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Sample records for automated dna sequencing

  1. Automated DNA Sequencing System

    SciTech Connect

    Armstrong, G.A.; Ekkebus, C.P.; Hauser, L.J.; Kress, R.L.; Mural, R.J.

    1999-04-25

    Oak Ridge National Laboratory (ORNL) is developing a core DNA sequencing facility to support biological research endeavors at ORNL and to conduct basic sequencing automation research. This facility is novel because its development is based on existing standard biology laboratory equipment; thus, the development process is of interest to the many small laboratories trying to use automation to control costs and increase throughput. Before automation, biology Laboratory personnel purified DNA, completed cycle sequencing, and prepared 96-well sample plates with commercially available hardware designed specifically for each step in the process. Following purification and thermal cycling, an automated sequencing machine was used for the sequencing. A technician handled all movement of the 96-well sample plates between machines. To automate the process, ORNL is adding a CRS Robotics A- 465 arm, ABI 377 sequencing machine, automated centrifuge, automated refrigerator, and possibly an automated SpeedVac. The entire system will be integrated with one central controller that will direct each machine and the robot. The goal of this system is to completely automate the sequencing procedure from bacterial cell samples through ready-to-be-sequenced DNA and ultimately to completed sequence. The system will be flexible and will accommodate different chemistries than existing automated sequencing lines. The system will be expanded in the future to include colony picking and/or actual sequencing. This discrete event, DNA sequencing system will demonstrate that smaller sequencing labs can achieve cost-effective the laboratory grow.

  2. Automated DNA sequencing.

    PubMed

    Wallis, Yvonne; Morrell, Natalie

    2011-01-01

    Fluorescent cycle sequencing of PCR products is a multistage process and several methodologies are available to perform each stage. This chapter will describe the more commonly utilised dye-terminator cycle sequencing approach using BigDye® terminator chemistry (Applied Biosystems) ready for analysis on a 3730 DNA genetic analyzer. Even though DNA sequencing is one of the most common and robust techniques performed in molecular laboratories it may not always produce desirable results. The causes of the most common problems will also be discussed in this chapter. PMID:20938839

  3. Use of an automated capillary DNA sequencer to investigate the interaction of cisplatin with telomeric DNA sequences.

    PubMed

    Paul, Moumita; Murray, Vincent

    2012-03-01

    The determination of the sequence selectivity of DNA-damaging agents is very important in elucidating the mechanism of action of anti-tumour drugs. The development of automated capillary DNA sequencers with fluorescent labelling has enabled a more precise method for DNA sequence specificity analysis. In this work we utilized the ABI 3730 capillary sequencer with laser-induced fluorescence to examine the sequence selectivity of cisplatin with purified DNA sequences. The use of this automated machine enabled a higher degree of precision of both position and intensity of cisplatin-DNA adducts than previously possible with manual and automated slab gel procedures. A problem with artefact bands was overcome by ethanol precipitation. It was found that cisplatin strongly formed adducts with telomeric DNA sequences. PMID:21678458

  4. Automated hybridization/imaging device for fluorescent multiplex DNA sequencing

    DOEpatents

    Weiss, R.B.; Kimball, A.W.; Gesteland, R.F.; Ferguson, F.M.; Dunn, D.M.; Di Sera, L.J.; Cherry, J.L.

    1995-11-28

    A method is disclosed for automated multiplex sequencing of DNA with an integrated automated imaging hybridization chamber system. This system comprises an hybridization chamber device for mounting a membrane containing size-fractionated multiplex sequencing reaction products, apparatus for fluid delivery to the chamber device, imaging apparatus for light delivery to the membrane and image recording of fluorescence emanating from the membrane while in the chamber device, and programmable controller apparatus for controlling operation of the system. The multiplex reaction products are hybridized with a probe, the enzyme (such as alkaline phosphatase) is bound to a binding moiety on the probe, and a fluorogenic substrate (such as a benzothiazole derivative) is introduced into the chamber device by the fluid delivery apparatus. The enzyme converts the fluorogenic substrate into a fluorescent product which, when illuminated in the chamber device with a beam of light from the imaging apparatus, excites fluorescence of the fluorescent product to produce a pattern of hybridization. The pattern of hybridization is imaged by a CCD camera component of the imaging apparatus to obtain a series of digital signals. These signals are converted by the controller apparatus into a string of nucleotides corresponding to the nucleotide sequence an automated sequence reader. The method and apparatus are also applicable to other membrane-based applications such as colony and plaque hybridization and Southern, Northern, and Western blots. 9 figs.

  5. Automated hybridization/imaging device for fluorescent multiplex DNA sequencing

    DOEpatents

    Weiss, Robert B.; Kimball, Alvin W.; Gesteland, Raymond F.; Ferguson, F. Mark; Dunn, Diane M.; Di Sera, Leonard J.; Cherry, Joshua L.

    1995-01-01

    A method is disclosed for automated multiplex sequencing of DNA with an integrated automated imaging hybridization chamber system. This system comprises an hybridization chamber device for mounting a membrane containing size-fractionated multiplex sequencing reaction products, apparatus for fluid delivery to the chamber device, imaging apparatus for light delivery to the membrane and image recording of fluorescence emanating from the membrane while in the chamber device, and programmable controller apparatus for controlling operation of the system. The multiplex reaction products are hybridized with a probe, then an enzyme (such as alkaline phosphatase) is bound to a binding moiety on the probe, and a fluorogenic substrate (such as a benzothiazole derivative) is introduced into the chamber device by the fluid delivery apparatus. The enzyme converts the fluorogenic substrate into a fluorescent product which, when illuminated in the chamber device with a beam of light from the imaging apparatus, excites fluorescence of the fluorescent product to produce a pattern of hybridization. The pattern of hybridization is imaged by a CCD camera component of the imaging apparatus to obtain a series of digital signals. These signals are converted by the controller apparatus into a string of nucleotides corresponding to the nucleotide sequence an automated sequence reader. The method and apparatus are also applicable to other membrane-based applications such as colony and plaque hybridization and Southern, Northern, and Western blots.

  6. A new technique for determining the distribution of N7-methylguanine using an automated DNA sequencer.

    PubMed

    Shoukry, S; Anderson, M W; Glickman, B W

    1991-11-01

    We have developed a method to determine rapidly the sequence specificity of DNA alkylation resulting from chemical treatment. The utility of this approach is demonstrated here in a study of the sequence specificity of alkylation by dimethylsulphate (DMS). The method is independent of the sequence chosen and makes use of the polymerase chain reaction (PCR) to generate a fluorescently labelled DNA target. In this study, a 302 bp segment of the Escherichia coli lacI gene was amplified and the product purified by liquid chromatography on a Mono Q column. This DNA was alkylated with DMS and treated with hot piperidine to produce single-strand breaks at sites of N7 alkylation. The distribution of the break points, and hence the position and extent of alkylation, were determined on an Applied Biosystems 370A automated DNA sequencer. PMID:1682064

  7. Aptaligner: Automated Software for Aligning Pseudorandom DNA X-Aptamers from Next-Generation Sequencing Data

    PubMed Central

    2015-01-01

    Next-generation sequencing results from bead-based aptamer libraries have demonstrated that traditional DNA/RNA alignment software is insufficient. This is particularly true for X-aptamers containing specialty bases (W, X, Y, Z, ...) that are identified by special encoding. Thus, we sought an automated program that uses the inherent design scheme of bead-based X-aptamers to create a hypothetical reference library and Markov modeling techniques to provide improved alignments. Aptaligner provides this feature as well as length error and noise level cutoff features, is parallelized to run on multiple central processing units (cores), and sorts sequences from a single chip into projects and subprojects. PMID:24866698

  8. Aptaligner: automated software for aligning pseudorandom DNA X-aptamers from next-generation sequencing data.

    PubMed

    Lu, Emily; Elizondo-Riojas, Miguel-Angel; Chang, Jeffrey T; Volk, David E

    2014-06-10

    Next-generation sequencing results from bead-based aptamer libraries have demonstrated that traditional DNA/RNA alignment software is insufficient. This is particularly true for X-aptamers containing specialty bases (W, X, Y, Z, ...) that are identified by special encoding. Thus, we sought an automated program that uses the inherent design scheme of bead-based X-aptamers to create a hypothetical reference library and Markov modeling techniques to provide improved alignments. Aptaligner provides this feature as well as length error and noise level cutoff features, is parallelized to run on multiple central processing units (cores), and sorts sequences from a single chip into projects and subprojects. PMID:24866698

  9. Automation and integration of multiplexed on-line sample preparation with capillary electrophoresis for DNA sequencing

    SciTech Connect

    Tan, H.

    1999-03-31

    The purpose of this research is to develop a multiplexed sample processing system in conjunction with multiplexed capillary electrophoresis for high-throughput DNA sequencing. The concept from DNA template to called bases was first demonstrated with a manually operated single capillary system. Later, an automated microfluidic system with 8 channels based on the same principle was successfully constructed. The instrument automatically processes 8 templates through reaction, purification, denaturation, pre-concentration, injection, separation and detection in a parallel fashion. A multiplexed freeze/thaw switching principle and a distribution network were implemented to manage flow direction and sample transportation. Dye-labeled terminator cycle-sequencing reactions are performed in an 8-capillary array in a hot air thermal cycler. Subsequently, the sequencing ladders are directly loaded into a corresponding size-exclusion chromatographic column operated at {approximately} 60 C for purification. On-line denaturation and stacking injection for capillary electrophoresis is simultaneously accomplished at a cross assembly set at {approximately} 70 C. Not only the separation capillary array but also the reaction capillary array and purification columns can be regenerated after every run. DNA sequencing data from this system allow base calling up to 460 bases with accuracy of 98%.

  10. Automated DNA mutation detection using universal conditions direct sequencing: application to ten muscular dystrophy genes

    PubMed Central

    2009-01-01

    sequences are reported in this paper. Conclusion This automated process allows laboratories to discover DNA variations in a short time and at low cost. PMID:19835634

  11. Dna Sequencing

    DOEpatents

    Tabor, Stanley; Richardson, Charles C.

    1995-04-25

    A method for sequencing a strand of DNA, including the steps off: providing the strand of DNA; annealing the strand with a primer able to hybridize to the strand to give an annealed mixture; incubating the mixture with four deoxyribonucleoside triphosphates, a DNA polymerase, and at least three deoxyribonucleoside triphosphates in different amounts, under conditions in favoring primer extension to form nucleic acid fragments complementory to the DNA to be sequenced; labelling the nucleic and fragments; separating them and determining the position of the deoxyribonucleoside triphosphates by differences in the intensity of the labels, thereby to determine the DNA sequence.

  12. High-speed automated DNA sequencing utilizing from-the-side laser excitation

    NASA Astrophysics Data System (ADS)

    Westphall, Michael S.; Brumley, Robert L., Jr.; Buxton, Erin C.; Smith, Lloyd M.

    1995-04-01

    The Human Genome Initiative is an ambitious international effort to map and sequence the three billion bases of DNA encoded in the human genome. If successfully completed, the resultant sequence database will be a tool of unparalleled power for biomedical research. One of the major challenges of this project is in the area of DNA sequencing technology. At this time, virtually all DNA sequencing is based upon the separation of DNA fragments in high resolution polyacrylamide gels. This method, as generally practiced, is one to two orders of magnitude too slow and expensive for the successful completion of the Human Genome projection. One reasonable approach is improved sequencing of DNA fragments is to increase the performance of such gel-based sequencing methods. Decreased sequencing times may be obtained by increasing the magnitude of the electric field employed. This is not possible with conventional sequencing, due to the fact that the additional heat associated with the increased electric field cannot be adequately dissipated. Recent developments in the use of thin gels have addressed this problem. Performing electrophoresis in ultrathin (50 to 100 microns) gels greatly increases the heat transfer efficiency, thus allowing the benefits of larger electric fields to be obtained. An increase in separation speed of about an order of magnitude is readily achieved. Thin gels have successfully been used in capillary and slab formats. A detection system has been designed for use with a multiple fluorophore sequencing strategy in horizontal ultrathin slab gels. The system employs laser through-the-side excitation and a cooled CCD detector; this allows for the parallel detection of up to 24 sets of four fluorescently labeled DNA sequencing reactions during their electrophoretic separation in ultrathin (115 micrometers ) denaturing polyacrylamide gels. Four hundred bases of sequence information is obtained from 100 ng of M13 template DNA in an hour, corresponding to an

  13. DNA Sequencing apparatus

    DOEpatents

    Tabor, Stanley; Richardson, Charles C.

    1992-01-01

    An automated DNA sequencing apparatus having a reactor for providing at least two series of DNA products formed from a single primer and a DNA strand, each DNA product of a series differing in molecular weight and having a chain terminating agent at one end; separating means for separating the DNA products to form a series bands, the intensity of substantially all nearby bands in a different series being different, band reading means for determining the position an This invention was made with government support including a grant from the U.S. Public Health Service, contract number AI-06045. The U.S. government has certain rights in the invention.

  14. A fully automated 384 capillary array for DNA sequencer. Final report

    SciTech Connect

    Li, Qingbo; Kane, T

    2003-03-20

    Phase I SpectruMedix has successfully developed an automatic 96-capillary array DNA prototype based on the multiplexed capillary electrophoresis system originated from Ames Laboratory-USDOE, Iowa State University. With computer control of all steps involved in a 96-capillary array running cycle, the prototype instrument (the SCE9600) is now capable of sequencing 450 base pairs (bp) per capillary, or 48,000 bp per instrument run within 2 hrs. Phase II of this grant involved the advancement of the core 96 capillary technologies, as well as designing a high density 384 capillary prototype. True commercialization of the 96 capillary instrument involved finalization of the gel matrix, streamlining the instrument hardware, creating a more reliable capillary cartridge, and further advancement of the data processing software. Together these silos of technology create a truly commercializable product (the SCE9610) capable of meeting the operation needs of the sequencing centers.

  15. Algorithms for automated DNA assembly

    PubMed Central

    Densmore, Douglas; Hsiau, Timothy H.-C.; Kittleson, Joshua T.; DeLoache, Will; Batten, Christopher; Anderson, J. Christopher

    2010-01-01

    Generating a defined set of genetic constructs within a large combinatorial space provides a powerful method for engineering novel biological functions. However, the process of assembling more than a few specific DNA sequences can be costly, time consuming and error prone. Even if a correct theoretical construction scheme is developed manually, it is likely to be suboptimal by any number of cost metrics. Modular, robust and formal approaches are needed for exploring these vast design spaces. By automating the design of DNA fabrication schemes using computational algorithms, we can eliminate human error while reducing redundant operations, thus minimizing the time and cost required for conducting biological engineering experiments. Here, we provide algorithms that optimize the simultaneous assembly of a collection of related DNA sequences. We compare our algorithms to an exhaustive search on a small synthetic dataset and our results show that our algorithms can quickly find an optimal solution. Comparison with random search approaches on two real-world datasets show that our algorithms can also quickly find lower-cost solutions for large datasets. PMID:20335162

  16. Image analysis for DNA sequencing

    NASA Astrophysics Data System (ADS)

    Palaniappan, Kannappan; Huang, Thomas S.

    1991-07-01

    There is a great deal of interest in automating the process of DNA (deoxyribonucleic acid) sequencing to support the analysis of genomic DNA such as the Human and Mouse Genome projects. In one class of gel-based sequencing protocols autoradiograph images are generated in the final step and usually require manual interpretation to reconstruct the DNA sequence represented by the image. The need to handle a large volume of sequence information necessitates automation of the manual autoradiograph reading step through image analysis in order to reduce the length of time required to obtain sequence data and reduce transcription errors. Various adaptive image enhancement, segmentation and alignment methods were applied to autoradiograph images. The methods are adaptive to the local characteristics of the image such as noise, background signal, or presence of edges. Once the two-dimensional data is converted to a set of aligned one-dimensional profiles waveform analysis is used to determine the location of each band which represents one nucleotide in the sequence. Different classification strategies including a rule-based approach are investigated to map the profile signals, augmented with the original two-dimensional image data as necessary, to textual DNA sequence information.

  17. Initial analysis of non-typical Leber hereditary optic neuropathy (LHON) at onset and late developing demyelinating disease in Italian patients by SSCP and automated DNA sequence analysis

    SciTech Connect

    Sartore, M.; Semeraro, A.; Fortina, P.

    1994-09-01

    LHON is a mitochondrial genetic disease characterized by maternal inheritance and late onset of blindness caused by bilateral retinal degeneration. A number of molecular defects are known affecting expression of seven mitochondrial genes encoding subunits of respiratory chain complex I, III and IV. We screened genomic DNA from Italian patients for seven of the known point mutations in the ND-1, ND-4 and ND-6 subunits of complex I by PCR followed by SSCP and restriction enzyme digestion. Most of the patients had nonfamilial bilateral visual loss with partial or no recovery and normal neurological examination. Fundoscopic examination revealed that none of the patients had features typical of LHON. Nine of 21 patients (43%) showed multifocal CNS demyelination on MRI. Our results show aberrant SSCP patterns for a PCR product from the ND-4 subunit in one affected child and his mother. Sfa NI and Mae III digestions suggested the absence of a previously defined LHON mutation, and automated DNA sequence analysis revealed two A to G neutral sequence polymorphisms in the third position of codons 351 and 353. In addition, PCR products from the same two samples and an unrelated one showed abnormal SSCP patterns for the ND-1 subunit region of complex I due to the presence of a T to C change at nt 4,216 which was demonstrated after Nla III digestion of PCR products and further confirmed by DNA sequence analysis. Our results indicate that additional defects are present in the Italian population, and identification of abnormal SSCP patterns followed by targeted automated DNA sequence analysis is a reasonable strategy for delineation of new LHON mutations.

  18. j5 DNA assembly design automation.

    PubMed

    Hillson, Nathan J

    2014-01-01

    Modern standardized methodologies, described in detail in the previous chapters of this book, have enabled the software-automated design of optimized DNA construction protocols. This chapter describes how to design (combinatorial) scar-less DNA assembly protocols using the web-based software j5. j5 assists biomedical and biotechnological researchers construct DNA by automating the design of optimized protocols for flanking homology sequence as well as type IIS endonuclease-mediated DNA assembly methodologies. Unlike any other software tool available today, j5 designs scar-less combinatorial DNA assembly protocols, performs a cost-benefit analysis to identify which portions of an assembly process would be less expensive to outsource to a DNA synthesis service provider, and designs hierarchical DNA assembly strategies to mitigate anticipated poor assembly junction sequence performance. Software integrated with j5 add significant value to the j5 design process through graphical user-interface enhancement and downstream liquid-handling robotic laboratory automation. PMID:24395369

  19. Comparison of Boiling and Robotics Automation Method in DNA Extraction for Metagenomic Sequencing of Human Oral Microbes.

    PubMed

    Yamagishi, Junya; Sato, Yukuto; Shinozaki, Natsuko; Ye, Bin; Tsuboi, Akito; Nagasaki, Masao; Yamashita, Riu

    2016-01-01

    The rapid improvement of next-generation sequencing performance now enables us to analyze huge sample sets with more than ten thousand specimens. However, DNA extraction can still be a limiting step in such metagenomic approaches. In this study, we analyzed human oral microbes to compare the performance of three DNA extraction methods: PowerSoil (a method widely used in this field), QIAsymphony (a robotics method), and a simple boiling method. Dental plaque was initially collected from three volunteers in the pilot study and then expanded to 12 volunteers in the follow-up study. Bacterial flora was estimated by sequencing the V4 region of 16S rRNA following species-level profiling. Our results indicate that the efficiency of PowerSoil and QIAsymphony was comparable to the boiling method. Therefore, the boiling method may be a promising alternative because of its simplicity, cost effectiveness, and short handling time. Moreover, this method was reliable for estimating bacterial species and could be used in the future to examine the correlation between oral flora and health status. Despite this, differences in the efficiency of DNA extraction for various bacterial species were observed among the three methods. Based on these findings, there is no "gold standard" for DNA extraction. In future, we suggest that the DNA extraction method should be selected on a case-by-case basis considering the aims and specimens of the study. PMID:27104353

  20. Comparison of Boiling and Robotics Automation Method in DNA Extraction for Metagenomic Sequencing of Human Oral Microbes

    PubMed Central

    Shinozaki, Natsuko; Ye, Bin; Tsuboi, Akito; Nagasaki, Masao; Yamashita, Riu

    2016-01-01

    The rapid improvement of next-generation sequencing performance now enables us to analyze huge sample sets with more than ten thousand specimens. However, DNA extraction can still be a limiting step in such metagenomic approaches. In this study, we analyzed human oral microbes to compare the performance of three DNA extraction methods: PowerSoil (a method widely used in this field), QIAsymphony (a robotics method), and a simple boiling method. Dental plaque was initially collected from three volunteers in the pilot study and then expanded to 12 volunteers in the follow-up study. Bacterial flora was estimated by sequencing the V4 region of 16S rRNA following species-level profiling. Our results indicate that the efficiency of PowerSoil and QIAsymphony was comparable to the boiling method. Therefore, the boiling method may be a promising alternative because of its simplicity, cost effectiveness, and short handling time. Moreover, this method was reliable for estimating bacterial species and could be used in the future to examine the correlation between oral flora and health status. Despite this, differences in the efficiency of DNA extraction for various bacterial species were observed among the three methods. Based on these findings, there is no “gold standard” for DNA extraction. In future, we suggest that the DNA extraction method should be selected on a case-by-case basis considering the aims and specimens of the study. PMID:27104353

  1. DNA sequencing conference, 2

    SciTech Connect

    Cook-Deegan, R.M.; Venter, J.C.; Gilbert, W.; Mulligan, J.; Mansfield, B.K.

    1991-06-19

    This conference focused on DNA sequencing, genetic linkage mapping, physical mapping, informatics and bioethics. Several were used to study this sequencing and mapping. This article also discusses computer hardware and software aiding in the mapping of genes.

  2. A Microfluidic Device for Preparing Next Generation DNA Sequencing Libraries and for Automating Other Laboratory Protocols That Require One or More Column Chromatography Steps

    PubMed Central

    Tan, Swee Jin; Phan, Huan; Gerry, Benjamin Michael; Kuhn, Alexandre; Hong, Lewis Zuocheng; Min Ong, Yao; Poon, Polly Suk Yean; Unger, Marc Alexander; Jones, Robert C.; Quake, Stephen R.; Burkholder, William F.

    2013-01-01

    Library preparation for next-generation DNA sequencing (NGS) remains a key bottleneck in the sequencing process which can be relieved through improved automation and miniaturization. We describe a microfluidic device for automating laboratory protocols that require one or more column chromatography steps and demonstrate its utility for preparing Next Generation sequencing libraries for the Illumina and Ion Torrent platforms. Sixteen different libraries can be generated simultaneously with significantly reduced reagent cost and hands-on time compared to manual library preparation. Using an appropriate column matrix and buffers, size selection can be performed on-chip following end-repair, dA tailing, and linker ligation, so that the libraries eluted from the chip are ready for sequencing. The core architecture of the device ensures uniform, reproducible column packing without user supervision and accommodates multiple routine protocol steps in any sequence, such as reagent mixing and incubation; column packing, loading, washing, elution, and regeneration; capture of eluted material for use as a substrate in a later step of the protocol; and removal of one column matrix so that two or more column matrices with different functional properties can be used in the same protocol. The microfluidic device is mounted on a plastic carrier so that reagents and products can be aliquoted and recovered using standard pipettors and liquid handling robots. The carrier-mounted device is operated using a benchtop controller that seals and operates the device with programmable temperature control, eliminating any requirement for the user to manually attach tubing or connectors. In addition to NGS library preparation, the device and controller are suitable for automating other time-consuming and error-prone laboratory protocols requiring column chromatography steps, such as chromatin immunoprecipitation. PMID:23894273

  3. DNA Sequencing by Capillary Electrophoresis

    PubMed Central

    Karger, Barry L.; Guttman, Andras

    2009-01-01

    Sequencing of human and other genomes has been at the center of interest in the biomedical field over the past several decades and is now leading toward an era of personalized medicine. During this time, DNA sequencing methods have evolved from the labor intensive slab gel electrophoresis, through automated multicapillary electrophoresis systems using fluorophore labeling with multispectral imaging, to the “next generation” technologies of cyclic array, hybridization based, nanopore and single molecule sequencing. Deciphering the genetic blueprint and follow-up confirmatory sequencing of Homo sapiens and other genomes was only possible by the advent of modern sequencing technologies that was a result of step by step advances with a contribution of academics, medical personnel and instrument companies. While next generation sequencing is moving ahead at break-neck speed, the multicapillary electrophoretic systems played an essential role in the sequencing of the Human Genome, the foundation of the field of genomics. In this prospective, we wish to overview the role of capillary electrophoresis in DNA sequencing based in part of several of our articles in this journal. PMID:19517496

  4. DNA sequencing: chemical methods

    SciTech Connect

    Ambrose, B.J.B.; Pless, R.C.

    1987-01-01

    Limited base-specific or base-selective cleavage of a defined DNA fragment yields polynucleotide products, the length of which correlates with the positions of the particular base (or bases) in the original fragment. Sverdlov and co-workers recognized the possibility of using this principle for the determination of DNA sequences. In 1977 a fully elaborated method was introduced based on this principle, which allowed routine analysis of DNA sequences over distances greater than 100 nucleotide unite from a defined, radiolabeled terminus. Six procedures for partial cleavage were described. Simultaneous parallel resolution of an appropriate set of partial cleavage mixtures by polyacrylamide gel electrophoresis, followed by visualization of the radioactive bands by autoradiography, allows the deduction of nucleotide sequence.

  5. Complementary DNA sequencing: Expressed sequence tags and human genome project

    SciTech Connect

    Adams, M.D.; Kelley, J.M.; Gocayne, J.D.; Dubnick, M.; Wu, A.; Olde, B.; Moreno, R.F.; Kerlavage, A.R.; McCombie, W.R.; Venter, J.C. ); Polymeropoulos, M.H.; Hong Xiao; Merril, C.R. )

    1991-06-21

    Automated partial DNA sequencing was conducted on more than 600 randomly selected human brain complementary DNA (cDNA) clones to generate expressed sequence tags (ESTs). ESTs have applications in the discovery of new human genes, mapping of the human genome, and identification of coding regions in genomic sequences. Of the sequences generated, 337 represent new genes, including 48 with significant similarity to genes from other organisms, such as a yeast RNA polymerase II subunit; Drosophila kinesin, Notch, and Enhancer of split; and a murine tyrosine kinase receptor. Forty-six ESTs were mapped to chromosomes after amplification by the polymerase chain reaction. This fast approach to cDNA characterization will facilitate the tagging of most human genes in a few years at a fraction of the cost of complete genomic sequencing, provide new genetic markers, and serve as a resource in diverse biological research fields.

