kb, including the MSH2 gene and 120 kb of flanking DNA. We show that the QuadMAPH probes report copy number with equivalent accuracy to simplex MAPH, reliably demonstrating diploid copy number in control samples and accurately detecting deletions in Hereditary Non-Polyposis Colorectal Cancer (HNPCC) samples. Conclusion QuadMAPH is an accurate, high-resolution method that allows targeted screening of large numbers of subjects without the expense of genome-wide approaches. Whilst we have applied this technique to a region of the human genome, it is equally applicable to the genomes of other organisms. PMID:19785739

  5. Genome-Wide High-Resolution Mapping by Recurrent Intermating Using Arabidopsis Thaliana as a Model

    PubMed Central

    Liu, S. C.; Kowalski, S. P.; Lan, T. H.; Feldmann, K. A.; Paterson, A. H.

    1996-01-01

    We demonstrate a method for developing populations suitable for genome-wide high-resolution genetic linkage mapping, by recurrent intermating among F(2) individuals derived from crosses between homozygous parents. Comparison of intermated progenies to F(2) and ``recombinant inbred'' (RI) populations from the same pedigree corroborate theoretical expectations that progenies intermated for four generations harbor about threefold more information for estimating recombination fraction between closely linked markers than either RI-selfed or F(2) individuals (which are, in fact, equivalent in this regard). Although intermated populations are heterozygous, homozygous ``intermated recombinant inbred'' (IRI) populations can readily be generated, combining additional information afforded by intermating with the permanence of RI populations. Intermated populations permit fine-mapping of genetic markers throughout a genome, helping to bridge the gap between genetic map resolution and the DNA-carrying capacity of modern cloning vectors, thus facilitating merger of genetic and physical maps. Intermating can also facilitate high-resolution mapping of genes and QTLs, accelerating map-based cloning. Finally, intermated populations will facilitate investigation of other fundamental genetic questions requiring a genome-wide high-resolution analysis, such as comparative mapping of distantly related species, and the genetic basis of heterosis. PMID:8770602

  6. High-resolution climate simulation of the last glacial maximum

    SciTech Connect

    Erickson III, David J

    2008-01-01

    The climate of the last glacial maximum (LGM) is simulated with a high-resolution atmospheric general circulation model, the NCAR CCM3 at spectral truncation of T170, corresponding to a grid cell size of roughly 75 km. The purpose of the study is to assess whether there are significant benefits from the higher resolution simulation compared to the lower resolution simulation associated with the role of topography. The LGM simulations were forced with modified CLIMAP sea ice distribution and sea surface temperatures (SST) reduced by 1 C, ice sheet topography, reduced CO{sub 2}, and 21,000 BP orbital parameters. The high-resolution model captures modern climate reasonably well, in particular the distribution of heavy precipitation in the tropical Pacific. For the ice age case, surface temperature simulated by the high-resolution model agrees better with those of proxy estimates than does the low-resolution model. Despite the fact that tropical SSTs were only 2.1 C less than the control run, there are many lowland tropical land areas 4-6 C colder than present. Comparison of T170 model results with the best constrained proxy temperature estimates (noble gas concentrations in groundwater) now yield no significant differences between model and observations. There are also significant upland temperature changes in the best resolved tropical mountain belt (the Andes). We provisionally attribute this result in part as resulting from decreased lateral mixing between ocean and land in a model with more model grid cells. A longstanding model-data discrepancy therefore appears to be resolved without invoking any unusual model physics. The response of the Asian summer monsoon can also be more clearly linked to local geography in the high-resolution model than in the low-resolution model; this distinction should enable more confident validation of climate proxy data with the high-resolution model. Elsewhere, an inferred salinity increase in the subtropical North Atlantic may have

  7. High-resolution DEM Effects on Geophysical Flow Models

    NASA Astrophysics Data System (ADS)

    Williams, M. R.; Bursik, M. I.; Stefanescu, R. E. R.; Patra, A. K.

    2014-12-01

    Geophysical mass flow models are numerical models that approximate pyroclastic flow events and can be used to assess the volcanic hazards certain areas may face. One such model, TITAN2D, approximates granular-flow physics based on a depth-averaged analytical model using inputs of basal and internal friction, material volume at a coordinate point, and a GIS in the form of a digital elevation model (DEM). The volume of modeled material propagates over the DEM in a way that is governed by the slope and curvature of the DEM surface and the basal and internal friction angles. Results from TITAN2D are highly dependent upon the inputs to the model. Here we focus on a single input: the DEM, which can vary in resolution. High resolution DEMs are advantageous in that they contain more surface details than lower-resolution models, presumably allowing modeled flows to propagate in a way more true to the real surface. However, very high resolution DEMs can create undesirable artifacts in the slope and curvature that corrupt flow calculations. With high-resolution DEMs becoming more widely available and preferable for use, determining the point at which high resolution data is less advantageous compared to lower resolution data becomes important. We find that in cases of high resolution, integer-valued DEMs, very high-resolution is detrimental to good model outputs when moderate-to-low (<10-15°) slope angles are involved. At these slope angles, multiple adjacent DEM cell elevation values are equal due to the need for the DEM to approximate the low slope with a limited set of integer values for elevation. The first derivative of the elevation surface thus becomes zero. In these cases, flow propagation is inhibited by these spurious zero-slope conditions. Here we present evidence for this "terracing effect" from 1) a mathematically defined simulated elevation model, to demonstrate the terracing effects of integer valued data, and 2) a real-world DEM where terracing must be

  8. High-resolution structure of the native histone octamer

    SciTech Connect

    Wood, Christopher M.; Nicholson, James M.; Lambert, Stanley J.; Chantalat, Laurent; Reynolds, Colin D.; Baldwin, John P.

    2005-06-01

    The high-resolution (1.90 Å) model of the native histone octamer allows structural comparisons to be made with the nucleosome-core particle, along with an identification of a likely core-histone binding site. Crystals of native histone octamers (H2A–H2B)–(H4–H3)–(H3′–H4′)–(H2B′–H2A′) from chick erythrocytes in 2 M KCl, 1.35 M potassium phosphate pH 6.9 diffract X-rays to 1.90 Å resolution, yielding a structure with an R{sub work} value of 18.7% and an R{sub free} of 22.2%. The crystal space group is P6{sub 5}, the asymmetric unit of which contains one complete octamer. This high-resolution model of the histone-core octamer allows further insight into intermolecular interactions, including water molecules, that dock the histone dimers to the tetramer in the nucleosome-core particle and have relevance to nucleosome remodelling. The three key areas analysed are the H2A′–H3–H4 molecular cluster (also H2A–H3′–H4′), the H4–H2B′ interaction (also H4′–H2B) and the H2A′–H4 β-sheet interaction (also H2A–H4′). The latter of these three regions is important to nucleosome remodelling by RNA polymerase II, as it is shown to be a likely core-histone binding site, and its disruption creates an instability in the nucleosome-core particle. A majority of the water molecules in the high-resolution octamer have positions that correlate to similar positions in the high-resolution nucleosome-core particle structure, suggesting that the high-resolution octamer model can be used for comparative studies with the high-resolution nucleosome-core particle.

  9. High resolution selective multilayer laser processing by nanosecond laser ablation of metal nanoparticle films

    SciTech Connect

    Ko, Seung H.; Pan Heng; Hwang, David J.; Chung, Jaewon; Ryu, Sangil; Grigoropoulos, Costas P.; Poulikakos, Dimos

    2007-11-01

    Ablation of gold nanoparticle films on polymer was explored using a nanosecond pulsed laser, with the goal to achieve feature size reduction and functionality not amenable with inkjet printing. The ablation threshold fluence for the unsintered nanoparticle deposit was at least ten times lower than the reported threshold for the bulk film. This could be explained by the combined effects of melting temperature depression, lower conductive heat transfer loss, strong absorption of the incident laser beam, and the relatively weak bonding between nanoparticles. The ablation physics were verified by the nanoparticle sintering characterization, ablation threshold measurement, time resolved ablation plume shadowgraphs, analysis of ablation ejecta, and the measurement and calculation of optical properties. High resolution and clean feature fabrication with small energy and selective multilayer processing are demonstrated.

  10. High-resolution transmission electron microscopy of grain-refining particles in amorphous aluminum alloys

    SciTech Connect

    Schumacher, P.; Greer, A.L.

    1996-10-01

    The nucleation mechanism of Al-Ti-B grain refiners is studied in an Al-based amorphous alloy. The ability to limit growth of {alpha}-Al in the amorphous alloy permits the microscopical observation of nucleation events on boride particles. Earlier studies of this kind are extended by using high-resolution electron microscopy. This shows that the efficient nucleation {alpha}-Al depends on the TiB{sub 2} particles being coated with a thin layer of Al{sub 3}Ti, which can form only when there is some excess titanium in the melt. The aluminide layer, stabilized by adsorption effects, can be as little as a few monolayers thick, and is coherent with the boride. The nature of this layer, and its importance for the nucleation mechanism are discussed. The fading of the grain refinement action is also considered.