  6. Automated DNA Base Pair Calling Algorithm

    Energy Science and Technology Software Center (ESTSC)

    1999-07-07

    The procedure solves the problem of calling the DNA base pair sequence from two channel electropherogram separations in an automated fashion. The core of the program involves a peak picking algorithm based upon first, second, and third derivative spectra for each electropherogram channel, signal levels as a function of time, peak spacing, base pair signal to noise sequence patterns, frequency vs ratio of the two channel histograms, and confidence levels generated during the run. Themore » ratios of the two channels at peak centers can be used to accurately and reproducibly determine the base pair sequence. A further enhancement is a novel Gaussian deconvolution used to determine the peak heights used in generating the ratio.« less

  7. Indexing Similar DNA Sequences

    NASA Astrophysics Data System (ADS)

    Huang, Songbo; Lam, T. W.; Sung, W. K.; Tam, S. L.; Yiu, S. M.

    To study the genetic variations of a species, one basic operation is to search for occurrences of patterns in a large number of very similar genomic sequences. To build an indexing data structure on the concatenation of all sequences may require a lot of memory. In this paper, we propose a new scheme to index highly similar sequences by taking advantage of the similarity among the sequences. To store r sequences with k common segments, our index requires only O(n + NlogN) bits of memory, where n is the total length of the common segments and N is the total length of the distinct regions in all texts. The total length of all sequences is rn + N, and any scheme to store these sequences requires Ω(n + N) bits. Searching for a pattern P of length m takes O(m + m logN + m log(rk)psc(P) + occlogn), where psc(P) is the number of prefixes of P that appear as a suffix of some common segments and occ is the number of occurrences of P in all sequences. In practice, rk ≤ N, and psc(P) is usually a small constant. We have implemented our solution and evaluated our solution using real DNA sequences. The experiments show that the memory requirement of our solution is much less than that required by BWT built on the concatenation of all sequences. When compared to the other existing solution (RLCSA), we use less memory with faster searching time.

  8. Transposon facilitated DNA sequencing

    SciTech Connect

    Berg, D.E.; Berg, C.M.; Huang, H.V.

    1990-01-01

    The purpose of this research is to investigate and develop methods that exploit the power of bacterial transposable elements for large scale DNA sequencing: Our premise is that the use of transposons to put primer binding sites randomly in target DNAs should provide access to all portions of large DNA fragments, without the inefficiencies of methods involving random subcloning and attendant repetitive sequencing, or of sequential synthesis of many oligonucleotide primers that are used to match systematically along a DNA molecule. Two unrelated bacterial transposons, Tn5 and {gamma}{delta}, are being used because they have both proven useful for molecular analyses, and because they differ sufficiently in mechanism and specificity of transposition to merit parallel development.

  9. Construction and evaluation of a capillary electrophoresis DNA sequencer

    SciTech Connect

    Drossman, H.

    1992-01-01

    This dissertation describes the construction and evaluation of an automated DNA sequencer using capillary gel electrophoresis (CGE) for separating single-strand DNA fragments and a fluorescence detector for analyzing labeled fragments. Theories governing the electrophoretic separation of DNA, dispersion processes in CGE and high sensitivity fluorescence detection are reviewed. The CGE DNA sequencer is compared with current DNA sequencing instruments and with projections of future DNA sequencing instruments. Parameters affecting the limits of detection, DNA sample loading, sample mobility and resolution are evaluated. Predictions for the future of capillary electrophoresis for large-scale sequencing projects are presented.

  10. Automated DNA extraction from pollen in honey.

    PubMed

    Guertler, Patrick; Eicheldinger, Adelina; Muschler, Paul; Goerlich, Ottmar; Busch, Ulrich

    2014-04-15

    In recent years, honey has become subject of DNA analysis due to potential risks evoked by microorganisms, allergens or genetically modified organisms. However, so far, only a few DNA extraction procedures are available, mostly time-consuming and laborious. Therefore, we developed an automated DNA extraction method from pollen in honey based on a CTAB buffer-based DNA extraction using the Maxwell 16 instrument and the Maxwell 16 FFS Nucleic Acid Extraction System, Custom-Kit. We altered several components and extraction parameters and compared the optimised method with a manual CTAB buffer-based DNA isolation method. The automated DNA extraction was faster and resulted in higher DNA yield and sufficient DNA purity. Real-time PCR results obtained after automated DNA extraction are comparable to results after manual DNA extraction. No PCR inhibition was observed. The applicability of this method was further successfully confirmed by analysis of different routine honey samples. PMID:24295710

  11. Pyrosequencing sheds light on DNA sequencing.

    PubMed

    Ronaghi, M

    2001-01-01

    DNA sequencing is one of the most important platforms for the study of biological systems today. Sequence determination is most commonly performed using dideoxy chain termination technology. Recently, pyrosequencing has emerged as a new sequencing methodology. This technique is a widely applicable, alternative technology for the detailed characterization of nucleic acids. Pyrosequencing has the potential advantages of accuracy, flexibility, parallel processing, and can be easily automated. Furthermore, the technique dispenses with the need for labeled primers, labeled nucleotides, and gel-electrophoresis. This article considers key features regarding different aspects of pyrosequencing technology, including the general principles, enzyme properties, sequencing modes, instrumentation, and potential applications. PMID:11156611

  12. A Bioluminometric Method of DNA Sequencing

    NASA Technical Reports Server (NTRS)

    Ronaghi, Mostafa; Pourmand, Nader; Stolc, Viktor; Arnold, Jim (Technical Monitor)

    2001-01-01

    Pyrosequencing is a bioluminometric single-tube DNA sequencing method that takes advantage of co-operativity between four enzymes to monitor DNA synthesis. In this sequencing-by-synthesis method, a cascade of enzymatic reactions yields detectable light, which is proportional to incorporated nucleotides. Pyrosequencing has the advantages of accuracy, flexibility and parallel processing. It can be easily automated. Furthermore, the technique dispenses with the need for labeled primers, labeled nucleotides and gel-electrophoresis. In this chapter, the use of this technique for different applications is discussed.

  13. Automated Identification of Nucleotide Sequences

    NASA Technical Reports Server (NTRS)

    Osman, Shariff; Venkateswaran, Kasthuri; Fox, George; Zhu, Dian-Hui

    2007-01-01

    STITCH is a computer program that processes raw nucleotide-sequence data to automatically remove unwanted vector information, perform reverse-complement comparison, stitch shorter sequences together to make longer ones to which the shorter ones presumably belong, and search against the user s choice of private and Internet-accessible public 16S rRNA databases. ["16S rRNA" denotes a ribosomal ribonucleic acid (rRNA) sequence that is common to all organisms.] In STITCH, a template 16S rRNA sequence is used to position forward and reverse reads. STITCH then automatically searches known 16S rRNA sequences in the user s chosen database(s) to find the sequence most similar to (the sequence that lies at the smallest edit distance from) each spliced sequence. The result of processing by STITCH is the identification of the most similar well-described bacterium. Whereas previously commercially available software for analyzing genetic sequences operates on one sequence at a time, STITCH can manipulate multiple sequences simultaneously to perform the aforementioned operations. A typical analysis of several dozen sequences (length of the order of 103 base pairs) by use of STITCH is completed in a few minutes, whereas such an analysis performed by use of prior software takes hours or days.

  14. The Dynamics of DNA Sequencing.

    ERIC Educational Resources Information Center

    Morvillo, Nancy

    1997-01-01

    Describes a paper-and-pencil activity that helps students understand DNA sequencing and expands student understanding of DNA structure, replication, and gel electrophoresis. Appropriate for advanced biology students who are familiar with the Sanger method. (DDR)

  15. Biosensors for DNA sequence detection

    NASA Technical Reports Server (NTRS)

    Vercoutere, Wenonah; Akeson, Mark

    2002-01-01

    DNA biosensors are being developed as alternatives to conventional DNA microarrays. These devices couple signal transduction directly to sequence recognition. Some of the most sensitive and functional technologies use fibre optics or electrochemical sensors in combination with DNA hybridization. In a shift from sequence recognition by hybridization, two emerging single-molecule techniques read sequence composition using zero-mode waveguides or electrical impedance in nanoscale pores.

  16. Graphene nanodevices for DNA sequencing.

    PubMed

    Heerema, Stephanie J; Dekker, Cees

    2016-02-01

    Fast, cheap, and reliable DNA sequencing could be one of the most disruptive innovations of this decade, as it will pave the way for personalized medicine. In pursuit of such technology, a variety of nanotechnology-based approaches have been explored and established, including sequencing with nanopores. Owing to its unique structure and properties, graphene provides interesting opportunities for the development of a new sequencing technology. In recent years, a wide range of creative ideas for graphene sequencers have been theoretically proposed and the first experimental demonstrations have begun to appear. Here, we review the different approaches to using graphene nanodevices for DNA sequencing, which involve DNA passing through graphene nanopores, nanogaps, and nanoribbons, and the physisorption of DNA on graphene nanostructures. We discuss the advantages and problems of each of these key techniques, and provide a perspective on the use of graphene in future DNA sequencing technology. PMID:26839258

  17. Graphene nanodevices for DNA sequencing

    NASA Astrophysics Data System (ADS)

    Heerema, Stephanie J.; Dekker, Cees

    2016-02-01

    Fast, cheap, and reliable DNA sequencing could be one of the most disruptive innovations of this decade, as it will pave the way for personalized medicine. In pursuit of such technology, a variety of nanotechnology-based approaches have been explored and established, including sequencing with nanopores. Owing to its unique structure and properties, graphene provides interesting opportunities for the development of a new sequencing technology. In recent years, a wide range of creative ideas for graphene sequencers have been theoretically proposed and the first experimental demonstrations have begun to appear. Here, we review the different approaches to using graphene nanodevices for DNA sequencing, which involve DNA passing through graphene nanopores, nanogaps, and nanoribbons, and the physisorption of DNA on graphene nanostructures. We discuss the advantages and problems of each of these key techniques, and provide a perspective on the use of graphene in future DNA sequencing technology.

  18. Automated cycle sequencing with Taquenase: protocols for internal labeling, dye primer and "doublex" simultaneous sequencing.

    PubMed

    Voss, H; Nentwich, U; Duthie, S; Wiemann, S; Benes, V; Zimmermann, J; Ansorge, W

    1997-08-01

    This paper describes automated cycle sequencing protocols for internal labeling, dye primer and "doublex" simultaneous sequencing using Taquenase, a new genetically modified DNA polymerase with increased thermostability. Sequencing performance both with labeled and unlabeled primer yields uniform unambiguous signals up to the resolution limit of the sequencing gels. Primer walking with internal labeling was successfully performed on Pl-derived artificial chromosome (PAC) constructs with 130-kb inserts. Taquenase, a commercially available modified thermostable sequencing enzyme (delta 280, F667Y Taq DNA polymerase), incorporates a variety of fluorescent dNTPs carrying fluorescein isothiocyanate, TexasRed or Cy5 labels during the cycle-sequencing process with higher efficiency than other thermostable DNA polymerases. Comparison to other modified Taq DNA polymerases suggests that the particular N-terminal deletion of Taquenase rather than the presence of the F667Y mutation is responsible for the efficient incorporation and extension of labeled dNTPs. Taquenase makes feasible highly accurate "doublex" simultaneous cylce sequencing on both strands of template DNA with two internal labels or two dye-labeled primers in combination with the EMBL-2-dye DNA sequencing system, ARAKIS, or with two commercial DNA sequencers. It allows up to 2000 bases at > 99% accuracy to be determined in a single reaction. PMID:9266089

  19. Sequence independent amplification of DNA

    DOEpatents

    Bohlander, S.K.

    1998-03-24

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

  20. Sequence independent amplification of DNA

    DOEpatents

    Bohlander, Stefan K.

    1998-01-01

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

  1. Chromosome specific repetitive DNA sequences

    DOEpatents

    Moyzis, Robert K.; Meyne, Julianne

    1991-01-01

    A method is provided for determining specific nucleotide sequences useful in forming a probe which can identify specific chromosomes, preferably through in situ hybridization within the cell itself. In one embodiment, chromosome preferential nucleotide sequences are first determined from a library of recombinant DNA clones having families of repetitive sequences. Library clones are identified with a low homology with a sequence of repetitive DNA families to which the first clones respectively belong and variant sequences are then identified by selecting clones having a pattern of hybridization with genomic DNA dissimilar to the hybridization pattern shown by the respective families. In another embodiment, variant sequences are selected from a sequence of a known repetitive DNA family. The selected variant sequence is classified as chromosome specific, chromosome preferential, or chromosome nonspecific. Sequences which are classified as chromosome preferential are further sequenced and regions are identified having a low homology with other regions of the chromosome preferential sequence or with known sequences of other family me This invention is the result of a contract with the Department of Energy (Contract No. W-7405-ENG-36).

  2. j5 DNA assembly design automation software.

    PubMed

    Hillson, Nathan J; Rosengarten, Rafael D; Keasling, Jay D

    2012-01-20

    Recent advances in Synthetic Biology have yielded standardized and automatable DNA assembly protocols that enable a broad range of biotechnological research and development. Unfortunately, the experimental design required for modern scar-less multipart DNA assembly methods is frequently laborious, time-consuming, and error-prone. Here, we report the development and deployment of a web-based software tool, j5, which automates the design of scar-less multipart DNA assembly protocols including SLIC, Gibson, CPEC, and Golden Gate. The key innovations of the j5 design process include cost optimization, leveraging DNA synthesis when cost-effective to do so, the enforcement of design specification rules, hierarchical assembly strategies to mitigate likely assembly errors, and the instruction of manual or automated construction of scar-less combinatorial DNA libraries. Using a GFP expression testbed, we demonstrate that j5 designs can be executed with the SLIC, Gibson, or CPEC assembly methods, used to build combinatorial libraries with the Golden Gate assembly method, and applied to the preparation of linear gene deletion cassettes for E. coli. The DNA assembly design algorithms reported here are generally applicable to broad classes of DNA construction methodologies and could be implemented to supplement other DNA assembly design tools. Taken together, these innovations save researchers time and effort, reduce the frequency of user design errors and off-target assembly products, decrease research costs, and enable scar-less multipart and combinatorial DNA construction at scales unfeasible without computer-aided design. PMID:23651006

  3. Automated Sequence Processor: Something Old, Something New

    NASA Technical Reports Server (NTRS)

    Streiffert, Barbara; Schrock, Mitchell; Fisher, Forest; Himes, Terry

    2012-01-01

    High productivity required for operations teams to meet schedules Risk must be minimized. Scripting used to automate processes. Scripts perform essential operations functions. Automated Sequence Processor (ASP) was a grass-roots task built to automate the command uplink process System engineering task for ASP revitalization organized. ASP is a set of approximately 200 scripts written in Perl, C Shell, AWK and other scripting languages.. ASP processes/checks/packages non-interactive commands automatically.. Non-interactive commands are guaranteed to be safe and have been checked by hardware or software simulators.. ASP checks that commands are non-interactive.. ASP processes the commands through a command. simulator and then packages them if there are no errors.. ASP must be active 24 hours/day, 7 days/week..

  4. Detecting seeded motifs in DNA sequences.

    PubMed

    Pizzi, Cinzia; Bortoluzzi, Stefania; Bisognin, Andrea; Coppe, Alessandro; Danieli, Gian Antonio

    2005-01-01

    The problem of detecting DNA motifs with functional relevance in real biological sequences is difficult due to a number of biological, statistical and computational issues and also because of the lack of knowledge about the structure of searched patterns. Many algorithms are implemented in fully automated processes, which are often based upon a guess of input parameters from the user at the very first step. In this paper, we present a novel method for the detection of seeded DNA motifs, composed by regions with a different extent of variability. The method is based on a multi-step approach, which was implemented in a motif searching web tool (MOST). Overrepresented exact patterns are extracted from input sequences and clustered to produce motifs core regions, which are then extended and scored to generate seeded motifs. The combination of automated pattern discovery algorithms and different display tools for the evaluation and selection of results at several analysis steps can potentially lead to much more meaningful results than complete automation can produce. Experimental results on different yeast and human real datasets proved the methodology to be a promising solution for finding seeded motifs. MOST web tool is freely available at http://telethon.bio.unipd.it/bioinfo/MOST. PMID:16141193

  5. Detecting seeded motifs in DNA sequences

    PubMed Central

    Pizzi, Cinzia; Bortoluzzi, Stefania; Bisognin, Andrea; Coppe, Alessandro; Danieli, Gian Antonio

    2005-01-01

    The problem of detecting DNA motifs with functional relevance in real biological sequences is difficult due to a number of biological, statistical and computational issues and also because of the lack of knowledge about the structure of searched patterns. Many algorithms are implemented in fully automated processes, which are often based upon a guess of input parameters from the user at the very first step. In this paper, we present a novel method for the detection of seeded DNA motifs, composed by regions with a different extent of variability. The method is based on a multi-step approach, which was implemented in a motif searching web tool (MOST). Overrepresented exact patterns are extracted from input sequences and clustered to produce motifs core regions, which are then extended and scored to generate seeded motifs. The combination of automated pattern discovery algorithms and different display tools for the evaluation and selection of results at several analysis steps can potentially lead to much more meaningful results than complete automation can produce. Experimental results on different yeast and human real datasets proved the methodology to be a promising solution for finding seeded motifs. MOST web tool is freely available at . PMID:16141193

  6. An automated hydrodynamic process for controlled, unbiased DNA shearing.

    PubMed

    Thorstenson, Y R; Hunicke-Smith, S P; Oefner, P J; Davis, R W

    1998-08-01

    An automated, inexpensive, easy-to-use, and reproducible technique for controlled, random DNA fragmentation has been developed. The technique is based on point-sink hydrodynamics that result when a DNA sample is forced through a small hole by a syringe pump. Commercially available components are used to reduce the cost and complexity of the instrument. The design is optimized to reduce the volume of sample required and to speed processing time. Shearing of the samples can be completely automated by computer control. Ninety percent of sheared DNA fragments fall within a twofold size distribution that is highly reproducible. Three parameters are critical: the flow geometry, the flow rate, and a minimum number of iterations. Shearing is reproducible over a wide range of temperatures, DNA concentrations, and initial DNA size. The cloning efficiency of the sheared DNA is very good even without end repair, the distribution of assembled sequences is random, and there is no sequence bias at the ends of sheared fragments that have been cloned. The instrument, called the Point-sink Shearer (PtS), has already been exported successfully to many other laboratories. PMID:9724331

  7. Characterization by automated DNA sequencing of mutations in the gene (rpoB) encoding the RNA polymerase beta subunit in rifampin-resistant Mycobacterium tuberculosis strains from New York City and Texas.

    PubMed Central

    Kapur, V; Li, L L; Iordanescu, S; Hamrick, M R; Wanger, A; Kreiswirth, B N; Musser, J M

    1994-01-01

    Automated DNA sequencing was used to characterize mutations associated with rifampin resistance in a 69-bp region of the gene, rpoB, encoding the beta subunit of RNA polymerase in Mycobacterium tuberculosis. The data confirmed that greater than 90% of rifampin-resistant strains have sequence alterations in this region and showed that most are missense mutations. The analysis also identified several mutant rpoB alleles not previously associated with resistant organisms and one short region of rpoB that had an unusually high frequency of insertions and deletions. Although many strains with an identical IS6110 restriction fragment length polymorphism pattern have the same variant rpoB allele, some do not, a result that suggests the occurrence of evolutionary divergence at the clone level. PMID:8027320

  8. Microchips for DNA sequencing

    NASA Astrophysics Data System (ADS)

    Mastrangelo, Carlos H.; Palaniappan, S.; Man, Piu Francis; Burns, Mark A.; Burke, David T.

    1999-08-01

    Genetic information is vital for understanding features and response of an organism. In humans, genetic errors are linked to the development of major diseases such as cancer and diabetes. In order to maximally exploit this information it is necessary to develop miniature sequencing assays that are rapid and inexpensive. In this paper we show how this could be attained with microfluidic chips that contain integrated assays. To date simple silicon/glass chips aimed for sequencing purpose have been realized; but these chips are not yet practical. Some of the solutions that are used to bring these devices closer to commercial applications are discussed.

  9. Statistical properties of DNA sequences

    NASA Technical Reports Server (NTRS)

    Peng, C. K.; Buldyrev, S. V.; Goldberger, A. L.; Havlin, S.; Mantegna, R. N.; Simons, M.; Stanley, H. E.

    1995-01-01

    We review evidence supporting the idea that the DNA sequence in genes containing non-coding regions is correlated, and that the correlation is remarkably long range--indeed, nucleotides thousands of base pairs distant are correlated. We do not find such a long-range correlation in the coding regions of the gene. We resolve the problem of the "non-stationarity" feature of the sequence of base pairs by applying a new algorithm called detrended fluctuation analysis (DFA). We address the claim of Voss that there is no difference in the statistical properties of coding and non-coding regions of DNA by systematically applying the DFA algorithm, as well as standard FFT analysis, to every DNA sequence (33301 coding and 29453 non-coding) in the entire GenBank database. Finally, we describe briefly some recent work showing that the non-coding sequences have certain statistical features in common with natural and artificial languages. Specifically, we adapt to DNA the Zipf approach to analyzing linguistic texts. These statistical properties of non-coding sequences support the possibility that non-coding regions of DNA may carry biological information.

  10. Statistical properties of DNA sequences

    NASA Astrophysics Data System (ADS)

    Peng, C.-K.; Buldyrev, S. V.; Goldberger, A. L.; Havlin, S.; Mantegna, R. N.; Simons, M.; Stanley, H. E.

    1995-02-01

    We review evidence supporting the idea that the DNA sequence in genese containing non-coding regions is correlated, and that the correlation is remarkably long range - indeed, nucleotides thousands of base pairs distant are correlated. We do not find such a long-range correlation in the coding regions of the gene. We resolve the problem of the “non-stationarity” feature of the sequence of base pairs by applying a new algorithm called detrended fluctuation analysis (DFA). We address the claim of Voss that there is no difference in the statistical properties of coding and non-coding regions of DNA by systematically applying the DFA algorithm, as well as standard FFT analysis, to every DNA sequence (33 301 coding and 29 453 non-coding) in the entire GenBank database. Finally, we describe briefly some recent work showing that the non-coding sequences have certain statistical features in common with natural and artificial languages. Specifically, we adapt to DNA the Zipf approach to analyzing linguistic texts. These statistical properties of non-coding sequences support the possibility that non-coding regions of DNA may carry biological information.

  11. Arduino-based automation of a DNA extraction system.

    PubMed

    Kim, Kyung-Won; Lee, Mi-So; Ryu, Mun-Ho; Kim, Jong-Won

    2015-01-01

    There have been many studies to detect infectious diseases with the molecular genetic method. This study presents an automation process for a DNA extraction system based on microfluidics and magnetic bead, which is part of a portable molecular genetic test system. This DNA extraction system consists of a cartridge with chambers, syringes, four linear stepper actuators, and a rotary stepper actuator. The actuators provide a sequence of steps in the DNA extraction process, such as transporting, mixing, and washing for the gene specimen, magnetic bead, and reagent solutions. The proposed automation system consists of a PC-based host application and an Arduino-based controller. The host application compiles a G code sequence file and interfaces with the controller to execute the compiled sequence. The controller executes stepper motor axis motion, time delay, and input-output manipulation. It drives the stepper motor with an open library, which provides a smooth linear acceleration profile. The controller also provides a homing sequence to establish the motor's reference position, and hard limit checking to prevent any over-travelling. The proposed system was implemented and its functionality was investigated, especially regarding positioning accuracy and velocity profile. PMID:26409535

  12. DNA Sequences at a Glance

    PubMed Central

    Pinho, Armando J.; Garcia, Sara P.; Pratas, Diogo; Ferreira, Paulo J. S. G.

    2013-01-01

    Data summarization and triage is one of the current top challenges in visual analytics. The goal is to let users visually inspect large data sets and examine or request data with particular characteristics. The need for summarization and visual analytics is also felt when dealing with digital representations of DNA sequences. Genomic data sets are growing rapidly, making their analysis increasingly more difficult, and raising the need for new, scalable tools. For example, being able to look at very large DNA sequences while immediately identifying potentially interesting regions would provide the biologist with a flexible exploratory and analytical tool. In this paper we present a new concept, the “information profile”, which provides a quantitative measure of the local complexity of a DNA sequence, independently of the direction of processing. The computation of the information profiles is computationally tractable: we show that it can be done in time proportional to the length of the sequence. We also describe a tool to compute the information profiles of a given DNA sequence, and use the genome of the fission yeast Schizosaccharomyces pombe strain 972 h− and five human chromosomes 22 for illustration. We show that information profiles are useful for detecting large-scale genomic regularities by visual inspection. Several discovery strategies are possible, including the standalone analysis of single sequences, the comparative analysis of sequences from individuals from the same species, and the comparative analysis of sequences from different organisms. The comparison scale can be varied, allowing the users to zoom-in on specific details, or obtain a broad overview of a long segment. Software applications have been made available for non-commercial use at http://bioinformatics.ua.pt/software/dna-at-glance. PMID:24278218

  13. DNA sequences at a glance.

    PubMed

    Pinho, Armando J; Garcia, Sara P; Pratas, Diogo; Ferreira, Paulo J S G

    2013-01-01

    Data summarization and triage is one of the current top challenges in visual analytics. The goal is to let users visually inspect large data sets and examine or request data with particular characteristics. The need for summarization and visual analytics is also felt when dealing with digital representations of DNA sequences. Genomic data sets are growing rapidly, making their analysis increasingly more difficult, and raising the need for new, scalable tools. For example, being able to look at very large DNA sequences while immediately identifying potentially interesting regions would provide the biologist with a flexible exploratory and analytical tool. In this paper we present a new concept, the "information profile", which provides a quantitative measure of the local complexity of a DNA sequence, independently of the direction of processing. The computation of the information profiles is computationally tractable: we show that it can be done in time proportional to the length of the sequence. We also describe a tool to compute the information profiles of a given DNA sequence, and use the genome of the fission yeast Schizosaccharomyces pombe strain 972 h(-) and five human chromosomes 22 for illustration. We show that information profiles are useful for detecting large-scale genomic regularities by visual inspection. Several discovery strategies are possible, including the standalone analysis of single sequences, the comparative analysis of sequences from individuals from the same species, and the comparative analysis of sequences from different organisms. The comparison scale can be varied, allowing the users to zoom-in on specific details, or obtain a broad overview of a long segment. Software applications have been made available for non-commercial use at http://bioinformatics.ua.pt/software/dna-at-glance. PMID:24278218

  14. Structural Complexity of DNA Sequence

    PubMed Central

    Liou, Cheng-Yuan; Cheng, Wei-Chen; Tsai, Huai-Ying

    2013-01-01

    In modern bioinformatics, finding an efficient way to allocate sequence fragments with biological functions is an important issue. This paper presents a structural approach based on context-free grammars extracted from original DNA or protein sequences. This approach is radically different from all those statistical methods. Furthermore, this approach is compared with a topological entropy-based method for consistency and difference of the complexity results. PMID:23662161

  15. Method enabling fast partial sequencing of cDNA clones.

    PubMed

    Nordström, T; Gharizadeh, B; Pourmand, N; Nyren, P; Ronaghi, M

    2001-05-15

    Pyrosequencing is a nonelectrophoretic single-tube DNA sequencing method that takes advantage of cooperativity between four enzymes to monitor DNA synthesis. To investigate the feasibility of the recently developed technique for tag sequencing, 64 colonies of a selected cDNA library from human were sequenced by both pyrosequencing and Sanger DNA sequencing. To determine the needed length for finding a unique DNA sequence, 100 sequence tags from human were retrieved from the database and different lengths from each sequence were randomly analyzed. An homology search based on 20 and 30 nucleotides produced 97 and 98% unique hits, respectively. An homology search based on 100 nucleotides could identify all searched genes. Pyrosequencing was employed to produce sequence data for 30 nucleotides. A similar search using BLAST revealed 16 different genes. Forty-six percent of the sequences shared homology with one gene at different positions. Two of the 64 clones had unique sequences. The search results from pyrosequencing were in 100% agreement with conventional DNA sequencing methods. The possibility of using a fully automated pyrosequencer machine for future high-throughput tag sequencing is discussed. PMID:11355860

  16. Apparatus for improved DNA sequencing

    DOEpatents

    Douthart, R.J.; Crowell, S.L.

    1996-05-07

    This invention is a means for the rapid sequencing of DNA samples. More specifically, it consists of a new design direct blotting electrophoresis unit. The DNA sequence is deposited on a membrane attached to a rotating drum. Initial data compaction is facilitated by the use of a machined multi-channeled plate called a ribbon channel plate. Each channel is an isolated mini gel system much like a gel filled capillary. The system as a whole, however, is in a slab gel like format with the advantages of uniformity and easy reusability. The system can be used in different embodiments. The drum system is unique in that after deposition the drum rotates the deposited DNA into a large non-buffer open space where processing and detection can occur. The drum can also be removed in toto to special workstations for downstream processing, multiplexing and detection. 18 figs.

  17. Apparatus for improved DNA sequencing

    DOEpatents

    Douthart, Richard J.; Crowell, Shannon L.

    1996-01-01

    This invention is a means for the rapid sequencing of DNA samples. More specifically, it consists of a new design direct blotting electrophoresis unit. The DNA sequence is deposited on a membrane attached to a rotating drum. Initial data compaction is facilitated by the use of a machined multi-channeled plate called a ribbon channel plate. Each channel is an isolated mini gel system much like a gel filled capillary. The system as a whole, however, is in a slab gel like format with the advantages of uniformity and easy reusability. The system can be used in different embodiments. The drum system is unique in that after deposition the drum rotates the deposited DNA into a large non-buffer open space where processing and detection can occur. The drum can also be removed in toto to special workstations for downstream processing, multiplexing and detection.

  18. Engineered DNA sequence syntax inspector.

    PubMed

    Hsiau, Timothy Hwei-Chung; Anderson, J Christopher

    2014-02-21

    DNAs encoding polypeptides often contain design errors that cause experiments to prematurely fail. One class of design errors is incorrect or missing elements in the DNA, here termed syntax errors. We have identified three major causes of syntax errors: point mutations from sequencing or manual data entry, gene structure misannotation, and unintended open reading frames (ORFs). The Engineered DNA Sequence Syntax Inspector (EDSSI) is an online bioinformatics pipeline that checks for syntax errors through three steps. First, ORF prediction in input DNA sequences is done by GeneMark; next, homologous sequences are retrieved by BLAST, and finally, syntax errors in the protein sequence are predicted by using the SIFT algorithm. We show that the EDSSI is able to identify previously published examples of syntactical errors and also show that our indel addition to the SIFT program is 97% accurate on a test set of Escherichia coli proteins. The EDSSI is available at http://andersonlab.qb3.berkeley.edu/Software/EDSSI/ . PMID:24364864

  19. The sequence of sequencers: The history of sequencing DNA

    PubMed Central

    Heather, James M.; Chain, Benjamin

    2016-01-01

    Determining the order of nucleic acid residues in biological samples is an integral component of a wide variety of research applications. Over the last fifty years large numbers of researchers have applied themselves to the production of techniques and technologies to facilitate this feat, sequencing DNA and RNA molecules. This time-scale has witnessed tremendous changes, moving from sequencing short oligonucleotides to millions of bases, from struggling towards the deduction of the coding sequence of a single gene to rapid and widely available whole genome sequencing. This article traverses those years, iterating through the different generations of sequencing technology, highlighting some of the key discoveries, researchers, and sequences along the way. PMID:26554401

  20. Automated DNA electrophoresis, hybridization and detection

    SciTech Connect

    Zapolski, E.J.; Gersten, D.M.; Golab, T.J.; Ledley, R.S.

    1986-05-01

    A fully automated, computer controlled system for nucleic acid hybridization analysis has been devised and constructed. In practice, DNA is digested with restriction endonuclease enzyme(s) and loaded into the system by pipette; /sup 32/P-labelled nucleic acid probe(s) is loaded into the nine hybridization chambers. Instructions for all the steps in the automated process are specified by answering questions that appear on the computer screen at the start of the experiment. Subsequent steps are performed automatically. The system performs horizontal electrophoresis in agarose gel, fixed the fragments to a solid phase matrix, denatures, neutralizes, prehybridizes, hybridizes, washes, dries and detects the radioactivity according to the specifications given by the operator. The results, printed out at the end, give the positions on the matrix to which radioactivity remains hybridized following stringent washing.

  1. Dynamical model for DNA sequences

    NASA Astrophysics Data System (ADS)

    Allegrini, P.; Barbi, M.; Grigolini, P.; West, B. J.

    1995-11-01

    We address the problem of DNA sequences, developing a ``dynamical'' method based on the assumption that the statistical properties of DNA paths are determined by the joint action of two processes, one deterministic with long-range correlations, and the other random and δ-function correlated. The generator of the deterministic evolution is a nonlinear map, belonging to a class of maps recently tailored to mimic the processes of weak chaos that are responsible for the birth of anomalous diffusion. It is assumed that the deterministic process corresponds to unknown biological rules that determine the DNA path, whereas the noise mimics the influence of an infinite-dimensional environment on the biological process under study. We prove that the resulting diffusion process, if the effect of the random process is neglected, is an α-stable Lévy process with 1<α<2. We also show that, if the diffusion process is determined by the joint action of the deterministic and the random process, the correlation effects of the ``deterministic dynamics'' are cancelled on the short-range scale, but show up in the long-range one. We denote our prescription to generate statistical sequences as the copying mistake map (CMM). We carry out our analysis of several DNA sequences and their CMM realizations with a variety of techniques, and we especially focus on a method of regression to equilibrium, which we call the Onsager analysis. With these techniques we establish the statistical equivalence of the real DNA sequences with their CMM realizations. We show that long-range correlations are present in exons as well as in introns, but are difficult to detect, since the exon ``dynamics'' is shown to be determined by the entanglement of three distinct and independent CMM's.

  2. Automation of a single-DNA molecule stretching device.

    PubMed

    Sørensen, Kristian Tølbøl; Lopacinska, Joanna M; Tommerup, Niels; Silahtaroglu, Asli; Kristensen, Anders; Marie, Rodolphe

    2015-06-01

    We automate the manipulation of genomic-length DNA in a nanofluidic device based on real-time analysis of fluorescence images. In our protocol, individual molecules are picked from a microchannel and stretched with pN forces using pressure driven flows. The millimeter-long DNA fragments free flowing in micro- and nanofluidics emit low fluorescence and change shape, thus challenging the image analysis for machine vision. We demonstrate a set of image processing steps that increase the intrinsically low signal-to-noise ratio associated with single-molecule fluorescence microscopy. Furthermore, we demonstrate how to estimate the length of molecules by continuous real-time image stitching and how to increase the effective resolution of a pressure controller by pulse width modulation. The sequence of image-processing steps addresses the challenges of genomic-length DNA visualization; however, they should also be general to other applications of fluorescence-based microfluidics. PMID:26133839

  3. Automated DNA diagnostics using an ELISA-based oligonucleotide ligation assay.

    PubMed Central

    Nickerson, D A; Kaiser, R; Lappin, S; Stewart, J; Hood, L; Landegren, U

    1990-01-01

    DNA diagnostics, the detection of specific DNA sequences, will play an increasingly important role in medicine as the molecular basis of human disease is defined. Here, we demonstrate an automated, nonisotopic strategy for DNA diagnostics using amplification of target DNA segments by the polymerase chain reaction (PCR) and the discrimination of allelic sequence variants by a colorimetric oligonucleotide ligation assay (OLA). We have applied the automated PCR/OLA procedure to diagnosis of common genetic diseases, such as sickle cell anemia and cystic fibrosis (delta F508 mutation), and to genetic linkage mapping of gene segments in the human T-cell receptor beta-chain locus. The automated PCR/OLA strategy provides a rapid system for diagnosis of genetic, malignant, and infectious diseases as well as a powerful approach to genetic linkage mapping of chromosomes and forensic DNA typing. Images PMID:2247466

  4. Channel plate for DNA sequencing

    DOEpatents

    Douthart, Richard J.; Crowell, Shannon L.

    1998-01-01

    This invention is a channel plate that facilitates data compaction in DNA sequencing. The channel plate has a length, a width and a thickness, and further has a plurality of channels that are parallel. Each channel has a depth partially through the thickness of the channel plate. Additionally an interface edge permits electrical communication across an interface through a buffer to a deposition membrane surface.

  5. Channel plate for DNA sequencing

    DOEpatents

    Douthart, R.J.; Crowell, S.L.

    1998-01-13

    This invention is a channel plate that facilitates data compaction in DNA sequencing. The channel plate has a length, a width and a thickness, and further has a plurality of channels that are parallel. Each channel has a depth partially through the thickness of the channel plate. Additionally an interface edge permits electrical communication across an interface through a buffer to a deposition membrane surface. 15 figs.

  6. DNA Sequencing Using capillary Electrophoresis

    SciTech Connect

    Dr. Barry Karger

    2011-05-09

    The overall goal of this program was to develop capillary electrophoresis as the tool to be used to sequence for the first time the Human Genome. Our program was part of the Human Genome Project. In this work, we were highly successful and the replaceable polymer we developed, linear polyacrylamide, was used by the DOE sequencing lab in California to sequence a significant portion of the human genome using the MegaBase multiple capillary array electrophoresis instrument. In this final report, we summarize our efforts and success. We began our work by separating by capillary electrophoresis double strand oligonucleotides using cross-linked polyacrylamide gels in fused silica capillaries. This work showed the potential of the methodology. However, preparation of such cross-linked gel capillaries was difficult with poor reproducibility, and even more important, the columns were not very stable. We improved stability by using non-cross linked linear polyacrylamide. Here, the entangled linear chains could move when osmotic pressure (e.g. sample injection) was imposed on the polymer matrix. This relaxation of the polymer dissipated the stress in the column. Our next advance was to use significantly lower concentrations of the linear polyacrylamide that the polymer could be automatically blown out after each run and replaced with fresh linear polymer solution. In this way, a new column was available for each analytical run. Finally, while testing many linear polymers, we selected linear polyacrylamide as the best matrix as it was the most hydrophilic polymer available. Under our DOE program, we demonstrated initially the success of the linear polyacrylamide to separate double strand DNA. We note that the method is used even today to assay purity of double stranded DNA fragments. Our focus, of course, was on the separation of single stranded DNA for sequencing purposes. In one paper, we demonstrated the success of our approach in sequencing up to 500 bases. Other

  7. Nanopore DNA sequencing with MspA

    PubMed Central

    Derrington, Ian M.; Butler, Tom Z.; Collins, Marcus D.; Manrao, Elizabeth; Pavlenok, Mikhail; Niederweis, Michael; Gundlach, Jens H.

    2010-01-01

    Nanopore sequencing has the potential to become a direct, fast, and inexpensive DNA sequencing technology. The simplest form of nanopore DNA sequencing utilizes the hypothesis that individual nucleotides of single-stranded DNA passing through a nanopore will uniquely modulate an ionic current flowing through the pore, allowing the record of the current to yield the DNA sequence. We demonstrate that the ionic current through the engineered Mycobacterium smegmatis porin A, MspA, has the ability to distinguish all four DNA nucleotides and resolve single-nucleotides in single-stranded DNA when double-stranded DNA temporarily holds the nucleotides in the pore constriction. Passing DNA with a series of double-stranded sections through MspA provides proof of principle of a simple DNA sequencing method using a nanopore. These findings highlight the importance of MspA in the future of nanopore sequencing. PMID:20798343

  8. Nanopore DNA sequencing with MspA.

    PubMed

    Derrington, Ian M; Butler, Tom Z; Collins, Marcus D; Manrao, Elizabeth; Pavlenok, Mikhail; Niederweis, Michael; Gundlach, Jens H

    2010-09-14

    Nanopore sequencing has the potential to become a direct, fast, and inexpensive DNA sequencing technology. The simplest form of nanopore DNA sequencing utilizes the hypothesis that individual nucleotides of single-stranded DNA passing through a nanopore will uniquely modulate an ionic current flowing through the pore, allowing the record of the current to yield the DNA sequence. We demonstrate that the ionic current through the engineered Mycobacterium smegmatis porin A, MspA, has the ability to distinguish all four DNA nucleotides and resolve single-nucleotides in single-stranded DNA when double-stranded DNA temporarily holds the nucleotides in the pore constriction. Passing DNA with a series of double-stranded sections through MspA provides proof of principle of a simple DNA sequencing method using a nanopore. These findings highlight the importance of MspA in the future of nanopore sequencing. PMID:20798343

  9. Scar-less multi-part DNA assembly design automation

    DOEpatents

    Hillson, Nathan J.

    2016-06-07

    The present invention provides a method of a method of designing an implementation of a DNA assembly. In an exemplary embodiment, the method includes (1) receiving a list of DNA sequence fragments to be assembled together and an order in which to assemble the DNA sequence fragments, (2) designing DNA oligonucleotides (oligos) for each of the DNA sequence fragments, and (3) creating a plan for adding flanking homology sequences to each of the DNA oligos. In an exemplary embodiment, the method includes (1) receiving a list of DNA sequence fragments to be assembled together and an order in which to assemble the DNA sequence fragments, (2) designing DNA oligonucleotides (oligos) for each of the DNA sequence fragments, and (3) creating a plan for adding optimized overhang sequences to each of the DNA oligos.

  10. Towards modeling DNA sequences as automata

    NASA Astrophysics Data System (ADS)

    Burks, Christian; Farmer, Doyne

    1984-01-01

    We seek to describe a starting point for modeling the evolution and role of DNA sequences within the framework of cellular automata by discussing the current understanding of genetic information storage in DNA sequences. This includes alternately viewing the role of DNA in living organisms as a simple scheme and as a complex scheme; a brief review of strategies for identifying and classifying patterns in DNA sequences; and finally, notes towards establishing DNA-like automata models, including a discussion of the extent of experimentally determined DNA sequence data present in the database at Los Alamos.

  11. Fluorescence-detected DNA sequencing

    SciTech Connect

    Haugland, R.P.

    1990-01-01

    Our research effort funded by this grant primarily focused on development of suitable fluorescent dyes for DNA sequencing studies. Prior to our efforts, the dyes being sued in commercial DNA sequencers were various versions of fluorescein dyes for the shorter wavelengths and of rhodamine dyes for the longer wavelengths. Our initial goal was to synthesize a set of four dyes that could all be excited by the 488 and 514 nm line of the argon laser lines and that have emission spectra that minimize spectral overlap. The specific result sought was higher fluorescent intensity, particularly of the longest wavelength dyes than was available using existing dyes. Another important property of the desired set of dyes was uniform ionic charge in order to have minimum interference on the electrophoretic mobility during the sequencing. During the period of this grant we prepared and characterized four types of dyes: fluorescent bifluorophores, derivatives of rhodamine dyes, derivatives of rhodol dyes and derivatives of boron dipyrromethene difluoride (BODIPY{trademark}) dyes.

  12. Particle sizer and DNA sequencer

    DOEpatents

    Olivares, Jose A.; Stark, Peter C.

    2005-09-13

    An electrophoretic device separates and detects particles such as DNA fragments, proteins, and the like. The device has a capillary which is coated with a coating with a low refractive index such as Teflon.RTM. AF. A sample of particles is fluorescently labeled and injected into the capillary. The capillary is filled with an electrolyte buffer solution. An electrical field is applied across the capillary causing the particles to migrate from a first end of the capillary to a second end of the capillary. A detector light beam is then scanned along the length of the capillary to detect the location of the separated particles. The device is amenable to a high throughput system by providing additional capillaries. The device can also be used to determine the actual size of the particles and for DNA sequencing.

  13. Genetic mapping and DNA sequencing

    SciTech Connect

    Speed, T.; Waterman, M.S.

    1996-12-31

    The Human Genome Initiative has as its primary objective the characterization of the human genome. High-resolution linkage maps of genetic markers will play an important role in completing the human genome project. This is one of two volumes based on the proceedings of the 1994 IMA Summer Program on Molecular Biology and comprises Weeks 1 and 2 of the four-week program. This volume focuses on genetic mapping and DNA sequencing. Selected papers are indexed separately for inclusion in the Energy Science and Technology Database.

  14. Laser desorption mass spectrometry for DNA analysis and sequencing

    SciTech Connect

    Chen, C.H.; Taranenko, N.I.; Tang, K.; Allman, S.L.

    1995-03-01

    Laser desorption mass spectrometry has been considered as a potential new method for fast DNA sequencing. Our approach is to use matrix-assisted laser desorption to produce parent ions of DNA segments and a time-of-flight mass spectrometer to identify the sizes of DNA segments. Thus, the approach is similar to gel electrophoresis sequencing using Sanger`s enzymatic method. However, gel, radioactive tagging, and dye labeling are not required. In addition, the sequencing process can possibly be finished within a few hundred microseconds instead of hours and days. In order to use mass spectrometry for fast DNA sequencing, the following three criteria need to be satisfied. They are (1) detection of large DNA segments, (2) sensitivity reaching the femtomole region, and (3) mass resolution good enough to separate DNA segments of a single nucleotide difference. It has been very difficult to detect large DNA segments by mass spectrometry before due to the fragile chemical properties of DNA and low detection sensitivity of DNA ions. We discovered several new matrices to increase the production of DNA ions. By innovative design of a mass spectrometer, we can increase the ion energy up to 45 KeV to enhance the detection sensitivity. Recently, we succeeded in detecting a DNA segment with 500 nucleotides. The sensitivity was 100 femtomole. Thus, we have fulfilled two key criteria for using mass spectrometry for fast DNA sequencing. The major effort in the near future is to improve the resolution. Different approaches are being pursued. When high resolution of mass spectrometry can be achieved and automation of sample preparation is developed, the sequencing speed to reach 500 megabases per year can be feasible.

  15. Variable copy number DNA sequences in rice.

    PubMed

    Kikuchi, S; Takaiwa, F; Oono, K

    1987-12-01

    We have cloned two types of variable copy number DNA sequences from the rice embryo genome. One of these sequences, which was cloned in pRB301, was amplified about 50-fold during callus formation and diminished in copy number to the embryonic level during regeneration. The other clone, named pRB401, showed the reciprocal pattern. The copy numbers of both sequences were changed even in the early developmental stage and eliminated from nuclear DNA along with growth of the plant. Sequencing analysis of the pRB301 insert revealed some open reading frames and direct repeat structures, but corresponding sequences were not identified in the EMBL and LASL DNA databases. Sequencing of the nuclear genomic fragment cloned in pRB401 revealed the presence of the 3'rps12-rps7 region of rice chloroplast DNA. Our observations suggest that during callus formation (dedifferentiation), regeneration and the growth process the copy numbers of some DNA sequences are variable and that nuclear integrated chloroplast DNA acts as a variable copy number sequence in the rice genome. Based on data showing a common sequence in mitochondria and chloroplast DNA of maize (Stern and Lonsdale 1982) and that the rps12 gene of tobacco chloroplast DNA is a divided gene (Torazawa et al. 1986), it is suggested that the sequence on the inverted repeat structure of chloroplast DNA may have the character of a movable genetic element. PMID:3481021

  16. The Value of DNA Sequencing - TCGA

    Cancer.gov

    DNA sequencing: what it tells us about DNA changes in cancer, how looking across many tumors will help to identify meaningful changes and potential drug targets, and how genomics is changing the way we think about cancer.

  17. Method for sequencing DNA base pairs

    DOEpatents

    Sessler, Andrew M.; Dawson, John

    1993-01-01

    The base pairs of a DNA structure are sequenced with the use of a scanning tunneling microscope (STM). The DNA structure is scanned by the STM probe tip, and, as it is being scanned, the DNA structure is separately subjected to a sequence of infrared radiation from four different sources, each source being selected to preferentially excite one of the four different bases in the DNA structure. Each particular base being scanned is subjected to such sequence of infrared radiation from the four different sources as that particular base is being scanned. The DNA structure as a whole is separately imaged for each subjection thereof to radiation from one only of each source.

  18. Fibonacci Sequence and Supramolecular Structure of DNA.

    PubMed

    Shabalkin, I P; Grigor'eva, E Yu; Gudkova, M V; Shabalkin, P I

    2016-05-01

    We proposed a new model of supramolecular DNA structure. Similar to the previously developed by us model of primary DNA structure [11-15], 3D structure of DNA molecule is assembled in accordance to a mathematic rule known as Fibonacci sequence. Unlike primary DNA structure, supramolecular 3D structure is assembled from complex moieties including a regular tetrahedron and a regular octahedron consisting of monomers, elements of the primary DNA structure. The moieties of the supramolecular DNA structure forming fragments of regular spatial lattice are bound via linker (joint) sequences of the DNA chain. The lattice perceives and transmits information signals over a considerable distance without acoustic aberrations. Linker sequences expand conformational space between lattice segments allowing their sliding relative to each other under the action of external forces. In this case, sliding is provided by stretching of the stacked linker sequences. PMID:27265133

  19. Sequence and Structure Dependent DNA-DNA Interactions

    NASA Astrophysics Data System (ADS)

    Kopchick, Benjamin; Qiu, Xiangyun

    Molecular forces between dsDNA strands are largely dominated by electrostatics and have been extensively studied. Quantitative knowledge has been accumulated on how DNA-DNA interactions are modulated by varied biological constituents such as ions, cationic ligands, and proteins. Despite its central role in biology, the sequence of DNA has not received substantial attention and ``random'' DNA sequences are typically used in biophysical studies. However, ~50% of human genome is composed of non-random-sequence DNAs, particularly repetitive sequences. Furthermore, covalent modifications of DNA such as methylation play key roles in gene functions. Such DNAs with specific sequences or modifications often take on structures other than the canonical B-form. Here we present series of quantitative measurements of the DNA-DNA forces with the osmotic stress method on different DNA sequences, from short repeats to the most frequent sequences in genome, and to modifications such as bromination and methylation. We observe peculiar behaviors that appear to be strongly correlated with the incurred structural changes. We speculate the causalities in terms of the differences in hydration shell and DNA surface structures.

  20. Atypical regions in large genomic DNA sequences

    SciTech Connect

    Scherer, S. |; McPeek, M.S.; Speed, T.P.

    1994-07-19

    Large genomic DNA sequences contain regions with distinctive patterns of sequence organization. The authors describe a method using logarithms of probabilities based on seventh-order Markov chains to rapidly identify genomic sequences that do not resemble models of genome organization built from compilations of octanucleotide usage. Data bases have been constructed from Escherichia coli and Saccharomyces cerevisiae DNA sequences of >1000 nt and human sequences of >10,000 nt. Atypical genes and clusters of genes have been located in bacteriophage, yeast, and primate DNA sequences. The authors consider criteria for statistical significance of the results, offer possible explanations for the observed variation in genome organization, and give additional applications of these methods in DNA sequence analysis.

  1. Sequence Affects the Cyclization of DNA Minicircles.

    PubMed

    Wang, Qian; Pettitt, B Montgomery

    2016-03-17

    Understanding how the sequence of a DNA molecule affects its dynamic properties is a central problem affecting biochemistry and biotechnology. The process of cyclizing short DNA, as a critical step in molecular cloning, lacks a comprehensive picture of the kinetic process containing sequence information. We have elucidated this process by using coarse-grained simulations, enhanced sampling methods, and recent theoretical advances. We are able to identify the types and positions of structural defects during the looping process at a base-pair level. Correlations along a DNA molecule dictate critical sequence positions that can affect the looping rate. Structural defects change the bending elasticity of the DNA molecule from a harmonic to subharmonic potential with respect to bending angles. We explore the subelastic chain as a possible model in loop formation kinetics. A sequence-dependent model is developed to qualitatively predict the relative loop formation time as a function of DNA sequence. PMID:26938490

  2. Using DNA looping to measure sequence dependent DNA elasticity

    NASA Astrophysics Data System (ADS)

    Kandinov, Alan; Raghunathan, Krishnan; Meiners, Jens-Christian

    2012-10-01

    We are using tethered particle motion (TPM) microscopy to observe protein-mediated DNA looping in the lactose repressor system in DNA constructs with varying AT / CG content. We use these data to determine the persistence length of the DNA as a function of its sequence content and compare the data to direct micromechanical measurements with constant-force axial optical tweezers. The data from the TPM experiments show a much smaller sequence effect on the persistence length than the optical tweezers experiments.