  11. Space to Think: Large, High-Resolution Displays for Sensemaking

    SciTech Connect

    Andrews, Christopher P.; Endert, Alexander; North, Chris

    2010-05-05

    Space supports human cognitive abilities in a myriad of ways. The note attached to the side of the monitor, the papers spread out on the desk, diagrams scrawled on a whiteboard, and even the keys left out on the counter are all examples of using space to recall, reveal relationships, and think. Technological advances have made it possible to construct large display environments in which space has real meaning. This paper examines how increased space affects the way displays are regarded and used within the context of the cognitively demanding task of sensemaking. A study was conducted observing analysts using a prototype large, high-resolution display to solve an analytic problem. This paper reports on the results of this study and suggests a number of potential design criteria for future sensemaking tools developed for large, high-resolution displays.

  12. High resolution map of light pollution over Poland

    NASA Astrophysics Data System (ADS)

    Netzel, Henryka; Netzel, Paweł

    2016-09-01

    In 1976 Berry introduced a simple mathematical equation to calculate artificial night sky brightness at zenith. In the original model cities, considered as points with given population, are only sources of light emission. In contrary to Berry's model, we assumed that all terrain surface can be a source of light. Emission of light depends on percent of built up area in a given cell. We based on Berry's model. Using field measurements and high-resolution data we obtained the map of night sky brightness over Poland in 100-m resolution. High resolution input data, combined with a very simple model, makes it possible to obtain detailed structures of the night sky brightness without complicating the calculations.

  13. Temperature-dependent high resolution absorption cross sections of propane

    NASA Astrophysics Data System (ADS)

    Beale, Christopher A.; Hargreaves, Robert J.; Bernath, Peter F.

    2016-10-01

    High resolution (0.005 cm-1) absorption cross sections have been measured for pure propane (C3H8). These cross sections cover the 2550-3500 cm-1 region at five temperatures (from 296 to 700 K) and were measured using a Fourier transform spectrometer and a quartz cell heated by a tube furnace. Calibrations were made by comparison to the integrated cross sections of propane from the Pacific Northwest National Laboratory. These are the first high resolution absorption cross sections of propane for the 3 μm region at elevated temperatures. The cross sections provided may be used to monitor propane in combustion environments and in astronomical sources such as the auroral regions of Jupiter, brown dwarfs and exoplanets.

  14. High-resolution imaging of cellular processes in Caenorhabditis elegans.

    PubMed

    Maddox, Amy S; Maddox, Paul S

    2012-01-01

    Differential interference contrast (DIC) imaging of Caenorhabditis elegans embryogenesis led to a Nobel Prize in Physiology or Medicine (Sulston et al., 1983) as did the first use of green fluorescent protein (GFP) in a transgenic C. elegans (Chalfie et al., 1994). Given that C. elegans is free living, does not require exceptional environmental control, and is optically clear, live imaging is a powerful tool in for this model system. Combining genetics with high-resolution imaging has continued to make important contributions to many fields. In this chapter, we discuss how certain aspects of high-resolution microscopy are implemented. This is not an exhaustive review of microscopy; it is meant to be a helpful guide and point of reference for some basic concepts in imaging. While these concepts are largely true for all biological imaging, they are chosen as particularly important for C. elegans. PMID:22226519

  15. Fabricating High-Resolution X-Ray Collimators

    NASA Technical Reports Server (NTRS)

    Appleby, Michael; Atkinson, James E.; Fraser, Iain; Klinger, Jill

    2008-01-01

    A process and method for fabricating multi-grid, high-resolution rotating modulation collimators for arcsecond and sub-arcsecond x-ray and gamma-ray imaging involves photochemical machining and precision stack lamination. The special fixturing and etching techniques that have been developed are used for the fabrication of multiple high-resolution grids on a single array substrate. This technology has application in solar and astrophysics and in a number of medical imaging applications including mammography, computed tomography (CT), single photon emission computed tomography (SPECT), and gamma cameras used in nuclear medicine. This collimator improvement can also be used in non-destructive testing, hydrodynamic weapons testing, and microbeam radiation therapy.

  16. Turbine component casting core with high resolution region

    DOEpatents

    Kamel, Ahmed; Merrill, Gary B.

    2014-08-26

    A hollow turbine engine component with complex internal features can include a first region and a second, high resolution region. The first region can be defined by a first ceramic core piece formed by any conventional process, such as by injection molding or transfer molding. The second region can be defined by a second ceramic core piece formed separately by a method effective to produce high resolution features, such as tomo lithographic molding. The first core piece and the second core piece can be joined by interlocking engagement that once subjected to an intermediate thermal heat treatment process thermally deform to form a three dimensional interlocking joint between the first and second core pieces by allowing thermal creep to irreversibly interlock the first and second core pieces together such that the joint becomes physically locked together providing joint stability through thermal processing.

  17. Progressive display of very high resolution images using wavelets.

    PubMed Central

    Zhang, Ya; Wang, James Z.

    2002-01-01

    Digital or digitized biomedical images often have very high resolutions', which make them difficult or impossible to display on computer screens. Therefore, it is desirable to develop a multiresolution display method with which users can freely browse the contents of those high resolution images. In this paper, we present an improved wavelet-based progressive image display algorithm by stressing on the encoding and decoding process. The encoder, which dynamically determines levels of transform and partition of coefficients, is based on a modified Haar wavelet transform. The decoder retrieves the necessary data and reconstructs the requested region at a scale specified by the user. A prototype system, which enables virtually any size of images to be displayed progressively, has been implemented based on this algorithm. The system has low computational complexity for both encoding and decoding process. Images Figure 2 PMID:12476909

  18. The theory and practice of high resolution scanning electron microscopy

    SciTech Connect

    Joy, D.C. Oak Ridge National Lab., TN )

    1990-01-01

    Recent advances in instrumentation have produced the first commercial examples of what can justifiably be called High Resolution Scanning Electron Microscopes. The key components of such instruments are a cold field emission gun, a small-gap immersion probe-forming lens, and a clean dry-pumped vacuum. The performance of these microscopes is characterized by several major features including a spatial resolution, in secondary electron mode on solid specimens, which can exceed 1nm on a routine basis; an incident probe current density of the order of 10{sup 6} amps/cm{sup 2}; and the ability to maintain these levels of performance over an accelerating voltage range of from 1 to 30keV. This combination of high resolution, high probe current, low contamination and flexible electron-optical conditions provides many new opportunitites for the application of the SEM to materials science, physics, and the life sciences. 27 refs., 14 figs.

  19. High-resolution array processing using implicit eigenvector weighting techniques

    SciTech Connect

    Steele, A.K. ); Byrne, C.L. )

    1990-01-01

    Many high-resolution bearing estimators require the explicit calculation of the eigenvectors and eigenvalues of the cross-spectral matrix of the sensor outputs. Once the eigenvectors have been calculated, various different estimators can be derived by altering the eigenvalues to give a re-weighing of the eigenvectors. For example, in the MUSIC method the eigenvalues corresponding to those eigenvectors in the noise subspace are set to unity, while the eigenvalues corresponding to those eigenvectors in the signal subspace are set to zero. These weighing functions are reminiscent of ideal filter responses in analog filter theory, where practical filters are designed by using polynomial approximations to the ideal desired response. In this paper, the approximation theory developed for filter design is used to derive high-resolution bearing estimators that do not require explicit calculation of the eigenvectors.

  20. High Resolution Scanning Electron Microscopy of Cells Using Dielectrophoresis

    PubMed Central

    Tang, Shi-Yang; Zhang, Wei; Soffe, Rebecca; Nahavandi, Sofia; Shukla, Ravi; Khoshmanesh, Khashayar

    2014-01-01

    Ultrastructural analysis of cells can reveal valuable information about their morphological, physiological, and biochemical characteristics. Scanning electron microscopy (SEM) has been widely used to provide high-resolution images from the surface of biological samples. However, samples need to be dehydrated and coated with conductive materials for SEM imaging. Besides, immobilizing non-adherent cells during processing and analysis is challenging and requires complex fixation protocols. In this work, we developed a novel dielectrophoresis based microfluidic platform for interfacing non-adherent cells with high-resolution SEM at low vacuum mode. The system enables rapid immobilization and dehydration of samples without deposition of chemical residues over the cell surface. Moreover, it enables the on-chip chemical stimulation and fixation of immobilized cells with minimum dislodgement. These advantages were demonstrated for comparing the morphological changes of non-budding and budding yeast cells following Lyticase treatment. PMID:25089528

  1. High-resolution x-ray phase tomography

    NASA Astrophysics Data System (ADS)

    Peele, Andrew G.; Thomas, C. David L.; Clement, John G.; Arhatari, Benedicta D.; Hannah, Kevin M.; Doshi, Chandni; Putkunz, Corey T.; Clark, Jesse N.