  3. Multiple tag labeling method for DNA sequencing

    DOEpatents

    Mathies, R.A.; Huang, X.C.; Quesada, M.A.

    1995-07-25

    A DNA sequencing method is described which uses single lane or channel electrophoresis. Sequencing fragments are separated in the lane and detected using a laser-excited, confocal fluorescence scanner. Each set of DNA sequencing fragments is separated in the same lane and then distinguished using a binary coding scheme employing only two different fluorescent labels. Also described is a method of using radioisotope labels. 5 figs.

  4. Multiple tag labeling method for DNA sequencing

    DOEpatents

    Mathies, Richard A.; Huang, Xiaohua C.; Quesada, Mark A.

    1995-01-01

    A DNA sequencing method described which uses single lane or channel electrophoresis. Sequencing fragments are separated in said lane and detected using a laser-excited, confocal fluorescence scanner. Each set of DNA sequencing fragments is separated in the same lane and then distinguished using a binary coding scheme employing only two different fluorescent labels. Also described is a method of using radio-isotope labels.

  5. Small scale sequence automation pays big dividends

    NASA Technical Reports Server (NTRS)

    Nelson, Bill

    1994-01-01

    Galileo sequence design and integration are supported by a suite of formal software tools. Sequence review, however, is largely a manual process with reviewers scanning hundreds of pages of cryptic computer printouts to verify sequence correctness. Beginning in 1990, a series of small, PC based sequence review tools evolved. Each tool performs a specific task but all have a common 'look and feel'. The narrow focus of each tool means simpler operation, and easier creation, testing, and maintenance. Benefits from these tools are (1) decreased review time by factors of 5 to 20 or more with a concomitant reduction in staffing, (2) increased review accuracy, and (3) excellent returns on time invested.

  6. Fractal analysis of DNA sequence data

    SciTech Connect

    Berthelsen, C.L.

    1993-01-01

    DNA sequence databases are growing at an almost exponential rate. New analysis methods are needed to extract knowledge about the organization of nucleotides from this vast amount of data. Fractal analysis is a new scientific paradigm that has been used successfully in many domains including the biological and physical sciences. Biological growth is a nonlinear dynamic process and some have suggested that to consider fractal geometry as a biological design principle may be most productive. This research is an exploratory study of the application of fractal analysis to DNA sequence data. A simple random fractal, the random walk, is used to represent DNA sequences. The fractal dimension of these walks is then estimated using the [open quote]sandbox method[close quote]. Analysis of 164 human DNA sequences compared to three types of control sequences (random, base-content matched, and dimer-content matched) reveals that long-range correlations are present in DNA that are not explained by base or dimer frequencies. The study also revealed that the fractal dimension of coding sequences was significantly lower than sequences that were primarily noncoding, indicating the presence of longer-range correlations in functional sequences. The multifractal spectrum is used to analyze fractals that are heterogeneous and have a different fractal dimension for subsets with different scalings. The multifractal spectrum of the random walks of twelve mitochondrial genome sequences was estimated. Eight vertebrate mtDNA sequences had uniformly lower spectra values than did four invertebrate mtDNA sequences. Thus, vertebrate mitochondria show significantly longer-range correlations than to invertebrate mitochondria. The higher multifractal spectra values for invertebrate mitochondria suggest a more random organization of the sequences. This research also includes considerable theoretical work on the effects of finite size, embedding dimension, and scaling ranges.

  7. Fractal Analysis of DNA Sequence Data

    NASA Astrophysics Data System (ADS)

    Berthelsen, Cheryl Lynn

    DNA sequence databases are growing at an almost exponential rate. New analysis methods are needed to extract knowledge about the organization of nucleotides from this vast amount of data. Fractal analysis is a new scientific paradigm that has been used successfully in many domains including the biological and physical sciences. Biological growth is a nonlinear dynamic process and some have suggested that to consider fractal geometry as a biological design principle may be most productive. This research is an exploratory study of the application of fractal analysis to DNA sequence data. A simple random fractal, the random walk, is used to represent DNA sequences. The fractal dimension of these walks is then estimated using the "sandbox method." Analysis of 164 human DNA sequences compared to three types of control sequences (random, base -content matched, and dimer-content matched) reveals that long-range correlations are present in DNA that are not explained by base or dimer frequencies. The study also revealed that the fractal dimension of coding sequences was significantly lower than sequences that were primarily noncoding, indicating the presence of longer-range correlations in functional sequences. The multifractal spectrum is used to analyze fractals that are heterogeneous and have a different fractal dimension for subsets with different scalings. The multifractal spectrum of the random walks of twelve mitochondrial genome sequences was estimated. Eight vertebrate mtDNA sequences had uniformly lower spectra values than did four invertebrate mtDNA sequences. Thus, vertebrate mitochondria show significantly longer-range correlations than do invertebrate mitochondria. The higher multifractal spectra values for invertebrate mitochondria suggest a more random organization of the sequences. This research also includes considerable theoretical work on the effects of finite size, embedding dimension, and scaling ranges.

  8. 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

  9. DNA sequencing: bench to bedside and beyond†

    PubMed Central

    Hutchison, Clyde A.

    2007-01-01

    Fifteen years elapsed between the discovery of the double helix (1953) and the first DNA sequencing (1968). Modern DNA sequencing began in 1977, with development of the chemical method of Maxam and Gilbert and the dideoxy method of Sanger, Nicklen and Coulson, and with the first complete DNA sequence (phage ϕX174), which demonstrated that sequence could give profound insights into genetic organization. Incremental improvements allowed sequencing of molecules >200 kb (human cytomegalovirus) leading to an avalanche of data that demanded computational analysis and spawned the field of bioinformatics. The US Human Genome Project spurred sequencing activity. By 1992 the first ‘sequencing factory’ was established, and others soon followed. The first complete cellular genome sequences, from bacteria, appeared in 1995 and other eubacterial, archaebacterial and eukaryotic genomes were soon sequenced. Competition between the public Human Genome Project and Celera Genomics produced working drafts of the human genome sequence, published in 2001, but refinement and analysis of the human genome sequence will continue for the foreseeable future. New ‘massively parallel’ sequencing methods are greatly increasing sequencing capacity, but further innovations are needed to achieve the ‘thousand dollar genome’ that many feel is prerequisite to personalized genomic medicine. These advances will also allow new approaches to a variety of problems in biology, evolution and the environment. PMID:17855400

  10. Automated shielding analysis sequences for spent fuel casks

    SciTech Connect

    Tang, J.S.; Parks, C.V.; Hermann, O.W.

    1987-01-01

    Two important Shielding Analysis Sequences (SAS) have recently been developed within the SCALE computational system. These sequences significantly enhance the existing SCALE system capabilities for evaluating radiation doses exterior to spent fuel casks. These new control module sequences (SAS1 and SAS4) and their capabilities are discussed and demonstrated, together with the existing SAS2 sequence that is used to generate radiation sources for spent fuel. Particular attention is given to the new SAS4 sequence which provides an automated scheme for generating and using biasing parameters in a subsequent Monte Carlo analysis of a cask.

  11. Applications of mass spectrometry to DNA fingerprinting and DNA sequencing

    SciTech Connect

    Jacobson, K.B.; Buchanan, M.V.; Chen, C.H.; Doktycz, M.J.; McLuckey, S.A. ); Arlinghaus, H.F. )

    1993-01-01

    DNA fingerprinting and sequencing rely on polyacrylamide gel electrophoresis to determine the sizes of the DNA fragments. Innovative altematives to polyacrylamide gel electrophoresis are under investigation for characterization of such fingerprinting and sequencing. One method uses stable isotopes of tin and other elements to label the DNAwhereas other procedures do not require labels. The detectors in each case are mass spectrometers that detect either the stable isotopes or the DNA fragments themselves. If successful, these methods will speed up the rate of DNA analysis by one or two orders of magnitude.

  12. Applications of mass spectrometry to DNA fingerprinting and DNA sequencing

    SciTech Connect

    Jacobson, K.B.; Buchanan, M.V.; Chen, C.H.; Doktycz, M.J.; McLuckey, S.A.; Arlinghaus, H.F.

    1993-06-01

    DNA fingerprinting and sequencing rely on polyacrylamide gel electrophoresis to determine the sizes of the DNA fragments. Innovative altematives to polyacrylamide gel electrophoresis are under investigation for characterization of such fingerprinting and sequencing. One method uses stable isotopes of tin and other elements to label the DNAwhereas other procedures do not require labels. The detectors in each case are mass spectrometers that detect either the stable isotopes or the DNA fragments themselves. If successful, these methods will speed up the rate of DNA analysis by one or two orders of magnitude.

  13. Data management for re-sequencing DNA

    SciTech Connect

    Ying Jiahsu; Gilson, H.; Long, K.; Gibbs, R.A.

    1993-12-31

    The human genome project has greatly stimulated the advancement of techniques to sequence large fragments of DNA. The development of improved molecular methods has also simplified the process of comparing shorter, homologous DNA sequences from different individuals and species. This process of `re-sequencing` DNA has applications in medical genetics, in evolutionary studies, and for the identification of complex molecular variation that may explain multifactorial traits. Intrinsic differences in the processes of `sequencing` and `re-sequencing` suggest new requirements for data management tools. A data management scheme for a `re-sequencing` project is demonstrated using the Virtual Notebook System, a flexible multi-user tool designed as a metaphor of the laboratory notebook.

  14. Automated Selection Of Pictures In Sequences

    NASA Technical Reports Server (NTRS)

    Rorvig, Mark E.; Shelton, Robert O.

    1995-01-01

    Method of automated selection of film or video motion-picture frames for storage or examination developed. Beneficial in situations in which quantity of visual information available exceeds amount stored or examined by humans in reasonable amount of time, and/or necessary to reduce large number of motion-picture frames to few conveying significantly different information in manner intermediate between movie and comic book or storyboard. For example, computerized vision system monitoring industrial process programmed to sound alarm when changes in scene exceed normal limits.

  15. Automated correction of genome sequence errors

    PubMed Central

    Gajer, Pawel; Schatz, Michael; Salzberg, Steven L.

    2004-01-01

    By using information from an assembly of a genome, a new program called AutoEditor significantly improves base calling accuracy over that achieved by previous algorithms. This in turn improves the overall accuracy of genome sequences and facilitates the use of these sequences for polymorphism discovery. We describe the algorithm and its application in a large set of recent genome sequencing projects. The number of erroneous base calls in these projects was reduced by 80%. In an analysis of over one million corrections, we found that AutoEditor made just one error per 8828 corrections. By substantially increasing the accuracy of base calling, AutoEditor can dramatically accelerate the process of finishing genomes, which involves closing all gaps and ensuring minimum quality standards for the final sequence. It also greatly improves our ability to discover single nucleotide polymorphisms (SNPs) between closely related strains and isolates of the same species. PMID:14744981

  16. Method for sequencing DNA base pairs

    DOEpatents

    Sessler, A.M.; Dawson, J.

    1993-12-14

    The base pairs of a DNA structure are sequenced with the use of a scanning tunneling microscope (STM). The DNA structure is scanned by the STM probe tip, and, as it is being scanned, the DNA structure is separately subjected to a sequence of infrared radiation from four different sources, each source being selected to preferentially excite one of the four different bases in the DNA structure. Each particular base being scanned is subjected to such sequence of infrared radiation from the four different sources as that particular base is being scanned. The DNA structure as a whole is separately imaged for each subjection thereof to radiation from one only of each source. 6 figures.

  17. Nucleotide sequence of bacteriophage fd DNA.

    PubMed Central

    Beck, E; Sommer, R; Auerswald, E A; Kurz, C; Zink, B; Osterburg, G; Schaller, H; Sugimoto, K; Sugisaki, H; Okamoto, T; Takanami, M

    1978-01-01

    The sequence of the 6,408 nucleotides of bacteriophage fd DNA has been determined. This allows to deduce the exact organisation of the filamentous phage genome and provides easy access to DNA segments of known structure and function. PMID:745987

  18. Streamlining DNA Barcoding Protocols: Automated DNA Extraction and a New cox1 Primer in Arachnid Systematics

    PubMed Central

    Vidergar, Nina; Toplak, Nataša; Kuntner, Matjaž

    2014-01-01

    Background DNA barcoding is a popular tool in taxonomic and phylogenetic studies, but for most animal lineages protocols for obtaining the barcoding sequences—mitochondrial cytochrome C oxidase subunit I (cox1 AKA CO1)—are not standardized. Our aim was to explore an optimal strategy for arachnids, focusing on the species-richest lineage, spiders by (1) improving an automated DNA extraction protocol, (2) testing the performance of commonly used primer combinations, and (3) developing a new cox1 primer suitable for more efficient alignment and phylogenetic analyses. Methodology We used exemplars of 15 species from all major spider clades, processed a range of spider tissues of varying size and quality, optimized genomic DNA extraction using the MagMAX Express magnetic particle processor—an automated high throughput DNA extraction system—and tested cox1 amplification protocols emphasizing the standard barcoding region using ten routinely employed primer pairs. Results The best results were obtained with the commonly used Folmer primers (LCO1490/HCO2198) that capture the standard barcode region, and with the C1-J-2183/C1-N-2776 primer pair that amplifies its extension. However, C1-J-2183 is designed too close to HCO2198 for well-interpreted, continuous sequence data, and in practice the resulting sequences from the two primer pairs rarely overlap. We therefore designed a new forward primer C1-J-2123 60 base pairs upstream of the C1-J-2183 binding site. The success rate of this new primer (93%) matched that of C1-J-2183. Conclusions The use of C1-J-2123 allows full, indel-free overlap of sequences obtained with the standard Folmer primers and with C1-J-2123 primer pair. Our preliminary tests suggest that in addition to spiders, C1-J-2123 will also perform in other arachnids and several other invertebrates. We provide optimal PCR protocols for these primer sets, and recommend using them for systematic efforts beyond DNA barcoding. PMID:25415202

  19. DNA sequencing using fluorescence background electroblotting membrane

    DOEpatents

    Caldwell, K.D.; Chu, T.J.; Pitt, W.G.

    1992-05-12

    A method for the multiplex sequencing on DNA is disclosed which comprises the electroblotting or specific base terminated DNA fragments, which have been resolved by gel electrophoresis, onto the surface of a neutral non-aromatic polymeric microporous membrane exhibiting low background fluorescence which has been surface modified to contain amino groups. Polypropylene membranes are preferably and the introduction of amino groups is accomplished by subjecting the membrane to radio or microwave frequency plasma discharge in the presence of an aminating agent, preferably ammonia. The membrane, containing physically adsorbed DNA fragments on its surface after the electroblotting, is then treated with crosslinking means such as UV radiation or a glutaraldehyde spray to chemically bind the DNA fragments to the membrane through amino groups contained on the surface. The DNA fragments chemically bound to the membrane are subjected to hybridization probing with a tagged probe specific to the sequence of the DNA fragments. The tagging may be by either fluorophores or radioisotopes. The tagged probes hybridized to the target DNA fragments are detected and read by laser induced fluorescence detection or autoradiograms. The use of aminated low fluorescent background membranes allows the use of fluorescent detection and reading even when the available amount of DNA to be sequenced is small. The DNA bound to the membranes may be reprobed numerous times. No Drawings

  20. DNA sequencing using fluorescence background electroblotting membrane

    DOEpatents

    Caldwell, Karin D.; Chu, Tun-Jen; Pitt, William G.

    1992-01-01

    A method for the multiplex sequencing on DNA is disclosed which comprises the electroblotting or specific base terminated DNA fragments, which have been resolved by gel electrophoresis, onto the surface of a neutral non-aromatic polymeric microporous membrane exhibiting low background fluorescence which has been surface modified to contain amino groups. Polypropylene membranes are preferably and the introduction of amino groups is accomplished by subjecting the membrane to radio or microwave frequency plasma discharge in the presence of an aminating agent, preferably ammonia. The membrane, containing physically adsorbed DNA fragments on its surface after the electroblotting, is then treated with crosslinking means such as UV radiation or a glutaraldehyde spray to chemically bind the DNA fragments to the membrane through said smino groups contained on the surface thereof. The DNA fragments chemically bound to the membrane are subjected to hybridization probing with a tagged probe specific to the sequence of the DNA fragments. The tagging may be by either fluorophores or radioisotopes. The tagged probes hybridized to said target DNA fragments are detected and read by laser induced fluorescence detection or autoradiograms. The use of aminated low fluorescent background membranes allows the use of fluorescent detection and reading even when the available amount of DNA to be sequenced is small. The DNA bound to the membrances may be reprobed numerous times.

  1. Automated Sequence Preprocessing in a Large-Scale Sequencing Environment

    PubMed Central

    Wendl, Michael C.; Dear, Simon; Hodgson, Dave; Hillier, LaDeana

    1998-01-01

    A software system for transforming fragments from four-color fluorescence-based gel electrophoresis experiments into assembled sequence is described. It has been developed for large-scale processing of all trace data, including shotgun and finishing reads, regardless of clone origin. Design considerations are discussed in detail, as are programming implementation and graphic tools. The importance of input validation, record tracking, and use of base quality values is emphasized. Several quality analysis metrics are proposed and applied to sample results from recently sequenced clones. Such quantities prove to be a valuable aid in evaluating modifications of sequencing protocol. The system is in full production use at both the Genome Sequencing Center and the Sanger Centre, for which combined weekly production is ∼100,000 sequencing reads per week. PMID:9750196

  2. Model annotation for synthetic biology: automating model to nucleotide sequence conversion

    PubMed Central

    Misirli, Goksel; Hallinan, Jennifer S.; Yu, Tommy; Lawson, James R.; Wimalaratne, Sarala M.; Cooling, Michael T.; Wipat, Anil

    2011-01-01

    Motivation: The need for the automated computational design of genetic circuits is becoming increasingly apparent with the advent of ever more complex and ambitious synthetic biology projects. Currently, most circuits are designed through the assembly of models of individual parts such as promoters, ribosome binding sites and coding sequences. These low level models are combined to produce a dynamic model of a larger device that exhibits a desired behaviour. The larger model then acts as a blueprint for physical implementation at the DNA level. However, the conversion of models of complex genetic circuits into DNA sequences is a non-trivial undertaking due to the complexity of mapping the model parts to their physical manifestation. Automating this process is further hampered by the lack of computationally tractable information in most models. Results: We describe a method for automatically generating DNA sequences from dynamic models implemented in CellML and Systems Biology Markup Language (SBML). We also identify the metadata needed to annotate models to facilitate automated conversion, and propose and demonstrate a method for the markup of these models using RDF. Our algorithm has been implemented in a software tool called MoSeC. Availability: The software is available from the authors' web site http://research.ncl.ac.uk/synthetic_biology/downloads.html. Contact: anil.wipat@ncl.ac.uk Supplementary information: Supplementary data are available at Bioinformatics online. PMID:21296753

  3. Automated serial extraction of DNA and RNA from biobanked tissue specimens

    PubMed Central

    2013-01-01

    Background With increasing biobanking of biological samples, methods for large scale extraction of nucleic acids are in demand. The lack of such techniques designed for extraction from tissues results in a bottleneck in downstream genetic analyses, particularly in the field of cancer research. We have developed an automated procedure for tissue homogenization and extraction of DNA and RNA into separate fractions from the same frozen tissue specimen. A purpose developed magnetic bead based technology to serially extract both DNA and RNA from tissues was automated on a Tecan Freedom Evo robotic workstation. Results 864 fresh-frozen human normal and tumor tissue samples from breast and colon were serially extracted in batches of 96 samples. Yields and quality of DNA and RNA were determined. The DNA was evaluated in several downstream analyses, and the stability of RNA was determined after 9 months of storage. The extracted DNA performed consistently well in processes including PCR-based STR analysis, HaloPlex selection and deep sequencing on an Illumina platform, and gene copy number analysis using microarrays. The RNA has performed well in RT-PCR analyses and maintains integrity upon storage. Conclusions The technology described here enables the processing of many tissue samples simultaneously with a high quality product and a time and cost reduction for the user. This reduces the sample preparation bottleneck in cancer research. The open automation format also enables integration with upstream and downstream devices for automated sample quantitation or storage. PMID:23957867

  4. The expanding scope of DNA sequencing

    PubMed Central

    Shendure, Jay; Aiden, Erez Lieberman

    2014-01-01

    In just seven years, next-generation technologies have reduced the cost and increased the speed of DNA sequencing by four orders of magnitude, and experiments requiring many millions of sequencing reads are now routine. In research, sequencing is being applied not only to assemble genomes and to investigate the genetic basis of human disease, but also to explore myriad phenomena in organismic and cellular biology. In the clinic, the utility of sequence data is being intensively evaluated in diverse contexts, including reproductive medicine, oncology and infectious disease. A recurrent theme in the development of new sequencing applications is the creative ‘recombination’ of existing experimental building blocks. However, there remain many potentially high-impact applications of next-generation DNA sequencing that are not yet fully realized. PMID:23138308

  5. Sequencing Intractable DNA to Close Microbial Genomes

    SciTech Connect

    Hurt, Jr., Richard Ashley; Brown, Steven D; Podar, Mircea; Palumbo, Anthony Vito; Elias, Dwayne A

    2012-01-01

    Advancement in high throughput DNA sequencing technologies has supported a rapid proliferation of microbial genome sequencing projects, providing the genetic blueprint for for in-depth studies. Oftentimes, difficult to sequence regions in microbial genomes are ruled intractable resulting in a growing number of genomes with sequence gaps deposited in databases. A procedure was developed to sequence such difficult regions in the non-contiguous finished Desulfovibrio desulfuricans ND132 genome (6 intractable gaps) and the Desulfovibrio africanus genome (1 intractable gap). The polynucleotides surrounding each gap formed GC rich secondary structures making the regions refractory to amplification and sequencing. Strand-displacing DNA polymerases used in concert with a novel ramped PCR extension cycle supported amplification and closure of all gap regions in both genomes. These developed procedures support accurate gene annotation, and provide a step-wise method that reduces the effort required for genome finishing.

  6. Osmylated DNA, a novel concept for sequencing DNA using nanopores

    NASA Astrophysics Data System (ADS)

    Kanavarioti, Anastassia

    2015-03-01

    Saenger sequencing has led the advances in molecular biology, while faster and cheaper next generation technologies are urgently needed. A newer approach exploits nanopores, natural or solid-state, set in an electrical field, and obtains base sequence information from current variations due to the passage of a ssDNA molecule through the pore. A hurdle in this approach is the fact that the four bases are chemically comparable to each other which leads to small differences in current obstruction. ‘Base calling’ becomes even more challenging because most nanopores sense a short sequence and not individual bases. Perhaps sequencing DNA via nanopores would be more manageable, if only the bases were two, and chemically very different from each other; a sequence of 1s and 0s comes to mind. Osmylated DNA comes close to such a sequence of 1s and 0s. Osmylation is the addition of osmium tetroxide bipyridine across the C5-C6 double bond of the pyrimidines. Osmylation adds almost 400% mass to the reactive base, creates a sterically and electronically notably different molecule, labeled 1, compared to the unreactive purines, labeled 0. If osmylated DNA were successfully sequenced, the result would be a sequence of osmylated pyrimidines (1), and purines (0), and not of the actual nucleobases. To solve this problem we studied the osmylation reaction with short oligos and with M13mp18, a long ssDNA, developed a UV-vis assay to measure extent of osmylation, and designed two protocols. Protocol A uses mild conditions and yields osmylated thymidines (1), while leaving the other three bases (0) practically intact. Protocol B uses harsher conditions and effectively osmylates both pyrimidines, but not the purines. Applying these two protocols also to the complementary of the target polynucleotide yields a total of four osmylated strands that collectively could define the actual base sequence of the target DNA.

  7. Dynamics and control of DNA sequence amplification

    SciTech Connect

    Marimuthu, Karthikeyan; Chakrabarti, Raj E-mail: rajc@andrew.cmu.edu

    2014-10-28

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

  8. Dynamics and control of DNA sequence amplification

    NASA Astrophysics Data System (ADS)

    Marimuthu, Karthikeyan; Chakrabarti, Raj

    2014-10-01

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

  9. Quadruplex DNA: sequence, topology and structure

    PubMed Central

    Burge, Sarah; Parkinson, Gary N.; Hazel, Pascale; Todd, Alan K.; Neidle, Stephen

    2006-01-01

    G-quadruplexes are higher-order DNA and RNA structures formed from G-rich sequences that are built around tetrads of hydrogen-bonded guanine bases. Potential quadruplex sequences have been identified in G-rich eukaryotic telomeres, and more recently in non-telomeric genomic DNA, e.g. in nuclease-hypersensitive promoter regions. The natural role and biological validation of these structures is starting to be explored, and there is particular interest in them as targets for therapeutic intervention. This survey focuses on the folding and structural features on quadruplexes formed from telomeric and non-telomeric DNA sequences, and examines fundamental aspects of topology and the emerging relationships with sequence. Emphasis is placed on information from the high-resolution methods of X-ray crystallography and NMR, and their scope and current limitations are discussed. Such information, together with biological insights, will be important for the discovery of drugs targeting quadruplexes from particular genes. PMID:17012276

  10. A test matrix sequencer for research test facility automation

    NASA Technical Reports Server (NTRS)

    Mccartney, Timothy P.; Emery, Edward F.

    1990-01-01

    The hardware and software configuration of a Test Matrix Sequencer, a general purpose test matrix profiler that was developed for research test facility automation at the NASA Lewis Research Center, is described. The system provides set points to controllers and contact closures to data systems during the course of a test. The Test Matrix Sequencer consists of a microprocessor controlled system which is operated from a personal computer. The software program, which is the main element of the overall system is interactive and menu driven with pop-up windows and help screens. Analog and digital input/output channels can be controlled from a personal computer using the software program. The Test Matrix Sequencer provides more efficient use of aeronautics test facilities by automating repetitive tasks that were once done manually.

  11. Sequencing mitochondrial DNA polymorphisms by hybridization

    SciTech Connect

    Chee, M.S.; Lockhart, D.J.; Hubbell, E.

    1994-09-01

    We have investigated the use of DNA chips for genetic analysis, using human mitochondrial DNA (mtDNA) as a model. The DNA chips are made up of ordered arrays of DNA oligonucleotide probes, synthesized on a glass substrate using photolithographic techniques. The synthesis site for each different probe is specifically addressed by illumination of the substrate through a photolithographic mask, achieving selective deprotection Nucleoside phosphoramidites bearing photolabile protecting groups are coupled only to exposed sites. Repeated cycles of deprotection and coupling generate all the probes in parallel. The set of 4{sup N} N-mer probes can be synthesized in only 4N steps. Any subset can be synthesized in 4N steps. Any subset can be synthesized in 4N or fewer steps. Sequences amplified from the D-loop region of human mitochondrial DNA (mtDNA) were fluorescently labelled and hybridized to DNA chips containing probes specific for mtDNA. Each nucleotide of a 1.3 kb region spanning the D loop is represented by four probes on the chip. Each probe has a different base at the position of interest: together they comprise a set of A, C, G and T probes which are otherwise identical. In principle, only one probe-target hybrid will be a perfect match. The other three will be single base mismatches. Fluorescence imaging of the hybridized chip allows quantification of hybridization signals. Heterozygous mixtures of sequences can also be characterized. We have developed software to quantitate and interpret the hybridization signals, and to call the sequence automatically. Results of sequence analysis of human mtDNAs will be presented.

  12. Quantum-Sequencing: Fast electronic single DNA molecule sequencing

    NASA Astrophysics Data System (ADS)

    Casamada Ribot, Josep; Chatterjee, Anushree; Nagpal, Prashant

    2014-03-01

    A major goal of third-generation sequencing technologies is to develop a fast, reliable, enzyme-free, high-throughput and cost-effective, single-molecule sequencing method. Here, we present the first demonstration of unique ``electronic fingerprint'' of all nucleotides (A, G, T, C), with single-molecule DNA sequencing, using Quantum-tunneling Sequencing (Q-Seq) at room temperature. We show that the electronic state of the nucleobases shift depending on the pH, with most distinct states identified at acidic pH. We also demonstrate identification of single nucleotide modifications (methylation here). Using these unique electronic fingerprints (or tunneling data), we report a partial sequence of beta lactamase (bla) gene, which encodes resistance to beta-lactam antibiotics, with over 95% success rate. These results highlight the potential of Q-Seq as a robust technique for next-generation sequencing.

  13. Automated screening for small organic ligands using DNA-encoded chemical libraries.

    PubMed

    Decurtins, Willy; Wichert, Moreno; Franzini, Raphael M; Buller, Fabian; Stravs, Michael A; Zhang, Yixin; Neri, Dario; Scheuermann, Jörg

    2016-04-01

    DNA-encoded chemical libraries (DECLs) are collections of organic compounds that are individually linked to different oligonucleotides, serving as amplifiable identification barcodes. As all compounds in the library can be identified by their DNA tags, they can be mixed and used in affinity-capture experiments on target proteins of interest. In this protocol, we describe the screening process that allows the identification of the few binding molecules within the multiplicity of library members. First, the automated affinity selection process physically isolates binding library members. Second, the DNA codes of the isolated binders are PCR-amplified and subjected to high-throughput DNA sequencing. Third, the obtained sequencing data are evaluated using a C++ program and the results are displayed using MATLAB software. The resulting selection fingerprints facilitate the discrimination of binding from nonbinding library members. The described procedures allow the identification of small organic ligands to biological targets from a DECL within 10 d. PMID:26985574

  14. Automated carboxy-terminal sequence analysis of peptides.

    PubMed Central

    Bailey, J. M.; Shenoy, N. R.; Ronk, M.; Shively, J. E.

    1992-01-01

    Proteins and peptides can be sequenced from the carboxy-terminus with isothiocyanate reagents to produce amino acid thiohydantoin derivatives. Previous studies in our laboratory have focused on solution phase conditions for formation of the peptidylthiohydantoins with trimethylsilylisothiocyanate (TMS-ITC) and for hydrolysis of these peptidylthiohydantoins into an amino acid thiohydantoin derivative and a new shortened peptide capable of continued degradation (Bailey, J. M. & Shively, J. E., 1990, Biochemistry 29, 3145-3156). The current study is a continuation of this work and describes the construction of an instrument for automated C-terminal sequencing, the application of the thiocyanate chemistry to peptides covalently coupled to a novel polyethylene solid support (Shenoy, N. R., Bailey, J. M., & Shively, J. E., 1992, Protein Sci. I, 58-67), the use of sodium trimethylsilanolate as a novel reagent for the specific cleavage of the derivatized C-terminal amino acid, and the development of methodology to sequence through the difficult amino acid, aspartate. Automated programs are described for the C-terminal sequencing of peptides covalently attached to carboxylic acid-modified polyethylene. The chemistry involves activation with acetic anhydride, derivatization with TMS-ITC, and cleavage of the derivatized C-terminal amino acid with sodium trimethylsilanolate. The thiohydantoin amino acid is identified by on-line high performance liquid chromatography using a Phenomenex Ultracarb 5 ODS(30) column and a triethylamine/phosphoric acid buffer system containing pentanesulfonic acid. The generality of our automated C-terminal sequencing methodology was examined by sequencing model peptides containing all 20 of the common amino acids. All of the amino acids were found to sequence in high yield (90% or greater) except for asparagine and aspartate, which could be only partially removed, and proline, which was found not be capable of derivatization. In spite of these

  15. Fluorogenic DNA Sequencing in PDMS Microreactors

    PubMed Central

    Sims, Peter A.; Greenleaf, William J.; Duan, Haifeng; Xie, X. Sunney

    2012-01-01

    We have developed a multiplex sequencing-by-synthesis method combining terminal-phosphate labeled fluorogenic nucleotides (TPLFNs) and resealable microreactors. In the presence of phosphatase, the incorporation of a non-fluorescent TPLFN into a DNA primer by DNA polymerase results in a fluorophore. We immobilize DNA templates within polydimethylsiloxane (PDMS) microreactors, sequentially introduce one of the four identically labeled TPLFNs, seal the microreactors, allow template-directed TPLFN incorporation, and measure the signal from the fluorophores trapped in the microreactors. This workflow allows sequencing in a manner akin to pyrosequencing but without constant monitoring of each microreactor. With cycle times of <10 minutes, we demonstrate 30 base reads with ∼99% raw accuracy. “Fluorogenic pyrosequencing” combines benefits of pyrosequencing, such as rapid turn-around, native DNA generation, and single-color detection, with benefits of fluorescence-based approaches, such as highly sensitive detection and simple parallelization. PMID:21666670

  16. Statistical and linguistic features of DNA sequences

    NASA Technical Reports Server (NTRS)

    Havlin, S.; Buldyrev, S. V.; Goldberger, A. L.; Mantegna, R. N.; Peng, C. K.; Simons, M.; Stanley, H. E.

    1995-01-01

    We present evidence supporting the idea that the DNA sequence in genes containing noncoding regions is correlated, and that the correlation is remarkably long range--indeed, base pairs thousands of base pairs distant are correlated. We do not find such a long-range correlation in the coding regions of the gene. We resolve the problem of the "non-stationary" feature of the sequence of base pairs by applying a new algorithm called Detrended Fluctuation Analysis (DFA). We address the claim of Voss that there is no difference in the statistical properties of coding and noncoding regions of DNA by systematically applying the DFA algorithm, as well as standard FFT analysis, to all eukaryotic DNA sequences (33 301 coding and 29 453 noncoding) in the entire GenBank database. We describe a simple model to account for the presence of long-range power-law correlations which is based upon a generalization of the classic Levy walk. Finally, we describe briefly some recent work showing that the noncoding sequences have certain statistical features in common with natural languages. Specifically, we adapt to DNA the Zipf approach to analyzing linguistic texts, and the Shannon approach to quantifying the "redundancy" of a linguistic text in terms of a measurable entropy function. We suggest that noncoding regions in plants and invertebrates may display a smaller entropy and larger redundancy than coding regions, further supporting the possibility that noncoding regions of DNA may carry biological information.

  17. Nanopore Technology: A Simple, Inexpensive, Futuristic Technology for DNA Sequencing.

    PubMed

    Gupta, P D

    2016-10-01

    In health care, importance of DNA sequencing has been fully established. Sanger's Capillary Electrophoresis DNA sequencing methodology is time consuming, cumbersome, hence become more expensive. Lately, because of its versatility DNA sequencing became house hold name, and therefore, there is an urgent need of simple, fast, inexpensive, DNA sequencing technology. In the beginning of this century efforts were made, and Nanopore DNA sequencing technology was developed; still it is infancy, nevertheless, it is the futuristic technology. PMID:27605732

  18. Replication pattern of human repeated DNA sequences.

    PubMed

    Meneveri, R; Agresti, A; Breviario, D; Ginelli, E

    1984-10-01

    Either aphidicolin- or thymidine-synchronized human HL-60 cells were used to study the replication pattern of a family of human repetitive DNA sequences, the Eco RI 340 bp family (alpha RI-DNA), and of the ladders of fragments generated in total human DNA after digestion with XbaI and HaeIII (alpha satellite sequences). DNAs replicated in early, middle-early, middle-late and late S periods were labelled with BUdR or with [3H]thymidine. The efficiency of the cell synchronization procedure was confirmed by the transition from a high-GC to a high-AT average base composition of the DNA synthesized going from early to late S periods. By hybridizing EcoRI 340 bp repetitive fragments to BUdR-DNAs it was found that this family of sequences is replicated throughout the entire S period. Comparing fluorograph densitometric scans of [3H]DNAs to the scans of ethidium bromide patterns of total HL-60 DNA digested with XbaI and HaeIII, it was observed that DNA synthesized in different S periods is characterized by approximately the same ladder of fragments, while the intensity of each band may vary through the S phase; in particular, the XbaI 2.4 kb fragment becomes undetectable in late S. PMID:6089891

  19. Sequence change and phylogenetic signal in muscoid COII DNA sequences.

    PubMed

    Szalanski, Allen L; Owens, Carrie B

    2003-08-01

    The complete DNA sequence of the mtDNA cytochrome oxidase II gene from house fly, Musca domestica, face fly, Musca autumnalis, stable fly, Stomoxys calcitrans, horn fly, Haematobia irritans, and black garbage fly, Hydrotaea aenescens, are reported. The nucleotide sequence codes for a 229 amino acid peptide. The COII sequence is A + T rich (74.1%), with up to 12.3% nucleotide and 8.4% amino acid divergence among the five taxa. Of the 688 nucleotides encoding for the gene, 135 nucleotide sites (19.6%) are variable, and 55 (8.0%) are phylogenetically informative. A phylogenetic analysis using three calliphorids as the outgroup taxa, indicates that the two haematophagus species, horn fly and stable fly, form a sister group. PMID:14631656

  20. DNA Sequencing in Cultural Heritage.

    PubMed

    Vai, Stefania; Lari, Martina; Caramelli, David

    2016-02-01

    During the last three decades, DNA analysis on degraded samples revealed itself as an important research tool in anthropology, archaeozoology, molecular evolution, and population genetics. Application on topics such as determination of species origin of prehistoric and historic objects, individual identification of famous personalities, characterization of particular samples important for historical, archeological, or evolutionary reconstructions, confers to the paleogenetics an important role also for the enhancement of cultural heritage. A really fast improvement in methodologies in recent years led to a revolution that permitted recovering even complete genomes from highly degraded samples with the possibility to go back in time 400,000 years for samples from temperate regions and 700,000 years for permafrozen remains and to analyze even more recent material that has been subjected to hard biochemical treatments. Here we propose a review on the different methodological approaches used so far for the molecular analysis of degraded samples and their application on some case studies. PMID:27572991

  1. A microchannel electrophoresis DNA sequencing system

    SciTech Connect

    Madabhushi, R S; Warth, T; Balch, J W; Bass, M; Brewer, L R; Copeland, A C; Davidson, J C; Fitch, J P; Kegelmeyer, L M; Kimbrough, J R; McCready, P; Nelson, D; Pastrone, R L; Richardson, P M; Swierkowski, S P; Tarte, L A; Vainer, M

    1999-01-01

    In order to increase the DNA sequencing throughput of the Joint Genome Institute, we have developed a microchannel electrophoresis system. The critical new and unique elements of this system include 1) a process for the production of arrays of 96 and 384 microchannels on bonded glass substrates up to 14 x 58 cm and 2) new sieving media for high resolution and high speed separations. With custom fabrication apparatus, microchannels are etched in a borosilicate substrate, and then fusion bonded to a top substrate 1.1 mm thick that has access holes formed in it. SEM examination shows a typical microchannel to be 40 micrometers deep x 180 micrometers wide by 46 cm long. This technology offers significant advantages over discrete capillaries or conventional slab-gel approaches. High throughput DNA sequencing with over 550 base pairs resolution has been achieved in roughly half the time of conventional sequencers. In February 1999, we begin a pre-production evaluation protocol for the microchannel and for three glass capillary electrophoresis systems (two from industry and one developed by Lawrence Berkeley National Laboratory for the Joint Genome Institute). In order to utilize these instruments for DNA production sequencing, we have been evaluating and implementing software to convert raw electropherograms into called DNA bases with an associated probability of error. Our original intent was to utilize the DNA base calling software known as Plan and Phred developed by the University of Washington. This software has been outstanding for our slab gel electrophoresis systems currently in the production facility. In our tests and evaluations of this software applied to microchannel data, we observed that the electropherograms are of a different statistical and underlying signal structure compared to slab gels. Even with substantial modifications to the software, base calling performance was not satisfactory for the microchannel data. In this paper, we will present o The

  2. Detection and quantitation of single nucleotide polymorphisms, DNA sequence variations, DNA mutations, DNA damage and DNA mismatches

    DOEpatents

    McCutchen-Maloney, Sandra L.

    2002-01-01

    DNA mutation binding proteins alone and as chimeric proteins with nucleases are used with solid supports to detect DNA sequence variations, DNA mutations and single nucleotide polymorphisms. The solid supports may be flow cytometry beads, DNA chips, glass slides or DNA dips sticks. DNA molecules are coupled to solid supports to form DNA-support complexes. Labeled DNA is used with unlabeled DNA mutation binding proteins such at TthMutS to detect DNA sequence variations, DNA mutations and single nucleotide length polymorphisms by binding which gives an increase in signal. Unlabeled DNA is utilized with labeled chimeras to detect DNA sequence variations, DNA mutations and single nucleotide length polymorphisms by nuclease activity of the chimera which gives a decrease in signal.

  3. Automated synthesis of distillation sequences using fuzzy logic and simulation

    SciTech Connect

    Flowers, T.L.; Harrison, B.K.; Niccolai, M.J. )

    1994-08-01

    An automated distillation sequencing system (DSEQSYS) is presented, which consists of three components: a control program, a fuzzy heuristic synthesis program, and a process simulator. DSEQSYS, when applied to problems previously reported in the literature, overcomes some of the disadvantages of using heuristics or mathematical programming alone. DSEQSYS can address problems involving nonsharp separations, nonideal chemical behavior, and conflicting heuristics. A simple approach for converting the traditional separation heuristics into corresponding fuzzy heuristics is also demonstrated.

  4. DNA Sequence Alignment during Homologous Recombination.

    PubMed

    Greene, Eric C

    2016-05-27

    Homologous recombination allows for the regulated exchange of genetic information between two different DNA molecules of identical or nearly identical sequence composition, and is a major pathway for the repair of double-stranded DNA breaks. A key facet of homologous recombination is the ability of recombination proteins to perfectly align the damaged DNA with homologous sequence located elsewhere in the genome. This reaction is referred to as the homology search and is akin to the target searches conducted by many different DNA-binding proteins. Here I briefly highlight early investigations into the homology search mechanism, and then describe more recent research. Based on these studies, I summarize a model that includes a combination of intersegmental transfer, short-distance one-dimensional sliding, and length-specific microhomology recognition to efficiently align DNA sequences during the homology search. I also suggest some future directions to help further our understanding of the homology search. Where appropriate, I direct the reader to other recent reviews describing various issues related to homologous recombination. PMID:27129270

  5. DNA sequencing via transverse electronic transport

    NASA Astrophysics Data System (ADS)

    Lagerqvist, Johan; Zwolak, Michael; di Ventra, Massimiliano

    2006-03-01

    Recently, it was theoretically shown that transverse current measurements could be used to distinguish the different bases of single stranded DNA. [1] If electrodes are embedded in a device, e.g., a nanopore, which allows translocation of ss-DNA, the strand can be sequenced by continuous measurement of the current in the direction perpendicular to the DNA backbone. [1] However, variations of the electronic signatures of each base in a real device due to structural fluctuations, counter-ions, water and other sources of noise will be important obstacles to overcome in order to make this theoretical proposal a reality. In order to explore these effects we have coupled molecular dynamics simulations with transport calculations to obtain the real time transverse current of ss-DNA translocating into a nanopore. We find that distributions of currents for each base are indeed different even in the presence of all the sources of noise discussed above. These results support even more the original proposal [1] that fast DNA sequencing could be done using transverse current measurements. Work supported by the National Humane Genome Research Institute. [1] M. Zwolak and M. Di Ventra, ``Electronic Signature of DNA Nucleotides via Transverse Transport'', Nano Lett. 5, 421 (2005).

  6. Imaging of DNA sequences with chemiluminescence

    SciTech Connect

    Tizard, R.; Cate, R.L.; Ramachandran, K.L.; Wysk, M.; Voyta, J.C.; Murphy, O.J.; Bronstein, I. )

    1990-06-01

    We have coupled a chemiluminescent detection method that uses an alkaline phosphatase label to the genomic DNA sequencing protocol of Church and Gilbert . Images of sequence ladders are obtained on x-ray film with exposure times of less than 30 min, as compared to 40 h required for a similar exposure with a 32P-labeled oligomer. Chemically cleaved DNA from a sequencing gel is transferred to a nylon membrane, and specific sequence ladders are selected by hybridization to DNA oligonucleotides labeled with alkaline phosphatase or with biotin, leading directly or indirectly to deposition of enzyme. If a biotinylated probe is used, an incubation with avidin-alkaline phosphatase conjugate follows. The membrane is soaked in the chemiluminescent substrate (AMPPD) and is exposed to film. Dephosphorylation of AMPPD leads in a two-step pathway to a highly localized emission of visible light. The demonstrated shorter exposure times may improve the efficiency of a serial reprobing strategy such as the multiplex sequencing approach of Church and Kieffer-Higgins.

  7. The DNA sequence of human chromosome 7.

    PubMed

    Hillier, Ladeana W; Fulton, Robert S; Fulton, Lucinda A; Graves, Tina A; Pepin, Kymberlie H; Wagner-McPherson, Caryn; Layman, Dan; Maas, Jason; Jaeger, Sara; Walker, Rebecca; Wylie, Kristine; Sekhon, Mandeep; Becker, Michael C; O'Laughlin, Michelle D; Schaller, Mark E; Fewell, Ginger A; Delehaunty, Kimberly D; Miner, Tracie L; Nash, William E; Cordes, Matt; Du, Hui; Sun, Hui; Edwards, Jennifer; Bradshaw-Cordum, Holland; Ali, Johar; Andrews, Stephanie; Isak, Amber; Vanbrunt, Andrew; Nguyen, Christine; Du, Feiyu; Lamar, Betty; Courtney, Laura; Kalicki, Joelle; Ozersky, Philip; Bielicki, Lauren; Scott, Kelsi; Holmes, Andrea; Harkins, Richard; Harris, Anthony; Strong, Cynthia Madsen; Hou, Shunfang; Tomlinson, Chad; Dauphin-Kohlberg, Sara; Kozlowicz-Reilly, Amy; Leonard, Shawn; Rohlfing, Theresa; Rock, Susan M; Tin-Wollam, Aye-Mon; Abbott, Amanda; Minx, Patrick; Maupin, Rachel; Strowmatt, Catrina; Latreille, Phil; Miller, Nancy; Johnson, Doug; Murray, Jennifer; Woessner, Jeffrey P; Wendl, Michael C; Yang, Shiaw-Pyng; Schultz, Brian R; Wallis, John W; Spieth, John; Bieri, Tamberlyn A; Nelson, Joanne O; Berkowicz, Nicolas; Wohldmann, Patricia E; Cook, Lisa L; Hickenbotham, Matthew T; Eldred, James; Williams, Donald; Bedell, Joseph A; Mardis, Elaine R; Clifton, Sandra W; Chissoe, Stephanie L; Marra, Marco A; Raymond, Christopher; Haugen, Eric; Gillett, Will; Zhou, Yang; James, Rose; Phelps, Karen; Iadanoto, Shawn; Bubb, Kerry; Simms, Elizabeth; Levy, Ruth; Clendenning, James; Kaul, Rajinder; Kent, W James; Furey, Terrence S; Baertsch, Robert A; Brent, Michael R; Keibler, Evan; Flicek, Paul; Bork, Peer; Suyama, Mikita; Bailey, Jeffrey A; Portnoy, Matthew E; Torrents, David; Chinwalla, Asif T; Gish, Warren R; Eddy, Sean R; McPherson, John D; Olson, Maynard V; Eichler, Evan E; Green, Eric D; Waterston, Robert H; Wilson, Richard K

    2003-07-10

    Human chromosome 7 has historically received prominent attention in the human genetics community, primarily related to the search for the cystic fibrosis gene and the frequent cytogenetic changes associated with various forms of cancer. Here we present more than 153 million base pairs representing 99.4% of the euchromatic sequence of chromosome 7, the first metacentric chromosome completed so far. The sequence has excellent concordance with previously established physical and genetic maps, and it exhibits an unusual amount of segmentally duplicated sequence (8.2%), with marked differences between the two arms. Our initial analyses have identified 1,150 protein-coding genes, 605 of which have been confirmed by complementary DNA sequences, and an additional 941 pseudogenes. Of genes confirmed by transcript sequences, some are polymorphic for mutations that disrupt the reading frame. PMID:12853948

  8. Nanopore-CMOS Interfaces for DNA Sequencing.

    PubMed

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

    2016-01-01

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

  9. Repetitive DNA sequences in Mycoplasma pneumoniae.

    PubMed Central

    Wenzel, R; Herrmann, R

    1988-01-01

    Two types of different repetitive DNA sequences called RepMP1 and RepMP2 were identified in the genome of Mycoplasma pneumoniae. The number of these repeated elements, their nucleotide sequence and their localization on a physical map of the M. pneumoniae genome were determined. The results show that RepMP1 appears at least 10 times and RepMP2 at least 8 times in the genome. The repeated elements are dispersed on the chromosome and, in three cases, linked to each other by a homologous DNA sequence of 400 bp. The elements themselves are 300 bp (for RepMP1) and 150 bp (for RepMP2) long showing a high degree of homology. One copy of RepMP2 is a translated part of the gene for the major cytadhesin protein P1 which is responsible for the adsorption of M. pneumoniae to its host cell. Images PMID:3138660

  10. Sequence-specific DNA nicking endonucleases.

    PubMed

    Xu, Shuang-yong

    2015-08-01

    A group of small HNH nicking endonucleases (NEases) was discovered recently from phage or prophage genomes that nick double-stranded DNA sites ranging from 3 to 5 bp in the presence of Mg2+ or Mn2+. The cosN site of phage HK97 contains a gp74 nicking site AC↑CGC, which is similar to AC↑CGR (R=A/G) of N.ϕGamma encoded by Bacillus phage Gamma. A minimal nicking domain of 76 amino acid residues from N.ϕGamma could be fused to other DNA binding partners to generate chimeric NEases with new specificities. The biological roles of a few small HNH endonucleases (HNHE, gp74 of HK97, gp37 of ϕSLT, ϕ12 HNHE) have been demonstrated in phage and pathogenicity island DNA packaging. Another group of NEases with 3- to 7-bp specificities are either natural components of restriction systems or engineered from type IIS restriction endonucleases. A phage group I intron-encoded HNH homing endonucleases, I-PfoP3I was found to nick DNA sites of 14-16 bp. I-TslI encoded by T7-like ΦI appeared to nick DNA sites with a 9-bp core sequence. DNA nicking and labeling have been applied to optical mapping to aid genome sequence assembly and detection of large insertion/deletion mutations in genomic DNA of cancer cells. Nicking enzyme-mediated amplification reaction has been applied to rapid diagnostic testing of influenza A and B in clinical setting and for construction of DNA-based Boolean logic gates. The clustered regularly interspaced short palindromic repeats-ribonucleoprotein complex consisting of engineered Cas9 nickases in conjunction with tracerRNA:crRNA or a single-guide RNA have been successfully used in genome modifications. PMID:26352356

  11. Toward a visualization of DNA sequences.

    PubMed

    Cox, David N; Tharp, Alan L

    2010-01-01

    Most biologists associate pattern discovery in DNA with finding repetitive sequences or commonalities across several sequences. However, pattern discovery is not limited to finding repetitions and commonalities. Pattern discovery also involves identifying objects and distinguishing objects from one another. Human vision is unmatched in its ability to identify and distinguish objects. Considerable research into human vision has revealed to a fair degree the visual cues that our brains use to segment an image into separate regions and entities. In this paper, we consider some of these visual cues to construct a novel graphical representation of a DNA sequence. We exploit one of these cues, proximity, to segment DNA into visibly distinct regions and structures. We also demonstrate how to manipulate proximity to identify motifs visually. Lastly, we demonstrate how an additional cue, color, can be used to visualize the Shannon entropy associated with different structures. The presence of large numbers of such regions and structures in DNA suggests that they likely play some important biological role and would be interesting targets for further research. PMID:20865527

  12. DNA Sequencing Using an Engineered Protein Nanopore

    NASA Astrophysics Data System (ADS)

    Gundlach, Jens H.