    2010-09-01

    X-ray tomography is a workhorse tool of non-destructive imaging. It is used to probe three-dimensional structures across a wide range of length scales for objects that offer good absorption contrast to x-rays. In recent years extremely high resolution imaging (on the order of tens of nanometres) has become possible due to technological advances in x-ray optics. At the same time the requirement for strong absorption contrast has been relaxed thanks to the advent of new experimental and algorithmic techniques in phase imaging. Advances in both resolution and phase imaging can be combined to image biological samples at the sub-cellular level. I will report on recent advances in our work including improvements to the current approaches in extracting phase information at high resolution from measurements of the diffracted intensity from a sample. I will also discuss our current experimental status.

  2. [Development of a high resolution simultaneous microwave plasma torch spectrometer].

    PubMed

    Jiang, Jie; Huan, Yan-Fu; Jin, Wei; Feng, Guo-Dong; Fei, Qiang; Cao, Yan-Bo; Jin, Qin-Han

    2007-11-01

    A unique high resolution simultaneous microwave plasma torch (MPT) atomic emission spectrometer was developed and studied preliminarily. Some advanced technologies were applied to the spectrometer, such as echelle grating, UV-intensified CCD array detector, adjustable microwave generator, and water cooling system for the generator, etc. The detection limits of the spectrometer for some elements were determined, the spectral resolution and pixel resolution of the spectrometer were calculated, and an analysis of a practical sample was carried out. The preliminary results demonstrate that such simultaneous spectrometer has advantages of saving sample and time, possessing high sensitivity and resolution, and low-cost for the purchase and maintenance. Taking analytical figures of merit into consideration, the high resolution simultaneous MPT spectrometer will have extended application areas and greater competition potential as compared with sequential MPT spectrometers.

  3. Note: Differential amplified high-resolution tilt angle measurement system.

    PubMed

    Zhao, Shijie; Li, Yan; Zhang, Enyao; Huang, Pei; Wei, Haoyun

    2014-09-01

    A high-resolution tilt angle measurement system is presented in this paper. In this system, the measurement signal is amplified by two steps: (1) amplified by operational amplifier and (2) differential amplified by two MEMS-based inclinometers. The novel application not only amplifies the signal but, more importantly, substantially reduces the electrical interference and common-mode noise among the same circuit design. Thus, both the extremely high resolution and great long-term stability are achieved in this system. Calibrated by an autocollimator, the system shows a resolution of 2 arc sec. The accuracy is better than ±1.5 arc sec. The zero-drift error is below ±1 arc sec and ±2 arc sec in the short and long term, respectively.

  4. Note: Differential amplified high-resolution tilt angle measurement system

    NASA Astrophysics Data System (ADS)

    Zhao, Shijie; Li, Yan; Zhang, Enyao; Huang, Pei; Wei, Haoyun

    2014-09-01

    A high-resolution tilt angle measurement system is presented in this paper. In this system, the measurement signal is amplified by two steps: (1) amplified by operational amplifier and (2) differential amplified by two MEMS-based inclinometers. The novel application not only amplifies the signal but, more importantly, substantially reduces the electrical interference and common-mode noise among the same circuit design. Thus, both the extremely high resolution and great long-term stability are achieved in this system. Calibrated by an autocollimator, the system shows a resolution of 2 arc sec. The accuracy is better than ±1.5 arc sec. The zero-drift error is below ±1 arc sec and ±2 arc sec in the short and long term, respectively.

  5. Parallelization and Algorithmic Enhancements of High Resolution IRAS Image Construction

    NASA Technical Reports Server (NTRS)

    Cao, Yu; Prince, Thomas A.; Tereby, Susan; Beichman, Charles A.

    1996-01-01

    The Infrared Astronomical Satellite caried out a nearly complete survey of the infrared sky, and the survey data are important for the study of many astrophysical phenomena. However, many data sets at other wavelengths have higher resolutions than that of the co-added IRAS maps, and high resolution IRAS images are strongly desired both for their own information content and their usefulness in correlation. The HIRES program was developed by the Infrared Processing and Analysis Center (IPAC) to produce high resolution (approx. 1') images from IRAS data using the Maximum Correlation Method (MCM). We describe the port of HIRES to the Intel Paragon, a massively parallel supercomputer, other software developments for mass production of HIRES images, and the IRAS Galaxy Atlas, a project to map the Galactic plane at 60 and 100(micro)m.