    2010-03-01

    Inexpensive and fast sequencing of DNA is of paramount importance to medicine, the life sciences and to many other applications. Because of the nanometer diameter of DNA a nanometer-scale reader directly interfaced to macroscopic observables seems particularly attractive. We are working on a new single molecule technique based on a biological pore embedded in a lipid bilayer. When a voltage is applied across the bilayer an ion current is measured that flows through the nanometer opening of the pore. Poly-negatively charged single stranded DNA passes through the pore and reduces the ion current with the remaining ion current being indicative of the nucleotide type in the constriction of the pore. The protein pore that we introduced to the field, MspA, has a shape ideally suited to nanopore sequencing, has robustness comparable to solid state devices, is easily reproduced with sub-nanometer level precision and is engineerable using genetic mutations. I will present proof-of-principle data showing that this technique can lead to a direct very inexpensive and fast sequencing technology. The experimental electronic signatures of the DNA translocation process provide an ideal test bed for molecular dynamics simulations, which in turn allows developing intuition and prediction of nanoscale dynamics.

  13. "Doublex" fluorescent DNA sequencing: two independent sequences obtained simultaneously in one reaction with internal labeling and unlabeled primers.

    PubMed

    Wiemann, S; Stegemann, J; Zimmermann, J; Voss, H; Benes, V; Ansorge, W

    1996-02-15

    The novel "doublex" DNA sequencing technique that makes it possible to obtain simultaneously two independent sequences from one sequencing reaction with the use of unlabeled primers and internal labeling is described. The different sequencing products are labeled in parallel with fluorescein-15-dATP and Texas red-5-dCTP present in the same tube. The characteristics of T7 DNA polymerase are exploited to ensure that only either of the labeled dNTPs is incorporated into the corresponding sequencing products. Specificity of labeling is ensured by the selection of primers. One of the unlabeled primers is chosen to be followed by an "A," the other by a "C" to be incorporated immediately downstream from the primer binding site. The doublex sequencing technique is applicable to the simultaneous sequencing of either the same DNA template/strand or a mixture of different templates. Combinations of unlabeled and labeled primers in the same sequencing reaction are also possible. The two sequences can be determined in parallel and on-line in the same lanes of a gel with a novel automated DNA sequencer, which was previously described for use with labeled primers. PMID:8714594

  14. Local Renyi entropic profiles of DNA sequences

    PubMed Central

    Vinga, Susana; Almeida, Jonas S

    2007-01-01

    Background In a recent report the authors presented a new measure of continuous entropy for DNA sequences, which allows the estimation of their randomness level. The definition therein explored was based on the Rényi entropy of probability density estimation (pdf) using the Parzen's window method and applied to Chaos Game Representation/Universal Sequence Maps (CGR/USM). Subsequent work proposed a fractal pdf kernel as a more exact solution for the iterated map representation. This report extends the concepts of continuous entropy by defining DNA sequence entropic profiles using the new pdf estimations to refine the density estimation of motifs. Results The new methodology enables two results. On the one hand it shows that the entropic profiles are directly related with the statistical significance of motifs, allowing the study of under and over-representation of segments. On the other hand, by spanning the parameters of the kernel function it is possible to extract important information about the scale of each conserved DNA region. The computational applications, developed in Matlab m-code, the corresponding binary executables and additional material and examples are made publicly available at . Conclusion The ability to detect local conservation from a scale-independent representation of symbolic sequences is particularly relevant for biological applications where conserved motifs occur in multiple, overlapping scales, with significant future applications in the recognition of foreign genomic material and inference of motif structures. PMID:17939871

  15. Linguistic features of noncoding DNA sequences

    NASA Astrophysics Data System (ADS)

    Mantegna, R. N.; Buldyrev, S. V.; Goldberger, A. L.; Havlin, S.; Peng, C.-K.; Simons, M.; Stanley, H. E.

    1994-12-01

    We extend the Zipf approach to analyzing linguistic texts to the statistical study of DNA base pair sequences, and find that the noncoding regions are more similar to natural languages than the coding regions. We also adapt the Shannon approach to quantifying the ``redundancy'' of a linguistic text in terms of a measurable entropy function, and demonstrate that noncoding regions in eukaryotes display a smaller entropy and larger redundancy B than coding regions, supporting the possibility that noncoding regions of DNA may carry biological information.

  16. Metagenomics: DNA sequencing of environmental samples

    SciTech Connect

    Tringe, Susannah Green; Rubin, Edward M.

    2005-09-01

    While genomics has classically focused on pure,easy-to-obtain samples, such as microbes that grow readily in culture orlarge animals and plants, these organisms represent but a fraction of theliving or once living organisms of interest. Many species are difficultto study in isolation, because they fail to grow in laboratory culture,depend on other organisms for critical processes, or have become extinct.DNA sequence-based methods circumvent these obstacles, as DNA can bedirectly isolated from live or dead cells in a variety of contexts, andhave led to the emergence of a new field referred to asmetagenomics.

  17. Compilation of DNA sequences of Escherichia coli

    PubMed Central

    Kröger, Manfred

    1989-01-01

    We have compiled the DNA sequence data for E.coli K12 available from the GENBANK and EMBO databases and over a period of several years independently from the literature. We have introduced all available genetic map data and have arranged the sequences accordingly. As far as possible the overlaps are deleted and a total of 940,449 individual bp is found to be determined till the beginning of 1989. This corresponds to a total of 19.92% of the entire E.coli chromosome consisting of about 4,720 kbp. This number may actually be higher by some extra 2% derived from the sequence of lysogenic bacteriophage lambda and the various insertion sequences. This compilation may be available in machine readable form from one of the international databanks in some future. PMID:2654890

  18. Highly multiplexed DNA sequencing by capillary electrophoresis

    SciTech Connect

    Yeung, E.S.; Ueno, K.; Chang, H.T.

    1994-12-31

    It is obvious that irrespective of whichever basic technology is eventually selected to sequence the entire human genome there are substantial gains to be made if a high degree of multiplexing of parallel runs can be implemented. Such multiplexing should not involve expensive instrumentation and should not require additional personnel, or else the main objective of cost reduction will not be satisfied even though the total time for sequencing is reduced. In the last two years, several research groups have shown that capillary electrophoresis (CE) is an attractive alternative for DNA sequencing. Part of the improvement in sequencing speed in CE is counteracted by the inherent ability of slab gels for accommodating multiple lanes in a single run. Recently, the authors have developed several excitation schemes for highly multiplexed capillary electrophoresis. Detection at the pM level was demonstrated. The authors report here the use of a novel excitation geometry to simultaneously monitor 100 capillary tubes during electrophoresis. This represents a truly parallel multiplexing scheme for high-speed DNA sequencing.

  19. Effective Automated Feature Construction and Selection for Classification of Biological Sequences

    PubMed Central

    Kamath, Uday; De Jong, Kenneth; Shehu, Amarda

    2014-01-01

    Background Many open problems in bioinformatics involve elucidating underlying functional signals in biological sequences. DNA sequences, in particular, are characterized by rich architectures in which functional signals are increasingly found to combine local and distal interactions at the nucleotide level. Problems of interest include detection of regulatory regions, splice sites, exons, hypersensitive sites, and more. These problems naturally lend themselves to formulation as classification problems in machine learning. When classification is based on features extracted from the sequences under investigation, success is critically dependent on the chosen set of features. Methodology We present an algorithmic framework (EFFECT) for automated detection of functional signals in biological sequences. We focus here on classification problems involving DNA sequences which state-of-the-art work in machine learning shows to be challenging and involve complex combinations of local and distal features. EFFECT uses a two-stage process to first construct a set of candidate sequence-based features and then select a most effective subset for the classification task at hand. Both stages make heavy use of evolutionary algorithms to efficiently guide the search towards informative features capable of discriminating between sequences that contain a particular functional signal and those that do not. Results To demonstrate its generality, EFFECT is applied to three separate problems of importance in DNA research: the recognition of hypersensitive sites, splice sites, and ALU sites. Comparisons with state-of-the-art algorithms show that the framework is both general and powerful. In addition, a detailed analysis of the constructed features shows that they contain valuable biological information about DNA architecture, allowing biologists and other researchers to directly inspect the features and potentially use the insights obtained to assist wet-laboratory studies on retainment or

  20. ASTRAL, a hyperspectral imaging DNA sequencer

    NASA Astrophysics Data System (ADS)

    O'Brien, Kevin M.; Wren, Jonathan; Davé, Varshal K.; Bai, Diane; Anderson, Richard D.; Rayner, Simon; Evans, Glen A.; Dabiri, Ali E.; Garner, Harold R.

    1998-05-01

    We are developing a prototype automatic DNA sequencer which utilizes polyacrylamide slab gels imaged through a novel optical detection system. The design of this prototype sequencer allows the ability to perform direct optical coupling over the entire read area of the gel and hyperspectrographic separation and detection of the fluorescence emission. The machine has no moving parts. All the major components incorporated in this prototype are all currently available "off the shelf," thus reducing equipment development time and decreasing costs. Software developed for data acquisition, analysis, and conversion to other standard formats facilitates compatibility.

  1. DNA sequences, recombinant DNA molecules and processes producing human phospholipase inhibitor polypeptides

    SciTech Connect

    Wallner, B.P.; Pepinsky, R.B.; Garwin, J.L.

    1989-11-07

    This patent describes a recombinant DNA molecule. In comprises a DNA sequence coding for a phospholopase inhibitor polypeptide and being selected from the group consisting of: the cDNA insert of ALC, DNA sequences which code on expression for a phospholopase inhibitor, and DNA sequences which are degenerate as a result of the genetic code to either of the foregoing DNA sequences and which code on expression for a phospholipase inhibitor.

  2. Sequencing and Analysis of Neanderthal Genomic DNA

    PubMed Central

    Noonan, James P.; Coop, Graham; Kudaravalli, Sridhar; Smith, Doug; Krause, Johannes; Alessi, Joe; Chen, Feng; Platt, Darren; Pääbo, Svante; Pritchard, Jonathan K.; Rubin, Edward M.

    2008-01-01

    Our knowledge of Neanderthals is based on a limited number of remains and artifacts from which we must make inferences about their biology, behavior, and relationship to ourselves. Here, we describe the characterization of these extinct hominids from a new perspective, based on the development of a Neanderthal metagenomic library and its high-throughput sequencing and analysis. Several lines of evidence indicate that the 65,250 base pairs of hominid sequence so far identified in the library are of Neanderthal origin, the strongest being the ascertainment of sequence identities between Neanderthal and chimpanzee at sites where the human genomic sequence is different. These results enabled us to calculate the human-Neanderthal divergence time based on multiple randomly distributed autosomal loci. Our analyses suggest that on average the Neanderthal genomic sequence we obtained and the reference human genome sequence share a most recent common ancestor ~706,000 years ago, and that the human and Neanderthal ancestral populations split ~370,000 years ago, before the emergence of anatomically modern humans. Our finding that the Neanderthal and human genomes are at least 99.5% identical led us to develop and successfully implement a targeted method for recovering specific ancient DNA sequences from metagenomic libraries. This initial analysis of the Neanderthal genome advances our understanding of the evolutionary relationship of Homo sapiens and Homo neanderthalensis and signifies the dawn of Neanderthal genomics. PMID:17110569

  3. Identification of genes in anonymous DNA sequences. Annual performance report, February 1, 1991--January 31, 1992

    SciTech Connect

    Fields, C.A.

    1996-06-01

    The objective of this project is the development of practical software to automate the identification of genes in anonymous DNA sequences from the human, and other higher eukaryotic genomes. A software system for automated sequence analysis, gm (gene modeler) has been designed, implemented, tested, and distributed to several dozen laboratories worldwide. A significantly faster, more robust, and more flexible version of this software, gm 2.0 has now been completed, and is being tested by operational use to analyze human cosmid sequence data. A range of efforts to further understand the features of eukaryoyic gene sequences are also underway. This progress report also contains papers coming out of the project including the following: gm: a Tool for Exploratory Analysis of DNA Sequence Data; The Human THE-LTR(O) and MstII Interspersed Repeats are subfamilies of a single widely distruted highly variable repeat family; Information contents and dinucleotide compostions of plant intron sequences vary with evolutionary origin; Splicing signals in Drosophila: intron size, information content, and consensus sequences; Integration of automated sequence analysis into mapping and sequencing projects; Software for the C. elegans genome project.

  4. Imaging of DNA sequences with chemiluminescence

    SciTech Connect

    Tizard, R.; Cate, R.L.; Ramachandran, K.L.; Wysk, M.; Bronstein, I.; Voyta, J.C.; Murphy, O.J.

    1989-12-31

    We have coupled a chemiluminescent method for detecting oligonucleotides labeled with alkaline phosphatase to the genomic DNA sequencing protocol of Church and Gilbert. Images of sequence ladders obtained on x-ray film in a 30 minute exposure are comparable to those from a 40 hour exposure with 3000 Ci/mmol {sup 32}P probes. Chemically cleaved DNA from a sequencing gel is transferred to a nylon membrane, and specific sequence ladders are selected by hybridization to an oligonucleotide probe conjugated either to biotin or to alkaline phosphates. If biotinylated probe is used, then an avidin-alkaline phosphatase conjugate is subsequently bound. This membrane, bearing immobilized alkaline phosphatase, is incubated with the commercially available chemiluminescent substrate disodium 3-(4-methoxyspiro[1,2-dioxetone-3,2{prime}-tricyclo[3.3.1.1.{sup 3.7}]decan]-4-yl)phenyl phosphate. (AMPPD) Dephosphorylation of AMPPD leads in a two step pathway to a highly localized emission of visible light.

  5. Imaging of DNA sequences with chemiluminescence

    SciTech Connect

    Tizard, R.; Cate, R.L.; Ramachandran, K.L.; Wysk, M. ); Bronstein, I.; Voyta, J.C.; Murphy, O.J. )

    1989-01-01

    We have coupled a chemiluminescent method for detecting oligonucleotides labeled with alkaline phosphatase to the genomic DNA sequencing protocol of Church and Gilbert. Images of sequence ladders obtained on x-ray film in a 30 minute exposure are comparable to those from a 40 hour exposure with 3000 Ci/mmol {sup 32}P probes. Chemically cleaved DNA from a sequencing gel is transferred to a nylon membrane, and specific sequence ladders are selected by hybridization to an oligonucleotide probe conjugated either to biotin or to alkaline phosphates. If biotinylated probe is used, then an avidin-alkaline phosphatase conjugate is subsequently bound. This membrane, bearing immobilized alkaline phosphatase, is incubated with the commercially available chemiluminescent substrate disodium 3-(4-methoxyspiro(1,2-dioxetone-3,2{prime}-tricyclo(3.3.1.1.{sup 3.7})decan)-4-yl)phenyl phosphate. (AMPPD) Dephosphorylation of AMPPD leads in a two step pathway to a highly localized emission of visible light.

  6. Accurate restoration of DNA sequences. Progress report

    SciTech Connect

    Churchill, G.A.

    1994-05-01

    The primary of this project are the development of (1) a general stochastic model for DNA sequencing errors (2) algorithms to restore the original DNA sequence and (3) statistical methods to assess the accuracy of this restoration. A secondary objective is to develop new algorithms for fragment assembly. Initially a stochastic model that assumes errors are independent and uniformly distributed will be developed. Generalizations of the basic model will be developed to account for (1) decay of accuracy along fragments, (2) variable error rates among fragments, (3) sequence dependent errors (e.g. homopolymeric, runs), and (4) strand--specific systematic errors (e.g. compressions). The emphasis of this project will be the development of a theoretical basis for determining sequence accuracy. However, new algorithms are proposed and these will be implemented as software (in the C programming language). This software will be tested using real and simulated data. It will be modular in design and will be made available for distribution to the scientific community.

  7. A comprehensive list of cloned human DNA sequences

    PubMed Central

    Schmidtke, Jörg; Cooper, David N.

    1990-01-01

    A list of DNA sequences cloned from the human genome is presented. Intended as a guide to clone availability, this list includes published reports of cDNA, genomic and synthetic clones comprising gene and pseudogene sequences, uncharacterised DNA segments and repetitive DNA elements. PMID:2333227

  8. A comprehensive list of cloned human DNA sequences

    PubMed Central

    Schmidtke, Jörg; Cooper, David N.

    1988-01-01

    A list of DNA sequences cloned from the human genome is presented. Intended as a guide to clone availability, this list includes published reports of cDNA, genomic and synthetic clones comprising gene and pseudogene sequences, uncharacterised DNA segments and repetitive DNA elements. PMID:3368330

  9. A comprehensive list of cloned human DNA sequences

    PubMed Central

    Schmidtke, Jörg; Cooper, David N.

    1989-01-01

    A list of DNA sequences cloned from the human genome is presented. Intended as a guide to clone availability, this list includes published reports of cDNA, genomic and synthetic clones comprising gene and pseudogene sequences, uncharacterised DNA segments and repetitive DNA elements. PMID:2654889

  10. Automated mass spectrometric sequence determination of cyclic peptide library members.

    PubMed

    Redman, James E; Wilcoxen, Keith M; Ghadiri, M Reza

    2003-01-01

    Cyclic peptides have come under scrutiny as potential antimicrobial therapeutic agents. Combinatorial split-and-pool synthesis of cyclic peptides can afford single compound per well libraries for antimicrobial screening, new lead identification, and construction of quantitative structure-activity relationships (QSAR). Here, we report a new sequencing protocol for rapid identification of the members of a cyclic peptide library based on automated computer analysis of mass spectra, obviating the need for library encoding/decoding strategies. Furthermore, the software readily integrates with common spreadsheet and database packages to facilitate data visualization and archiving. The utility of the new MS-sequencing approach is demonstrated using sonic spray ionization ion trap MS and MS/MS spectrometry on a single compound per bead cyclic peptide library and validated with individually synthesized pure cyclic D,L-alpha-peptides. PMID:12523832

  11. Laser mass spectrometry for DNA sequencing, disease diagnosis, and fingerprinting

    SciTech Connect

    Winston Chen, C.H.; Taranenko, N.I.; Zhu, Y.F.; Chung, C.N.; Allman, S.L.

    1997-03-01

    Since laser mass spectrometry has the potential for achieving very fast DNA analysis, the authors recently applied it to DNA sequencing, DNA typing for fingerprinting, and DNA screening for disease diagnosis. Two different approaches for sequencing DNA have been successfully demonstrated. One is to sequence DNA with DNA ladders produced from Snager`s enzymatic method. The other is to do direct sequencing without DNA ladders. The need for quick DNA typing for identification purposes is critical for forensic application. The preliminary results indicate laser mass spectrometry can possibly be used for rapid DNA fingerprinting applications at a much lower cost than gel electrophoresis. Population screening for certain genetic disease can be a very efficient step to reducing medical costs through prevention. Since laser mass spectrometry can provide very fast DNA analysis, the authors applied laser mass spectrometry to disease diagnosis. Clinical samples with both base deletion and point mutation have been tested with complete success.

  12. Laser mass spectrometry for DNA sequencing, disease diagnosis, and fingerprinting

    NASA Astrophysics Data System (ADS)

    Chen, C. H. Winston; Taranenko, N. I.; Zhu, Y. F.; Chung, C. N.; Allman, S. L.

    1997-05-01

    Since laser mass spectrometry has the potential for achieving very fast DNA analysis, we recently applied it to DNA sequencing, DNA typing for fingerprinting, and DNA screening for disease diagnosis. Two different approaches for sequencing DNA have been successfully demonstrated. One is to sequence DNA with DNA ladders produced from Sanger's enzymatic method. The other is to do direct sequencing without DNA ladders. The need for quick DNA typing for identification purposes is critical for forensic application. Our preliminary results indicate laser mass spectrometry can possible be used for rapid DNA fingerprinting applications at a much lower cost than gel electrophoresis. Population screening for certain genetic disease can be a very efficient step to reducing medical costs through prevention. Since laser mass spectrometry can provide very fast DNA analysis, we applied laser mass spectrometry to disease diagnosis. Clinical samples with both base deletion and point mutation have been tested with complete success.

  13. DNA separations in microfabricated devices with automated capillary sample introduction.

    PubMed

    Smith, E M; Xu, H; Ewing, A G

    2001-01-01

    A novel method is presented for automated injection of DNA samples into microfabricated separation devices via capillary electrophoresis. A single capillary is used to electrokinetically inject discrete plugs of DNA into an array of separation lanes on a glass chip. A computer-controlled micromanipulator is used to automate this injection process and to repeat injections into five parallel lanes several times over the course of the experiment. After separation, labeled DNA samples are detected by laser-induced fluorescence. Five serial separations of 6-carboxyfluorescein (FAM)-labeled oligonucleotides in five parallel lanes are shown, resulting in the analysis of 25 samples in 25 min. It is estimated that approximately 550 separations of these same oligonucleotides could be performed in one hour by increasing the number of lanes to 37 and optimizing the rate of the manipulator movement. Capillary sample introduction into chips allows parallel separations to be continuously performed in serial, yielding high throughput and minimal need for operator intervention. PMID:11288906

  14. Porcine parvovirus: DNA sequence and genome organization.

    PubMed

    Ranz, A I; Manclús, J J; Díaz-Aroca, E; Casal, J I

    1989-10-01

    We have determined the nucleotide sequence of an almost full-length clone of porcine parvovirus (PPV). The sequence is 4973 nucleotides (nt) long. The 3' end of virion DNA shows a Y-shaped configuration homologous to rodent parvoviruses. The 5' end of virion DNA shows a repetition of 127 nt at the carboxy terminus of the capsid proteins. The overall organization of the PPV genome is similar to those of other autonomous parvoviruses. There are two large open reading frames (ORFs) that almost entirely cover the genome, both located in the same frame of the complementary strand. The left ORF encodes the non-structural protein NS1 and the right ORF encodes the capsid proteins (VP1, VP2 and VP3). Promoter analysis, location of splicing sites and putative amino acid sequences for the viral proteins show a high homology of PPV with feline panleukopenia virus and canine parvoviruses (FPV and CPV) and rodent parvovirus. Therefore we conclude that PPV is related to the Kilham rat virus (KRV) group of autonomous parvoviruses formed by KRV, minute virus of mice, Lu III, H-1, FPV and CPV. PMID:2794971

  15. Chimeric proteins for detection and quantitation of DNA mutations, DNA sequence variations, DNA damage and DNA mismatches

    DOEpatents

    McCutchen-Maloney, Sandra L.

    2002-01-01

    Chimeric proteins having both DNA mutation binding activity and nuclease activity are synthesized by recombinant technology. The proteins are of the general formula A-L-B and B-L-A where A is a peptide having DNA mutation binding activity, L is a linker and B is a peptide having nuclease activity. The chimeric proteins are useful for detection and identification of DNA sequence variations including DNA mutations (including DNA damage and mismatches) by binding to the DNA mutation and cutting the DNA once the DNA mutation is detected.

  16. Automated detection of point mutations using fluorescent sequence trace subtraction.

    PubMed Central

    Bonfield, J K; Rada, C; Staden, R

    1998-01-01

    The final step in the detection of mutations is to determine the sequence of the suspected mutant and to compare it with that of the wild-type, and for this fluorescence-based sequencing instruments are widely used. We describe some simple algorithms forcomparing sequence traces which, as part of our sequence assembly and analysis package, are proving useful for the discovery of mutations and which may also help to identify misplaced readings in sequence assembly projects. The mutations can be detected automatically by a new program called TRACE_DIFF and new types of trace display in our program GAP4 greatly simplify visual checking of the assigned changes. To assess the accuracy of the automatic mutation detection algorithm we analysed 214 sequence readings from hypermutating DNA comprising a total of 108 497 bases. After the readings were assembled there were 1232 base differences, including 392 Ns and 166 alignment characters. Visual inspection of the traces established that of the 1232 differences, 353 were real mutations while the rest were due to base calling errors. The TRACE_DIFF algorithm automatically identified all but 36, with 28 false positives. Further information about the software can be obtained from http://www.mrc-lmb.cam.ac.uk/pubseq/ PMID:9649626

  17. Poincaré recurrences of DNA sequences

    NASA Astrophysics Data System (ADS)

    Frahm, K. M.; Shepelyansky, D. L.

    2012-01-01

    We analyze the statistical properties of Poincaré recurrences of Homo sapiens, mammalian, and other DNA sequences taken from the Ensembl Genome data base with up to 15 billion base pairs. We show that the probability of Poincaré recurrences decays in an algebraic way with the Poincaré exponent β≈4 even if the oscillatory dependence is well pronounced. The correlations between recurrences decay with an exponent ν≈0.6 that leads to an anomalous superdiffusive walk. However, for Homo sapiens sequences, with the largest available statistics, the diffusion coefficient converges to a finite value on distances larger than one million base pairs. We argue that the approach based on Poncaré recurrences determines new proximity features between different species and sheds a new light on their evolution history.

  18. Recent advances in DNA sequencing techniques

    NASA Astrophysics Data System (ADS)

    Singh, Rama Shankar

    2013-06-01

    Successful mapping of the draft human genome in 2001 and more recent mapping of the human microbiome genome in 2012 have relied heavily on the parallel processing of the second generation/Next Generation Sequencing (NGS) DNA machines at a cost of several millions dollars and long computer processing times. These have been mainly biochemical approaches. Here a system analysis approach is used to review these techniques by identifying the requirements, specifications, test methods, error estimates, repeatability, reliability and trends in the cost reduction. The first generation, NGS and the Third Generation Single Molecule Real Time (SMART) detection sequencing methods are reviewed. Based on the National Human Genome Research Institute (NHGRI) data, the achieved cost reduction of 1.5 times per yr. from Sep. 2001 to July 2007; 7 times per yr., from Oct. 2007 to Apr. 2010; and 2.5 times per yr. from July 2010 to Jan 2012 are discussed.