  6. Airborne laser scanning for high-resolution mapping of Antarctica

    NASA Astrophysics Data System (ADS)

    Csatho, Bea; Schenk, Toni; Krabill, William; Wilson, Terry; Lyons, William; McKenzie, Garry; Hallam, Cheryl; Manizade, Serdar; Paulsen, Timothy

    In order to evaluate the potential of airborne laser scanning for topographic mapping in Antarctica and to establish calibration/validation sites for NASA's Ice, Cloud and land Elevation Satellite (ICESat) altimeter mission, NASA, the U.S. National Science Foundation (NSF), and the U.S. Geological Survey (USGS) joined forces to collect high-resolution airborne laser scanning data.In a two-week campaign during the 2001-2002 austral summer, NASA's Airborne Topographic Mapper (ATM) system was used to collect data over several sites in the McMurdo Sound area of Antarctica (Figure 1a). From the recorded signals, NASA computed laser points and The Ohio State University (OSU) completed the elaborate computation/verification of high-resolution Digital Elevation Models (DEMs) in 2003. This article reports about the DEM generation and some exemplary results from scientists using the geomorphologic information from the DEMs during the 2003-2004 field season.

  7. High resolution reservoir geological modelling using outcrop information

    SciTech Connect

    Zhang Changmin; Lin Kexiang; Liu Huaibo

    1997-08-01

    This is China`s first case study of high resolution reservoir geological modelling using outcrop information. The key of the modelling process is to build a prototype model and using the model as a geological knowledge bank. Outcrop information used in geological modelling including seven aspects: (1) Determining the reservoir framework pattern by sedimentary depositional system and facies analysis; (2) Horizontal correlation based on the lower and higher stand duration of the paleo-lake level; (3) Determining the model`s direction based on the paleocurrent statistics; (4) Estimating the sandbody communication by photomosaic and profiles; (6) Estimating reservoir properties distribution within sandbody by lithofacies analysis; and (7) Building the reservoir model in sandbody scale by architectural element analysis and 3-D sampling. A high resolution reservoir geological model of Youshashan oil field has been built by using this method.

  8. Effective Area of the AXAF High Resolution Camera (HRC)

    NASA Technical Reports Server (NTRS)

    Patnaude, Daniel; Pease, Deron; Donnelly, Hank; Juda, Mike; Jones, Christine; Murray, Steve; Zombeck, Martin; Kraft, Ralph; Kenter, Almus; Meehan, Gary; Meehan, Gary; Swartz, Doug; Elsner, Ron

    1998-01-01

    The AXAF High-Resolution Camera (HRC) was calibrated at NASA MSFC's X-Ray Calibration Facility (XRCF) during 1997 March and April. We have undertaken an analysis of the HRC effective area using all data presently available from the XRCF. We discuss our spectral fitting of the beam-normalization detectors (BNDs), our method of removing higher order contamination lines present in the spectra, and corrections for beam non-uniformities. We apply a model of photon absorption depth in order to fit a smooth curve to the quantum efficiency of the detector. This is then combined with the most recent model of the AXAF High-Resolution Mirror Assembly (HRMA) to determine the ensemble effective area versus energy for the HRC. We also address future goals and concerns.

  9. A Procedure for High Resolution Satellite Imagery Quality Assessment

    PubMed Central

    Crespi, Mattia; De Vendictis, Laura

    2009-01-01

    Data products generated from High Resolution Satellite Imagery (HRSI) are routinely evaluated during the so-called in-orbit test period, in order to verify if their quality fits the desired features and, if necessary, to obtain the image correction parameters to be used at the ground processing center. Nevertheless, it is often useful to have tools to evaluate image quality also at the final user level. Image quality is defined by some parameters, such as the radiometric resolution and its accuracy, represented by the noise level, and the geometric resolution and sharpness, described by the Modulation Transfer Function (MTF). This paper proposes a procedure to evaluate these image quality parameters; the procedure was implemented in a suitable software and tested on high resolution imagery acquired by the QuickBird, WorldView-1 and Cartosat-1 satellites. PMID:22412312

  10. PCR amplification of SNP loci from crude DNA for large-scale genotyping of oomycetes.

    PubMed

    Hu, Jian; Lyon, Rebecca; Zhou, Yuxin; Lamour, Kurt

    2014-01-01

    Similar to other eukaryotes, single nucleotide polymorphism (SNP) markers are abundant in many oomycete plant pathogen genomes. High resolution DNA melting analysis (HR-DMA) is a cost-effective method for SNP genotyping, but like many SNP marker technologies, is limited by the amount and quality of template DNA. We describe PCR preamplification of Phytophthora and Peronospora SNP loci from crude DNA extracted from a small amount of mycelium and/or infected plant tissue to produce sufficient template to genotype at least 10 000 SNPs. The approach is fast, inexpensive, requires minimal biological material and should be useful for many organisms in a variety of contexts. PMID:24871597