  19. Elucidating population histories using genomic DNA sequences.

    PubMed

    Vigilant, Linda

    2009-04-01

    In 1993, Cliff Jolly suggested that rather than debating species definitions and classifications, energy would be better spent investigating multidimensional patterns of variation and gene flow among populations. Until now, however, genetic studies of wild primate populations have been limited to very small portions of the genome. Access to complete genome sequences of humans, chimpanzees, macaques, and other primates makes it possible to design studies surveying substantial amounts of DNA sequence variation at multiple genetic loci in representatives of closely related but distinct wild primate populations. Such data can be analyzed with new approaches that estimate not only when populations diverged but also the relative amounts and directions of subsequent gene flow. These analyses will reemphasize the difficulty of achieving consistent species and subspecies definitions by revealing the extent of variation in the amount and duration of gene flow accompanying population divergences. PMID:19817223

  20. Direct Detection and Sequencing of Damaged DNA Bases

    PubMed Central

    2011-01-01

    Products of various forms of DNA damage have been implicated in a variety of important biological processes, such as aging, neurodegenerative diseases, and cancer. Therefore, there exists great interest to develop methods for interrogating damaged DNA in the context of sequencing. Here, we demonstrate that single-molecule, real-time (SMRT®) DNA sequencing can directly detect damaged DNA bases in the DNA template - as a by-product of the sequencing method - through an analysis of the DNA polymerase kinetics that are altered by the presence of a modified base. We demonstrate the sequencing of several DNA templates containing products of DNA damage, including 8-oxoguanine, 8-oxoadenine, O6-methylguanine, 1-methyladenine, O4-methylthymine, 5-hydroxycytosine, 5-hydroxyuracil, 5-hydroxymethyluracil, or thymine dimers, and show that these base modifications can be readily detected with single-modification resolution and DNA strand specificity. We characterize the distinct kinetic signatures generated by these DNA base modifications. PMID:22185597

  1. SNP discovery through de novo deep sequencing using the next generation of DNA sequencers

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The production of high volumes of DNA sequence data using new technologies has permitted more efficient identification of single nucleotide polymorphisms in vertebrate genomes. This chapter presented practical methodology for production and analysis of DNA sequence data for SNP discovery....

  2. Improving DNA sequencing accuracy and throughput

    SciTech Connect

    Nelson, D.O. |

    1996-12-31

    LLNL is beginning to explore statistical approaches to the problem of determining the DNA sequence underlying data obtained from fluorescence-based gel electrophoresis. Among the features of this problem that make it interesting to statisticians include: (1) the underlying mechanics of electrophoresis is quite complex and still not completely understood; (2) the yield of fragments of any given size can be quite small and variable; (3) the mobility of fragments of a given size can depend on the terminating base; (4) the data consists of samples from one or more continuous, non-stationary signals; (5) boundaries between segments generated by distinct elements of the underlying sequence are ill-defined or nonexistent in the signal; and (6) the sampling rate of the signal greatly exceeds the rate of evolution of the underlying discrete sequence. Current approaches to base calling address only some of these issues, and usually in a heuristic, ad hoc way. In this article we describe some of our initial efforts towards increasing base calling accuracy and throughput by providing a rational, statistical foundation to the process of deducing sequence from signal. 31 refs., 12 figs.

  3. Image correlation method for DNA sequence alignment.

    PubMed

    Curilem Saldías, Millaray; Villarroel Sassarini, Felipe; Muñoz Poblete, Carlos; Vargas Vásquez, Asticio; Maureira Butler, Iván

    2012-01-01

    The complexity of searches and the volume of genomic data make sequence alignment one of bioinformatics most active research areas. New alignment approaches have incorporated digital signal processing techniques. Among these, correlation methods are highly sensitive. This paper proposes a novel sequence alignment method based on 2-dimensional images, where each nucleic acid base is represented as a fixed gray intensity pixel. Query and known database sequences are coded to their pixel representation and sequence alignment is handled as object recognition in a scene problem. Query and database become object and scene, respectively. An image correlation process is carried out in order to search for the best match between them. Given that this procedure can be implemented in an optical correlator, the correlation could eventually be accomplished at light speed. This paper shows an initial research stage where results were "digitally" obtained by simulating an optical correlation of DNA sequences represented as images. A total of 303 queries (variable lengths from 50 to 4500 base pairs) and 100 scenes represented by 100 x 100 images each (in total, one million base pair database) were considered for the image correlation analysis. The results showed that correlations reached very high sensitivity (99.01%), specificity (98.99%) and outperformed BLAST when mutation numbers increased. However, digital correlation processes were hundred times slower than BLAST. We are currently starting an initiative to evaluate the correlation speed process of a real experimental optical correlator. By doing this, we expect to fully exploit optical correlation light properties. As the optical correlator works jointly with the computer, digital algorithms should also be optimized. The results presented in this paper are encouraging and support the study of image correlation methods on sequence alignment. PMID:22761742

  4. Detection of specific DNA sequences by fluorescence amplification: a color complementation assay.

    PubMed Central

    Chehab, F F; Kan, Y W

    1989-01-01

    We have developed a color complementation assay that allows rapid screening of specific genomic DNA sequences. It is based on the simultaneous amplification of two or more DNA segments with fluorescent oligonucleotide primers such that the generation of a color, or combination of colors, can be visualized and used for diagnosis. Color complementation assay obviates the need for gel electrophoresis and has been applied to the detection of a large and small gene deletion, a chromosomal translocation, an infectious agent, and a single-base substitution. DNA amplification with fluorescent oligonucleotide primers has also been used to multiplex and discriminate five different amplified DNA loci simultaneously. Each primer set is conjugated to a different dye, and the fluorescence of each dye respective to its amplified DNA locus is scored on a fluorometer. This method is valuable for DNA diagnostics of genetic, acquired, and infectious diseases, as well as in DNA forensics. It also lends itself to complete automation. Images PMID:2594760

  5. A Microfluidic DNA Library Preparation Platform for Next-Generation Sequencing

    PubMed Central

    Sinha, Anupama; Bent, Zachary W.; Solberg, Owen D.; Williams, Kelly P.; Langevin, Stanley A.; Renzi, Ronald F.; Van De Vreugde, James L.; Meagher, Robert J.; Schoeniger, Joseph S.; Lane, Todd W.; Branda, Steven S.; Bartsch, Michael S.; Patel, Kamlesh D.

    2013-01-01

    Next-generation sequencing (NGS) is emerging as a powerful tool for elucidating genetic information for a wide range of applications. Unfortunately, the surging popularity of NGS has not yet been accompanied by an improvement in automated techniques for preparing formatted sequencing libraries. To address this challenge, we have developed a prototype microfluidic system for preparing sequencer-ready DNA libraries for analysis by Illumina sequencing. Our system combines droplet-based digital microfluidic (DMF) sample handling with peripheral modules to create a fully-integrated, sample-in library-out platform. In this report, we use our automated system to prepare NGS libraries from samples of human and bacterial genomic DNA. E. coli libraries prepared on-device from 5 ng of total DNA yielded excellent sequence coverage over the entire bacterial genome, with >99% alignment to the reference genome, even genome coverage, and good quality scores. Furthermore, we produced a de novo assembly on a previously unsequenced multi-drug resistant Klebsiella pneumoniae strain BAA-2146 (KpnNDM). The new method described here is fast, robust, scalable, and automated. Our device for library preparation will assist in the integration of NGS technology into a wide variety of laboratories, including small research laboratories and clinical laboratories. PMID:23894387

  6. Automating the Photogrammetric Bridging Based on MMS Image Sequence Processing

    NASA Astrophysics Data System (ADS)

    Silva, J. F. C.; Lemes Neto, M. C.; Blasechi, V.

    2014-11-01

    The photogrammetric bridging or traverse is a special bundle block adjustment (BBA) for connecting a sequence of stereo-pairs and of determining the exterior orientation parameters (EOP). An object point must be imaged in more than one stereo-pair. In each stereo-pair the distance ratio between an object and its corresponding image point varies significantly. We propose to automate the photogrammetric bridging based on a fully automatic extraction of homologous points in stereo-pairs and on an arbitrary Cartesian datum to refer the EOP and tie points. The technique uses SIFT algorithm and the keypoint matching is given by similarity descriptors of each keypoint based on the smallest distance. All the matched points are used as tie points. The technique was applied initially to two pairs. The block formed by four images was treated by BBA. The process follows up to the end of the sequence and it is semiautomatic because each block is processed independently and the transition from one block to the next depends on the operator. Besides four image blocks (two pairs), we experimented other arrangements with block sizes of six, eight, and up to twenty images (respectively, three, four, five and up to ten bases). After the whole image pairs sequence had sequentially been adjusted in each experiment, a simultaneous BBA was run so to estimate the EOP set of each image. The results for classical ("normal case") pairs were analyzed based on standard statistics regularly applied to phototriangulation, and they show figures to validate the process.

  7. A compilation of partial sequences of randomly selected cDNA clones from the rat incisor.

    PubMed

    Matsuki, Y; Nakashima, M; Amizuka, N; Warshawsky, H; Goltzman, D; Yamada, K M; Yamada, Y

    1995-01-01

    The formation of tooth organs is regulated by a series of developmental programs. We have initiated a genome project with the ultimate goal of identifying novel genes important for tooth development. As an initial approach, we constructed a unidirectional cDNA library from the non-calcified portion of incisors of 3- to 4-week-old rats, sequenced cDNA clones, and classified their sequences by homology search through the GenBank data base and the PIR protein data base. Here, we report partial DNA sequences obtained by automated DNA sequencing on 400 cDNA clones randomly selected from the library. Of the sequences determined, 51% represented sequences of new genes that were not related to any previously reported gene. Twenty-six percent of the clones strongly matched genes and proteins in the data bases, including amelogenin, alpha 1(I) and alpha 2(I) collagen chains, osteonectin, and decorin. Nine percent of clones revealed partial sequence homology to known genes such as transcription factors and cell surface receptors. A significant number of the previously identified genes were expressed redundantly and were found to encode extracellular matrix proteins. Identification and cataloging of cDNA clones in these tissues are the first step toward identification of markers expressed in a tissue- or stage-specific manner, as well as the genetic linkage study of tooth anomalies. Further characterization of the clones described in this paper should lead to the discovery of novel genes important for tooth development. PMID:7876422

  8. A Molecular Fraction Collecting Tool for the ABI 310 Automated Sequencer

    PubMed Central

    Lin, Ming-Tseh; Rich, Roy G.; Shipley, Royce F.; Hafez, Michael J.; Tseng, Li-Hui; Murphy, Kathleen M.; Gocke, Christopher D.; Eshleman, James R.

    2007-01-01

    Several methods exist to retrieve and purify DNA fragments after agarose or polyacrylamide gel electrophoresis for subsequent analyses. However, molecules present in low concentration and molecules similar in size to their neighbors are difficult to purify. Capillary electrophoresis has become popular in molecular diagnostic laboratories because of its automation, excellent resolution, and high sensitivity. In the current study, the ABI Prism 310 Genetic Analyzer was reconfigured into a fraction collector by adapting the standard gel block to accommodate a collection tube at the distal end of capillary. The time to collect the desired peaks was estimated by extrapolating from standard capillary electrophoresis using the original gel block. Fraction collection from a mixture of DNA fragments amplified from wild type and several internal tandem duplication mutations of the FMS-like tyrosine kinase 3 (Flt3) gene yielded highly purified DNA fragments containing internal tandem duplication mutations and predictable electrokinetics using the reconstructed gel block. The reconfigured instrument could successfully isolate DNA amplicons from extremely low-amplitude peaks (110 relative fluorescent units), which were undetectable using polyacrylamide gel electrophoresis. In addition, we successfully isolated bands that were only three bases apart that comigrated on polyacrylamide gel electrophoresis. DNA sequencing was used to confirm that the correct peaks were recovered at sufficient purity. PMID:17916601

  9. (abstract) Automated Constraint Checking of Spacecraft Command Sequences

    NASA Technical Reports Server (NTRS)

    Horvath, Joan; Alkalaj, Leon; Schneider, Karl; Spitale, Joseph

    1994-01-01

    Making certain that spacecraft command sequences do not violate any constraints is often tedious and expensive in terms of both personnel and software development. To reduce this cost, we have pursued the development of a flexible system for specifying models of spacecraft behavior in response to commands as well as constraints on that behavior. The potential need for modeling complex spacecraft behavior required that the system be designed to be usable both on a conventional workstation and a parallel supercomputer. Finally, it needed to be intuitive enough for the the intended mission operations users to easily design sets of rules and models to automate tedious, resource-consuming constraint checking of commands. We have defined a Specification And Verification Environment (SAVE) for spacecraft flight rules.

  10. Determining orientation and direction of DNA sequences

    DOEpatents

    Goodwin, Edwin H.; Meyne, Julianne

    2000-01-01

    Determining orientation and direction of DNA sequences. A method by which fluorescence in situ hybridization can be made strand specific is described. Cell cultures are grown in a medium containing a halogenated nucleotide. The analog is partially incorporated in one DNA strand of each chromatid. This substitution takes place in opposite strands of the two sister chromatids. After staining with the fluorescent DNA-binding dye Hoechst 33258, cells are exposed to long-wavelength ultraviolet light which results in numerous strand nicks. These nicks enable the substituted strand to be denatured and solubilized by heat, treatment with high or low pH aqueous solutions, or by immersing the strands in 2.times.SSC (0.3M NaCl+0.03M sodium citrate), to name three procedures. It is unnecessary to enzymatically digest the strands using Exo III or another exonuclease in order to excise and solubilize nucleotides starting at the sites of the nicks. The denaturing/solubilizing process removes most of the substituted strand while leaving the prereplication strand largely intact. Hybridization of a single-stranded probe of a tandem repeat arranged in a head-to-tail orientation will result in hybridization only to the chromatid with the complementary strand present.

  11. Using Huffman coding method to visualize and analyze DNA sequences.

    PubMed

    Qi, Zhao-Hui; Li, Ling; Qi, Xiao-Qin

    2011-11-30

    On the basis of the Huffman coding method, we propose a new graphical representation of DNA sequence. The representation can avoid degeneracy and loss of information in the transfer of data from a DNA sequence to its graphical representation. Then a multicomponent vector from the representation is introduced to characterize quantitatively DNA sequences. The components of the vector are derived from the graphical representation of DNA primary sequence. The examination of similarities and dissimilarities among the complete coding sequences of β-globin gene of 11 species and six ND6 proteins shows the utility of the scheme. PMID:21953557

  12. Sequence-of-events-driven automation of the deep space network

    NASA Technical Reports Server (NTRS)

    Hill, R., Jr.; Fayyad, K.; Smyth, C.; Santos, T.; Chen, R.; Chien, S.; Bevan, R.

    1996-01-01

    In February 1995, sequence-of-events (SOE)-driven automation technology was demonstrated for a Voyager telemetry downlink track at DSS 13. This demonstration entailed automated generation of an operations procedure (in the form of a temporal dependency network) from project SOE information using artificial intelligence planning technology and automated execution of the temporal dependency network using the link monitor and control operator assistant system. This article describes the overall approach to SOE-driven automation that was demonstrated, identifies gaps in SOE definitions and project profiles that hamper automation, and provides detailed measurements of the knowledge engineering effort required for automation.

  13. Non-random DNA fragmentation in next-generation sequencing

    PubMed Central

    Poptsova, Maria S.; Il'icheva, Irina A.; Nechipurenko, Dmitry Yu.; Panchenko, Larisa A.; Khodikov, Mingian V.; Oparina, Nina Y.; Polozov, Robert V.; Nechipurenko, Yury D.; Grokhovsky, Sergei L.

    2014-01-01

    Next Generation Sequencing (NGS) technology is based on cutting DNA into small fragments, and their massive parallel sequencing. The multiple overlapping segments termed “reads” are assembled into a contiguous sequence. To reduce sequencing errors, every genome region should be sequenced several dozen times. This sequencing approach is based on the assumption that genomic DNA breaks are random and sequence-independent. However, previously we showed that for the sonicated restriction DNA fragments the rates of double-stranded breaks depend on the nucleotide sequence. In this work we analyzed genomic reads from NGS data and discovered that fragmentation methods based on the action of the hydrodynamic forces on DNA, produce similar bias. Consideration of this non-random DNA fragmentation may allow one to unravel what factors and to what extent influence the non-uniform coverage of various genomic regions. PMID:24681819

  14. Non-random DNA fragmentation in next-generation sequencing

    NASA Astrophysics Data System (ADS)

    Poptsova, Maria S.; Il'Icheva, Irina A.; Nechipurenko, Dmitry Yu.; Panchenko, Larisa A.; Khodikov, Mingian V.; Oparina, Nina Y.; Polozov, Robert V.; Nechipurenko, Yury D.; Grokhovsky, Sergei L.

    2014-03-01

    Next Generation Sequencing (NGS) technology is based on cutting DNA into small fragments, and their massive parallel sequencing. The multiple overlapping segments termed ``reads'' are assembled into a contiguous sequence. To reduce sequencing errors, every genome region should be sequenced several dozen times. This sequencing approach is based on the assumption that genomic DNA breaks are random and sequence-independent. However, previously we showed that for the sonicated restriction DNA fragments the rates of double-stranded breaks depend on the nucleotide sequence. In this work we analyzed genomic reads from NGS data and discovered that fragmentation methods based on the action of the hydrodynamic forces on DNA, produce similar bias. Consideration of this non-random DNA fragmentation may allow one to unravel what factors and to what extent influence the non-uniform coverage of various genomic regions.

  15. MG-Digger: An Automated Pipeline to Search for Giant Virus-Related Sequences in Metagenomes

    PubMed Central

    Verneau, Jonathan; Levasseur, Anthony; Raoult, Didier; La Scola, Bernard; Colson, Philippe

    2016-01-01

    The number of metagenomic studies conducted each year is growing dramatically. Storage and analysis of such big data is difficult and time-consuming. Interestingly, analysis shows that environmental and human metagenomes include a significant amount of non-annotated sequences, representing a ‘dark matter.’ We established a bioinformatics pipeline that automatically detects metagenome reads matching query sequences from a given set and applied this tool to the detection of sequences matching large and giant DNA viral members of the proposed order Megavirales or virophages. A total of 1,045 environmental and human metagenomes (≈ 1 Terabase) were collected, processed, and stored on our bioinformatics server. In addition, nucleotide and protein sequences from 93 Megavirales representatives, including 19 giant viruses of amoeba, and 5 virophages, were collected. The pipeline was generated by scripts written in Python language and entitled MG-Digger. Metagenomes previously found to contain megavirus-like sequences were tested as controls. MG-Digger was able to annotate 100s of metagenome sequences as best matching those of giant viruses. These sequences were most often found to be similar to phycodnavirus or mimivirus sequences, but included reads related to recently available pandoraviruses, Pithovirus sibericum, and faustoviruses. Compared to other tools, MG-Digger combined stand-alone use on Linux or Windows operating systems through a user-friendly interface, implementation of ready-to-use customized metagenome databases and query sequence databases, adjustable parameters for BLAST searches, and creation of output files containing selected reads with best match identification. Compared to Metavir 2, a reference tool in viral metagenome analysis, MG-Digger detected 8% more true positive Megavirales-related reads in a control metagenome. The present work shows that massive, automated and recurrent analyses of metagenomes are effective in improving knowledge about

  16. MG-Digger: An Automated Pipeline to Search for Giant Virus-Related Sequences in Metagenomes.

    PubMed

    Verneau, Jonathan; Levasseur, Anthony; Raoult, Didier; La Scola, Bernard; Colson, Philippe

    2016-01-01

    The number of metagenomic studies conducted each year is growing dramatically. Storage and analysis of such big data is difficult and time-consuming. Interestingly, analysis shows that environmental and human metagenomes include a significant amount of non-annotated sequences, representing a 'dark matter.' We established a bioinformatics pipeline that automatically detects metagenome reads matching query sequences from a given set and applied this tool to the detection of sequences matching large and giant DNA viral members of the proposed order Megavirales or virophages. A total of 1,045 environmental and human metagenomes (≈ 1 Terabase) were collected, processed, and stored on our bioinformatics server. In addition, nucleotide and protein sequences from 93 Megavirales representatives, including 19 giant viruses of amoeba, and 5 virophages, were collected. The pipeline was generated by scripts written in Python language and entitled MG-Digger. Metagenomes previously found to contain megavirus-like sequences were tested as controls. MG-Digger was able to annotate 100s of metagenome sequences as best matching those of giant viruses. These sequences were most often found to be similar to phycodnavirus or mimivirus sequences, but included reads related to recently available pandoraviruses, Pithovirus sibericum, and faustoviruses. Compared to other tools, MG-Digger combined stand-alone use on Linux or Windows operating systems through a user-friendly interface, implementation of ready-to-use customized metagenome databases and query sequence databases, adjustable parameters for BLAST searches, and creation of output files containing selected reads with best match identification. Compared to Metavir 2, a reference tool in viral metagenome analysis, MG-Digger detected 8% more true positive Megavirales-related reads in a control metagenome. The present work shows that massive, automated and recurrent analyses of metagenomes are effective in improving knowledge about the

  17. Automated Analysis of Dynamic Ca2+ Signals in Image Sequences

    PubMed Central

    Francis, Michael; Waldrup, Josh; Qian, Xun; Taylor, Mark S.

    2014-01-01

    Intracellular Ca2+ signals are commonly studied with fluorescent Ca2+ indicator dyes and microscopy techniques. However, quantitative analysis of Ca2+ imaging data is time consuming and subject to bias. Automated signal analysis algorithms based on region of interest (ROI) detection have been implemented for one-dimensional line scan measurements, but there is no current algorithm which integrates optimized identification and analysis of ROIs in two-dimensional image sequences. Here an algorithm for rapid acquisition and analysis of ROIs in image sequences is described. It utilizes ellipses fit to noise filtered signals in order to determine optimal ROI placement, and computes Ca2+ signal parameters of amplitude, duration and spatial spread. This algorithm was implemented as a freely available plugin for ImageJ (NIH) software. Together with analysis scripts written for the open source statistical processing software R, this approach provides a high-capacity pipeline for performing quick statistical analysis of experimental output. The authors suggest that use of this analysis protocol will lead to a more complete and unbiased characterization of physiologic Ca2+ signaling. PMID:24962784

  18. SeqTrace: A Graphical Tool for Rapidly Processing DNA Sequencing Chromatograms

    PubMed Central

    Stucky, Brian J.

    2012-01-01

    Modern applications of Sanger DNA sequencing often require converting a large number of chromatogram trace files into high-quality DNA sequences for downstream analyses. Relatively few nonproprietary software tools are available to assist with this process. SeqTrace is a new, free, and open-source software application that is designed to automate the entire workflow by facilitating easy batch processing of large numbers of trace files. SeqTrace can identify, align, and compute consensus sequences from matching forward and reverse traces, filter low-quality base calls, and end-trim finished sequences. The software features a graphical interface that includes a full-featured chromatogram viewer and sequence editor. SeqTrace runs on most popular operating systems and is freely available, along with supporting documentation, at http://seqtrace.googlecode.com/. PMID:22942788

  19. DNA Shape versus Sequence Variations in the Protein Binding Process.

    PubMed

    Chen, Chuanying; Pettitt, B Montgomery

    2016-02-01

    The binding process of a protein with a DNA involves three stages: approach, encounter, and association. It has been known that the complexation of protein and DNA involves mutual conformational changes, especially for a specific sequence association. However, it is still unclear how the conformation and the information in the DNA sequences affects the binding process. What is the extent to which the DNA structure adopted in the complex is induced by protein binding, or is instead intrinsic to the DNA sequence? In this study, we used the multiscale simulation method to explore the binding process of a protein with DNA in terms of DNA sequence, conformation, and interactions. We found that in the approach stage the protein can bind both the major and minor groove of the DNA, but uses different features to locate the binding site. The intrinsic conformational properties of the DNA play a significant role in this binding stage. By comparing the specific DNA with the nonspecific in unbound, intermediate, and associated states, we found that for a specific DNA sequence, ∼40% of the bending in the association forms is intrinsic and that ∼60% is induced by the protein. The protein does not induce appreciable bending of nonspecific DNA. In addition, we proposed that the DNA shape variations induced by protein binding are required in the early stage of the binding process, so that the protein is able to approach, encounter, and form an intermediate at the correct site on DNA. PMID:26840719

  20. Inferring coalescence times from DNA sequence data.

    PubMed

    Tavaré, S; Balding, D J; Griffiths, R C; Donnelly, P

    1997-02-01

    The paper is concerned with methods for the estimation of the coalescence time (time since the most recent common ancestor) of a sample of intraspecies DNA sequences. The methods take advantage of prior knowledge of population demography, in addition to the molecular data. While some theoretical results are presented, a central focus is on computational methods. These methods are easy to implement, and, since explicit formulae tend to be either unavailable or unilluminating, they are also more useful and more informative in most applications. Extensions are presented that allow for the effects of uncertainty in our knowledge of population size and mutation rates, for variability in population sizes, for regions of different mutation rate, and for inference concerning the coalescence time of the entire population. The methods are illustrated using recent data from the human Y chromosome. PMID:9071603

  1. Development of an Automated DNA Detection System Using an Electrochemical DNA Chip Technology

    NASA Astrophysics Data System (ADS)

    Hongo, Sadato; Okada, Jun; Hashimoto, Koji; Tsuji, Koichi; Nikaido, Masaru; Gemma, Nobuhiro

    A new compact automated DNA detection system Genelyzer™ has been developed. After injecting a sample solution into a cassette with a built-in electrochemical DNA chip, processes from hybridization reaction to detection and analysis are all operated fully automatically. In order to detect a sample DNA, electrical currents from electrodes due to an oxidization reaction of electrochemically active intercalator molecules bound to hybridized DNAs are detected. The intercalator is supplied as a reagent solution by a fluid supply unit of the system. The feasibility test proved that the simultaneous typing of six single nucleotide polymorphisms (SNPs) associated with a rheumatoid arthritis (RA) was carried out within two hours and that all the results were consistent with those by conventional typing methods. It is expected that this system opens a new way to a DNA testing such as a test for infectious diseases, a personalized medicine, a food inspection, a forensic application and any other applications.

  2. On 2D graphical representation of DNA sequence of nondegeneracy

    NASA Astrophysics Data System (ADS)

    Zhang, Yusen; Liao, Bo; Ding, Kequan

    2005-08-01

    Some two-dimensional (2D) graphical representations of DNA sequences have been given by Gates, Nandy, Leong and Mogenthaler, Randić, and Liao et al., which give visual characterizations of DNA sequences. In this Letter, we introduce a nondegeneracy 2D graphical representation of DNA sequence, which is different from Randić's novel 2D representation and Liao's 2D representation. We also present the nondegeneracy forms corresponding to the representations of Gates, Nandy, Leong and Mogenthaler.