  11. High Resolution Measurements and Electronic Structure Calculations of a Diazanaphthalene

    NASA Astrophysics Data System (ADS)

    Gruet, Sébastien; Goubet, Manuel; Pirali, Olivier

    2014-06-01

    Polycyclic Aromatic Hydrocarbons (PAHs) have long been suspected to be the carriers of so called Unidentified Infrared Bands (UIBs). Most of the results published in the literature report rotationally unresolved spectra of pure carbon as well as heteroatom-containing PAHs species. To date for this class of molecules, the principal source of rotational informations is ruled by microwave (MW) spectroscopy while high resolution measurements reporting rotational structure of the infrared (IR) vibrational bands are very scarce. Recently, some high resolution techniques provided interesting new results to rotationally resolve the IR and far-IR bands of these large carbonated molecules of astrophysical interest. One of them is to use the bright synchrotron radiation as IR continuum source of a high resolution Fourier transform (FTIR) spectrometer. We report the very complementary analysis of the [1,6] naphthyridine (a N-bearing PAH) for which we recorded the microwave spectrum at the PhLAM laboratory (Lille) and the high resolution far-infrared spectrum on the AILES beamline at synchrotron facility SOLEIL. MW spectroscopy provided highly accurate rotational constants in the ground state to perform Ground State Combinations Differences (GSCD) allowing the analysis of the two most intense FT-FIR bands in the 50-900 wn range. Moreover, during this presentation the negative value of the inertial defect in the GS of the molecule will be discussed. A. Leger, J. L. Puget, Astron. Astrophys. 137, L5-L8 (1984) L. J. Allamandola et al. Astrophys. J. 290, L25-L28 (1985). Z. Kisiel et al. J. Mol. Spectrosc. 217, 115 (2003) S. Thorwirth et al. Astrophys. J. 662, 1309 (2007) D. McNaughton et al. J. Chem. Phys. 124, 154305 (2011). S. Albert et al. Faraday Discuss. 150, 71-99 (2011) B. E. Brumfield et al. Phys. Chem. Lett. 3, 1985-1988 (2012) O. Pirali et al. Phys. Chem. Chem. Phys. 15, 10141 (2013).

  12. High-resolution, cryogenic, side-entry type specimen stage

    DOEpatents

    King, Wayne E.; Merkle, Karl L.

    1979-01-01

    A high-resolution, cryogenic side-entry type specimen stage includes a copper block within which a specimen can be positioned in the electron beam of an electron microscope, one end of the copper block constituting a specimen heat exchanger, means for directing a flow of helium at cryogenic temperature into the heat exchanger, and electrical leads running from the specimen to the exterior of the microscope for four point D.C. electrical resistivity measurements.

  13. Achievement of a 920-MHz High Resolution NMR

    NASA Astrophysics Data System (ADS)

    Hashi, Kenjiro; Shimizu, Tadashi; Goto, Atsushi; Kiyoshi, Tsukasa; Matsumoto, Shinji; Wada, Hitoshi; Fujito, Teruaki; Hasegawa, Ken-ichi; Yoshikawa, Masatoshi; Miki, Takashi; Ito, Satoshi; Hamada, Mamoru; Hayashi, Seiji

    2002-06-01

    We have developed a 920-MHz NMR system and performed the proton NMR measurement of H 2O and ethylbenzene using the superconducting magnet operating at 21.6 T (920 MHz for proton), which is the highest field produced by a superconducting NMR magnet in the persistent mode. From the NMR measurements, it is verified that both homogeneity and stability of the magnet have a specification sufficient for a high resolution NMR.

  14. High resolution microwave spectrometer sounder (HIMSS), volume 1, book 2

    NASA Technical Reports Server (NTRS)

    1990-01-01

    The following topics are presented with respect to the high resolution microwave spectrometer sounder (HIMSS) that is to be used as an instrument for NASA's Earth Observing System (EOS): (1) preliminary program plans; (2) contract end item (CEI) specification; and (3) the instrument interface description document. Under the preliminary program plans section, plans dealing with the following subject areas are discussed: spares, performance assurance, configuration management, software implementation, contamination, calibration management, and verification.

  15. High-resolution schemes for hyperbolic conservation laws

    NASA Technical Reports Server (NTRS)

    Harten, A.

    1982-01-01

    A class of new explicit second order accurate finite difference schemes for the computation of weak solutions of hyperbolic conservation laws is presented. These highly nonlinear schemes are obtained by applying a nonoscillatory first order accurae scheme to an appropriately modified flux function. The so derived second order accurate schemes achieve high resolution while preserving the robustness of the original nonoscillatory first order accurate scheme.

  16. LandScan 2013 High Resolution Global Population Data Set

    SciTech Connect

    2014-07-01

    The LandScan data set is a worldwide population database compiled on a 30"x30" latitude/longitude grid. Census counts (at sub-national level) were apportioned to each grid cell based on likelihood coefficients, which are based on land cover, slope, road proximity, high-resolution imagery, and other data sets. The LandScan data set was developed as part of Oak Ridge National Laboratory (ORNL) Global Population Project for estimating ambient populations at risk.

  17. New Challenges in High-Resolution Modeling of Hurricanes

    NASA Astrophysics Data System (ADS)

    Chen, S. S.

    2006-12-01

    The extreme active Atlantic hurricane seasons in recent years have highlighted the urgent need for a better understanding of the factors that contribute to hurricane intensity and for development of the corresponding advanced hurricane prediction models to improve intensity forecasts. The lack of skill in present forecasts of hurricane structure and intensity may be attributed in part to deficiencies in the current prediction models: insufficient grid resolution, inadequate surface and boundary layer formulations, and the lack of full coupling to a dynamic ocean. The extreme high winds, intense rainfall, large ocean waves, and copious sea spray in hurricanes push the surface-exchange parameters for temperature, water vapor, and momentum into untested regimes. The recent modeling effort is to develop and test a fully coupled atmosphere-wave-ocean modeling system that is capable of resolving the eye and eyewall in a hurricane at ~1 km grid resolution. The new challenges for these very high resolution models are the corresponding physical representations at 1-km scale, including microphysics, sub-grid turbulence parameterization, atmospheric boundary layer, physical processes at the air-sea interface with surface waves among others. The lack of accurate initial conditions for high-resolution hurricane modeling presents another major challenge. Improvements in initial conditions rest on the use of more airborne and remotely sensed observations in high-resolution assimilation systems and on the application of advanced assimilation schemes to hurricanes. This study aimed to provide an overview of these new challenges using high-resolution model simulations of Hurricanes Isabel (2003), Frances (2004), Katrina and Rita (2005) that were observed extensively by two recent field programs, namely, the Coupled Boundary Layer Air-Sea Transfer (CBLAST)-Hurricane in 2003-2004 and the Hurricane Rainbands and Intensity Change Experiment (RAINEX) in 2005.

  18. High resolution microwave spectrometer sounder (HIMSS), volume 1, book 1

    NASA Technical Reports Server (NTRS)

    1990-01-01

    The following topics are presented with respect to the high resolution microwave spectrometer sounder (HIMSS) that is to be used as an instrument for NASA's Earth Observing System (EOS): (1) an instrument overview; (2) an instrument description; (3) the instrument's conceptual design; (4) technical risks and offsets; (5) instrument reliability; (6) commands and telemetry; (7) mass and power budgets; (8) integration and test program; (9) program implementation; and (10) phase CD schedule.

  19. Applications of high-resolution remote sensing image data

    NASA Technical Reports Server (NTRS)

    Strome, W. M.; Leckie, D.; Miller, J.; Buxton, R.

    1990-01-01

    There are many situations in which the image resolution of satellite data is insufficient to provide the detail required for resource management and environmental monitoring. This paper will focus on applications of high-resolution (0.4 to 10 m) airborne multispectral and imaging spectrometer data acquired in Canada using the MEIS II multispectral line imager and the PMI imaging spectrometer. Applications discussed will include forestry, mapping, and geobotany.

  20. Optical alignment of high resolution Fourier transform spectrometers

    NASA Technical Reports Server (NTRS)

    Breckinridge, J. B.; Ocallaghan, F. G.; Cassie, A. G.

    1980-01-01

    Remote sensing, high resolution FTS instruments often contain three primary optical subsystems: Fore-Optics, Interferometer Optics, and Post, or Detector Optics. We discuss the alignment of a double-pass FTS containing a cat's-eye retro-reflector. Also, the alignment of fore-optics containing confocal paraboloids with a reflecting field stop which relays a field image onto a camera is discussed.