  3. Duplication count distributions in DNA sequences

    NASA Astrophysics Data System (ADS)

    Sindi, Suzanne S.; Hunt, Brian R.; Yorke, James A.

    2008-12-01

    We study quantitative features of complex repetitive DNA in several genomes by studying sequences that are sufficiently long that they are unlikely to have repeated by chance. For each genome we study, we determine the number of identical copies, the “duplication count,” of each sequence of length 40, that is of each “40-mer.” We say a 40-mer is “repeated” if its duplication count is at least 2. We focus mainly on “complex” 40-mers, those without short internal repetitions. We find that we can classify most of the complex repeated 40-mers into two categories: one category has its copies clustered closely together on one chromosome, the other has its copies distributed widely across multiple chromosomes. For each genome and each of the categories above, we compute N(c) , the number of 40-mers that have duplication count c , for each integer c . In each case, we observe a power-law-like decay in N(c) as c increases from 3 to 50 or higher. In particular, we find that N(c) decays much more slowly than would be predicted by evolutionary models where each 40-mer is equally likely to be duplicated. We also analyze an evolutionary model that does reflect the slow decay of N(c) .

  4. What Advances Are Being Made in DNA Sequencing?

    MedlinePlus

    ... the future. For more information about DNA sequencing technologies and their use: Genetics Home Reference discusses whether ... the University of Washington describes the different sequencing technologies and what the new technologies have meant for ...

  5. DNA sequence determination by hybridization: A strategy for efficient large-scale sequencing

    SciTech Connect

    Drmanac, R.; Drmanac, S.; Strezoska, Z.; Paunesku, T.; Labat, I.; Zeremski, M.; Snoody, J.; Crkvenjakov, R. ); Funkhouser, W.K.; Koop, B.; Hood, L. )

    1993-06-11

    The concept of sequencing by hybridization (SBH) makes use of an array of all possible n-nucleotide oligomers (n-mers) to identify n-mers present in an unknown DNA sequence. Computational approaches can then be used to assemble the complete sequence. As a validation of this concept, the sequences of three DNA fragments, 343 base pairs in length, were determined with octamer oligonucleotides. Possible applications of SBH include physical mapping (ordering) of overlapping DNA clones, sequence checking, DNA fingerprinting comparisons of normal and disease-causing genes, and the identification of DNA fragments with particular sequence motifs in complementary DNA and genomic libraries. The SBH techniques may accelerate the mapping and sequencing phases of the human genome project. 22 refs., 3 figs.

  6. DNA Sequence Determination by Hybridization: A Strategy for Efficient Large-Scale Sequencing

    NASA Astrophysics Data System (ADS)

    Drmanac, R.; Drmanac, S.; Strezoska, Z.; Paunesku, T.; Labat, I.; Zeremski, M.; Snoddy, J.; Funkhouser, W. K.; Koop, B.; Hood, L.; Crkvenjakov, R.

    1993-06-01

    The concept of sequencing by hybridization (SBH) makes use of an array of all possible n-nucleotide oligomers (n-mers) to identify n-mers present in an unknown DNA sequence. Computational approaches can then be used to assemble the complete sequence. As a validation of this concept, the sequences of three DNA fragments, 343 base pairs in length, were determined with octamer oligonucleotides. Possible applications of SBH include physical mapping (ordering) of overlapping DNA clones, sequence checking, DNA fingerprinting comparisons of normal and disease-causing genes, and the identification of DNA fragments with particular sequence motifs in complementary DNA and genomic libraries. The SBH techniques may accelerate the mapping and sequencing phases of the human genome project.

  7. Kinetoplast DNA minicircles: regions of extensive sequence divergence.

    PubMed Central

    Rogers, W O; Wirth, D F

    1987-01-01

    Previous work has shown that the kinetoplast minicircle DNA of Leishmania species exhibits species-specific sequence divergence and this observation has led to the development of a DNA probe-based diagnostic test for leishmaniasis. In the work reported here, we demonstrate that the minicircle is composed of three types of DNA sequences with differing specificities reflecting different rates of DNA sequence change. A library of cloned fragments of kinetoplast DNA (kDNA) from Leishmania mexicana amazonensis was prepared and the cloned subfragments were found to contain DNA sequences with different taxonomic specificities based on hybridization analysis with various species of Leishmania. Four groups of subfragments were found, those that hybridized with a large number of Leishmania sp. as well as sequences unique to the species, subspecies, or isolate. Analysis of nested deletions of a single, full-length minicircle demonstrates that these different taxonomic specificities are contained within a single minicircle. This implies that different regions of a single minicircle have DNA sequences that diverge at different rates. These sequences represent potentially valuable tools in diagnostic, epidemiologic, and ecological studies of leishmaniasis and provide the basis for a model of kDNA sequence evolution. Images PMID:3025880

  8. A Novel Constraint for Thermodynamically Designing DNA Sequences

    PubMed Central

    Zhang, Qiang; Wang, Bin; Wei, Xiaopeng; Zhou, Changjun

    2013-01-01

    Biotechnological and biomolecular advances have introduced novel uses for DNA such as DNA computing, storage, and encryption. For these applications, DNA sequence design requires maximal desired (and minimal undesired) hybridizations, which are the product of a single new DNA strand from 2 single DNA strands. Here, we propose a novel constraint to design DNA sequences based on thermodynamic properties. Existing constraints for DNA design are based on the Hamming distance, a constraint that does not address the thermodynamic properties of the DNA sequence. Using a unique, improved genetic algorithm, we designed DNA sequence sets which satisfy different distance constraints and employ a free energy gap based on a minimum free energy (MFE) to gauge DNA sequences based on set thermodynamic properties. When compared to the best constraints of the Hamming distance, our method yielded better thermodynamic qualities. We then used our improved genetic algorithm to obtain lower-bound DNA sequence sets. Here, we discuss the effects of novel constraint parameters on the free energy gap. PMID:24015217

  9. A novel constraint for thermodynamically designing DNA sequences.

    PubMed

    Zhang, Qiang; Wang, Bin; Wei, Xiaopeng; Zhou, Changjun

    2013-01-01

    Biotechnological and biomolecular advances have introduced novel uses for DNA such as DNA computing, storage, and encryption. For these applications, DNA sequence design requires maximal desired (and minimal undesired) hybridizations, which are the product of a single new DNA strand from 2 single DNA strands. Here, we propose a novel constraint to design DNA sequences based on thermodynamic properties. Existing constraints for DNA design are based on the Hamming distance, a constraint that does not address the thermodynamic properties of the DNA sequence. Using a unique, improved genetic algorithm, we designed DNA sequence sets which satisfy different distance constraints and employ a free energy gap based on a minimum free energy (MFE) to gauge DNA sequences based on set thermodynamic properties. When compared to the best constraints of the Hamming distance, our method yielded better thermodynamic qualities. We then used our improved genetic algorithm to obtain lower-bound DNA sequence sets. Here, we discuss the effects of novel constraint parameters on the free energy gap. PMID:24015217

  10. Guanine-rich sequences inhibit proofreading DNA polymerases

    PubMed Central

    Zhu, Xiao-Jing; Sun, Shuhui; Xie, Binghua; Hu, Xuemei; Zhang, Zunyi; Qiu, Mengsheng; Dai, Zhong-Min

    2016-01-01

    DNA polymerases with proofreading activity are important for accurate amplification of target DNA. Despite numerous efforts have been made to improve the proofreading DNA polymerases, they are more susceptible to be failed in PCR than non-proofreading DNA polymerases. Here we showed that proofreading DNA polymerases can be inhibited by certain primers. Further analysis showed that G-rich sequences such as GGGGG and GGGGHGG can cause PCR failure using proofreading DNA polymerases but not Taq DNA polymerase. The inhibitory effect of these G-rich sequences is caused by G-quadruplex and is dose dependent. G-rich inhibitory sequence-containing primers can be used in PCR at a lower concentration to amplify its target DNA fragment. PMID:27349576

  11. Advances in high throughput DNA sequence data compression.

    PubMed

    Sardaraz, Muhammad; Tahir, Muhammad; Ikram, Ataul Aziz

    2016-06-01

    Advances in high throughput sequencing technologies and reduction in cost of sequencing have led to exponential growth in high throughput DNA sequence data. This growth has posed challenges such as storage, retrieval, and transmission of sequencing data. Data compression is used to cope with these challenges. Various methods have been developed to compress genomic and sequencing data. In this article, we present a comprehensive review of compression methods for genome and reads compression. Algorithms are categorized as referential or reference free. Experimental results and comparative analysis of various methods for data compression are presented. Finally, key challenges and research directions in DNA sequence data compression are highlighted. PMID:26846812

  12. Preparing DNA Libraries for Multiplexed Paired-End Deep Sequencing for Illumina GA Sequencers

    PubMed Central

    Son, Mike S.; Taylor, Ronald K.

    2011-01-01

    Whole genome sequencing, also known as deep sequencing, is becoming a more affordable and efficient way to identify SNP mutations, deletions and insertions in DNA sequences across several different strains. Two major obstacles preventing the widespread use of deep sequencers are the costs involved in services used to prepare DNA libraries for sequencing and the overall accuracy of the sequencing data. This Unit describes the preparation of DNA libraries for multiplexed paired-end sequencing using the Illumina GA series sequencer. Self-preparation of DNA libraries can help reduce overall expenses, especially if optimization is required for the different samples, and use of the Illumina GA Sequencer can improve the quality of the data. PMID:21400673

  13. Chimeric DNA methyltransferases target DNA methylation to specific DNA sequences and repress expression of target genes

    PubMed Central

    Li, Fuyang; Papworth, Monika; Minczuk, Michal; Rohde, Christian; Zhang, Yingying; Ragozin, Sergei; Jeltsch, Albert

    2007-01-01

    Gene silencing by targeted DNA methylation has potential applications in basic research and therapy. To establish targeted methylation in human cell lines, the catalytic domains (CDs) of mouse Dnmt3a and Dnmt3b DNA methyltransferases (MTases) were fused to different DNA binding domains (DBD) of GAL4 and an engineered Cys2His2 zinc finger domain. We demonstrated that (i) Dense DNA methylation can be targeted to specific regions in gene promoters using chimeric DNA MTases. (ii) Site-specific methylation leads to repression of genes controlled by various cellular or viral promoters. (iii) Mutations affecting any of the DBD, MTase or target DNA sequences reduce targeted methylation and gene silencing. (iv) Targeted DNA methylation is effective in repressing Herpes Simplex Virus type 1 (HSV-1) infection in cell culture with the viral titer reduced by at least 18-fold in the presence of an MTase fused to an engineered zinc finger DBD, which binds a single site in the promoter of HSV-1 gene IE175k. In short, we show here that it is possible to direct DNA MTase activity to predetermined sites in DNA, achieve targeted gene silencing in mammalian cell lines and interfere with HSV-1 propagation. PMID:17151075

  14. Affordable Hands-On DNA Sequencing and Genotyping: An Exercise for Teaching DNA Analysis to Undergraduates

    ERIC Educational Resources Information Center

    Shah, Kushani; Thomas, Shelby; Stein, Arnold

    2013-01-01

    In this report, we describe a 5-week laboratory exercise for undergraduate biology and biochemistry students in which students learn to sequence DNA and to genotype their DNA for selected single nucleotide polymorphisms (SNPs). Students use miniaturized DNA sequencing gels that require approximately 8 min to run. The students perform G, A, T, C…

  15. Next Generation Sequencing to Characterize Mitochondrial Genomic DNA Heteroplasmy

    PubMed Central

    Huang, Taosheng

    2015-01-01

    This protocol is to describe the methodology to characterize mitochondria DNA (mtDNA) heteroplasmy with parallel sequencing. Mitochondria play a very important role in important cellular functions. Each eukaryotic cell contains hundreds of mitochondria with hundreds of mitochondria genomes. The mutant mtDNA and the wild type may co-exist as heteroplasmy, and cause human disease. The purpose of this methodology is to simultaneously determine mtDNA sequence and to quantify the heteroplasmy level. The protocol includes two-fragment mitochondria genome DNA PCR amplification. The PCR product is then mixed at an equimolar ratio. The samples will be barcoded and sequenced with high-throughput next-generation sequencing technology. We found that this technology is highly sensitive, specific, and accurate in determining mtDNA mutations and the degree of heteroplasmic level. PMID:21975941

  16. DNA polymerases drive DNA sequencing-by-synthesis technologies: both past and present

    PubMed Central

    Chen, Cheng-Yao

    2014-01-01

    Next-generation sequencing (NGS) technologies have revolutionized modern biological and biomedical research. The engines responsible for this innovation are DNA polymerases; they catalyze the biochemical reaction for deriving template sequence information. In fact, DNA polymerase has been a cornerstone of DNA sequencing from the very beginning. Escherichia coli DNA polymerase I proteolytic (Klenow) fragment was originally utilized in Sanger’s dideoxy chain-terminating DNA sequencing chemistry. From these humble beginnings followed an explosion of organism-specific, genome sequence information accessible via public database. Family A/B DNA polymerases from mesophilic/thermophilic bacteria/archaea were modified and tested in today’s standard capillary electrophoresis (CE) and NGS sequencing platforms. These enzymes were selected for their efficient incorporation of bulky dye-terminator and reversible dye-terminator nucleotides respectively. Third generation, real-time single molecule sequencing platform requires slightly different enzyme properties. Enterobacterial phage ϕ29 DNA polymerase copies long stretches of DNA and possesses a unique capability to efficiently incorporate terminal phosphate-labeled nucleoside polyphosphates. Furthermore, ϕ29 enzyme has also been utilized in emerging DNA sequencing technologies including nanopore-, and protein-transistor-based sequencing. DNA polymerase is, and will continue to be, a crucial component of sequencing technologies. PMID:25009536

  17. Laser Desorption Mass Spectrometry for DNA Sequencing and Analysis

    NASA Astrophysics Data System (ADS)

    Chen, C. H. Winston; Taranenko, N. I.; Golovlev, V. V.; Isola, N. R.; Allman, S. L.

    1998-03-01

    Rapid DNA sequencing and/or analysis is critically important for biomedical research. In the past, gel electrophoresis has been the primary tool to achieve DNA analysis and sequencing. However, gel electrophoresis is a time-consuming and labor-extensive process. Recently, we have developed and used laser desorption mass spectrometry (LDMS) to achieve sequencing of ss-DNA longer than 100 nucleotides. With LDMS, we succeeded in sequencing DNA in seconds instead of hours or days required by gel electrophoresis. In addition to sequencing, we also applied LDMS for the detection of DNA probes for hybridization LDMS was also used to detect short tandem repeats for forensic applications. Clinical applications for disease diagnosis such as cystic fibrosis caused by base deletion and point mutation have also been demonstrated. Experimental details will be presented in the meeting. abstract.

  18. Next Generation Sequencing of Ancient DNA: Requirements, Strategies and Perspectives

    PubMed Central

    Knapp, Michael; Hofreiter, Michael

    2010-01-01

    The invention of next-generation-sequencing has revolutionized almost all fields of genetics, but few have profited from it as much as the field of ancient DNA research. From its beginnings as an interesting but rather marginal discipline, ancient DNA research is now on its way into the centre of evolutionary biology. In less than a year from its invention next-generation-sequencing had increased the amount of DNA sequence data available from extinct organisms by several orders of magnitude. Ancient DNA research is now not only adding a temporal aspect to evolutionary studies and allowing for the observation of evolution in real time, it also provides important data to help understand the origins of our own species. Here we review progress that has been made in next-generation-sequencing of ancient DNA over the past five years and evaluate sequencing strategies and future directions. PMID:24710043

  19. Nanopores: A journey towards DNA sequencing

    PubMed Central

    Wanunu, Meni

    2013-01-01

    Much more than ever, nucleic acids are recognized as key building blocks in many of life's processes, and the science of studying these molecular wonders at the single-molecule level is thriving. A new method of doing so has been introduced in the mid 1990's. This method is exceedingly simple: a nanoscale pore that spans across an impermeable thin membrane is placed between two chambers that contain an electrolyte, and voltage is applied across the membrane using two electrodes. These conditions lead to a steady stream of ion flow across the pore. Nucleic acid molecules in solution can be driven through the pore, and structural features of the biomolecules are observed as measurable changes in the trans-membrane ion current. In essence, a nanopore is a high-throughput ion microscope and a single-molecule force apparatus. Nanopores are taking center stage as a tool that promises to read a DNA sequence, and this promise has resulted in overwhelming academic, industrial, and national interest. Regardless of the fate of future nanopore applications, in the process of this 16-year-long exploration, many studies have validated the indispensability of nanopores in the toolkit of single-molecule biophysics. This review surveys past and current studies related to nucleic acid biophysics, and will hopefully provoke a discussion of immediate and future prospects for the field. PMID:22658507

  20. Food Fish Identification from DNA Extraction through Sequence Analysis

    ERIC Educational Resources Information Center

    Hallen-Adams, Heather E.

    2015-01-01

    This experiment exposed 3rd and 4th y undergraduates and graduate students taking a course in advanced food analysis to DNA extraction, polymerase chain reaction (PCR), and DNA sequence analysis. Students provided their own fish sample, purchased from local grocery stores, and the class as a whole extracted DNA, which was then subjected to PCR,…

  1. Characteristics of cloned repeated DNA sequences in the barley genome

    SciTech Connect

    Anan'ev, E.V.; Bochkanov, S.S.; Ryzhik, M.V.; Sonina, N.V.; Chernyshev, A.I.; Shchipkova, N.I.; Yakovleva, E.Yu.

    1986-12-01

    A partial clone library of barley DNA fragments based on plasmid pBR325 was created. The cloned EcoRI-fragments of chromosomal DNA are from 2 to 14 kbp in length. More than 95% of the barley DNA inserts comprise repeated sequences of different complexity and copy number. Certain of these DNA sequences are from families comprising at least 1% of the barley genome. A significant proportion of the clones hybridize with numerous sets of restriction fragments of genome DNA and they are dispersed throughout the barley chromosomes.

  2. Sequence-Specific DNA Binding by a Short Peptide Dimer

    NASA Astrophysics Data System (ADS)

    Talanian, Robert V.; McKnight, C. James; Kim, Peter S.

    1990-08-01

    A recently described class of DNA binding proteins is characterized by the "bZIP" motif, which consists of a basic region that contacts DNA and an adjacent "leucine zipper" that mediates protein dimerization. A peptide model for the basic region of the yeast transcriptional activator GCN4 has been developed in which the leucine zipper has been replaced by a disulfide bond. The 34-residue peptide dimer, but not the reduced monomer, binds DNA with nanomolar affinity at 4^circC. DNA binding is sequence-specific as judged by deoxyribonuclease I footprinting. Circular dichroism spectroscopy suggests that the peptide adopts a helical structure when bound to DNA. These results demonstrate directly that the GCN4 basic region is sufficient for sequence-specific DNA binding and suggest that a major function of the GCN4 leucine zipper is simply to mediate protein dimerization. Our approach provides a strategy for the design of short sequence-specific DNA binding peptides.

  3. Deconvolving the recognition of DNA shape from sequence.

    PubMed

    Abe, Namiko; Dror, Iris; Yang, Lin; Slattery, Matthew; Zhou, Tianyin; Bussemaker, Harmen J; Rohs, Remo; Mann, Richard S

    2015-04-01

    Protein-DNA binding is mediated by the recognition of the chemical signatures of the DNA bases and the 3D shape of the DNA molecule. Because DNA shape is a consequence of sequence, it is difficult to dissociate these modes of recognition. Here, we tease them apart in the context of Hox-DNA binding by mutating residues that, in a co-crystal structure, only recognize DNA shape. Complexes made with these mutants lose the preference to bind sequences with specific DNA shape features. Introducing shape-recognizing residues from one Hox protein to another swapped binding specificities in vitro and gene regulation in vivo. Statistical machine learning revealed that the accuracy of binding specificity predictions improves by adding shape features to a model that only depends on sequence, and feature selection identified shape features important for recognition. Thus, shape readout is a direct and independent component of binding site selection by Hox proteins. PMID:25843630

  4. Use of robotics in high-throughput DNA sequencing.

    PubMed

    Keeney, Stephen

    2011-01-01

    Until relatively recently, full sequencing of genes consisting of more than several exons was not considered practicable within a routine diagnostic context. As a result, many approaches to unknown mutation detection in a specific gene involved a mutation pre-screening step to limit the amount of DNA sequencing required. Protocols to pre-screen for mutations and limit the amount of DNA sequencing may not localise every base change present and/or require considerable levels of manual intervention. Advances in technology, allied with careful protocol design, now permit direct DNA sequencing to be applied to larger areas of gene sequence, allowing unequivocal mutation identification in the area of a gene being analysed. The protocol described below utilises robotic systems, allied to custom-designed PCR primers, to facilitate rapid DNA sequencing of multiple gene targets. The general approach is amenable to adaptation for use with multi-channel pipettes. PMID:20938842

  5. DNA polymerase having modified nucleotide binding site for DNA sequencing

    DOEpatents

    Tabor, S.; Richardson, C.

    1997-03-25

    A modified gene encoding a modified DNA polymerase is disclosed. The modified polymerase incorporates dideoxynucleotides at least 20-fold better compared to the corresponding deoxynucleotides as compared with the corresponding naturally-occurring DNA polymerase. 6 figs.

  6. DNA polymerase having modified nucleotide binding site for DNA sequencing

    DOEpatents

    Tabor, Stanley; Richardson, Charles

    1997-01-01

    Modified gene encoding a modified DNA polymerase wherein the modified polymerase incorporates dideoxynucleotides at least 20-fold better compared to the corresponding deoxynucleotides as compared with the corresponding naturally-occurring DNA polymerase.

  7. Spectral entropy criteria for structural segmentation in genomic DNA sequences

    NASA Astrophysics Data System (ADS)

    Chechetkin, V. R.; Lobzin, V. V.

    2004-07-01

    The spectral entropy is calculated with Fourier structure factors and characterizes the level of structural ordering in a sequence of symbols. It may efficiently be applied to the assessment and reconstruction of the modular structure in genomic DNA sequences. We present the relevant spectral entropy criteria for the local and non-local structural segmentation in DNA sequences. The results are illustrated with the model examples and analysis of intervening exon-intron segments in the protein-coding regions.

  8. Integrated platform for detection of DNA sequence variants using capillary array electrophoresis

    SciTech Connect

    Qingbro, Li; Liu, Zhaowei; Monroe, Heidi M; Culiat, Cymbeline T

    2002-08-01

    We have developed a highly versatile platform that performs temperature gradient capillary electrophoresis (TGCE) for mutation/single-nucleotide polymorphism (SNP) detection, sequencing and mutation/SNP genotyping for identification of sequence variants on an automated 24-, 96- or 192-capillary array instrument. In the first mode, multiple DNA samples consisting of homoduplexes and heteroduplexes are separated by CE, during which a temperature gradient is applied that covers all possible temperatures of 50% melting equilibrium (Tms) for the samples. The differences in Tms result in separation of homoduplexes from heteroduplexes, thereby identifying the presence of DNA variants. The sequencing mode is then used to determine the exact location of the mutation/SNPs in the DNA variants. The first two modes allow the rapid identification of variants from the screening of a large number of samples. Only the variants need to be sequenced. The third mode utilizes multiplexed single-base extensions (SBEs) to survey mutations and SNPs at the known sites of DNA sequence. The TGCE approach combined with sequencing and SBE is fast and cost-effective for high-throughput mutation/SNP detection.

  9. Progress towards DNA sequencing at the single molecule level

    SciTech Connect

    Goodwin, P.M.; Affleck, R.L.; Ambrose, W.P.

    1995-12-01

    We describe progress towards sequencing DNA at the single molecule level. Our technique involves incorporation of fluorescently tagged nucleotides into a targeted sequence, anchoring the labeled DNA strand in a flowing stream, sequential exonuclease digestion of the DNA strand, and efficient detection and identification of single tagged nucleotides. Experiments demonstrating strand specific exonuclease digestion of fluorescently labeled DNA anchored in flow as well as the detection of single cleaved fluorescently tagged nucleotides from a small number of anchored DNA fragments axe described. We find that the turnover rate of Esherichia coli exonuclease III on fluorescently labeled DNA in flow at 36{degree}C is {approximately}7 nucleotides per DNA strand per second, which is approximately the same as that measured for this enzyme on native DNA under static, saturated (excess enzyme) conditions. Experiments demonstrating the efficient detection of single fluorescent molecules delivered electrokinetically to a {approximately}3 pL probe volume are also described.

  10. Advanced microinstrumentation for rapid DNA sequencing and large DNA fragment separation

    SciTech Connect

    Balch, J.; Davidson, J.; Brewer, L.; Gingrich, J.; Koo, J.; Mariella, R.; Carrano, A.

    1995-01-25

    Our efforts to develop novel technology for a rapid DNA sequencer and large fragment analysis system based upon gel electrophoresis are described. We are using microfabrication technology to build dense arrays of high speed micro electrophoresis lanes that will ultimately increase the sequencing rate of DNA by at least 100 times the rate of current sequencers. We have demonstrated high resolution DNA fragment separation needed for sequencing in polyacrylamide microgels formed in glass microchannels. We have built prototype arrays of microchannels having up to 48 channels. Significant progress has also been made in developing a sensitive fluorescence detection system based upon a confocal microscope design that will enable the diagnostics and detection of DNA fragments in ultrathin microchannel gels. Development of a rapid DNA sequencer and fragment analysis system will have a major impact on future DNA instrumentation used in clinical, molecular and forensic analysis of DNA fragments.

  11. Advances in DNA sequencing technologies for high resolution HLA typing.

    PubMed

    Cereb, Nezih; Kim, Hwa Ran; Ryu, Jaejun; Yang, Soo Young

    2015-12-01

    This communication describes our experience in large-scale G group-level high resolution HLA typing using three different DNA sequencing platforms - ABI 3730 xl, Illumina MiSeq and PacBio RS II. Recent advances in DNA sequencing technologies, so-called next generation sequencing (NGS), have brought breakthroughs in deciphering the genetic information in all living species at a large scale and at an affordable level. The NGS DNA indexing system allows sequencing multiple genes for large number of individuals in a single run. Our laboratory has adopted and used these technologies for HLA molecular testing services. We found that each sequencing technology has its own strengths and weaknesses, and their sequencing performances complement each other. HLA genes are highly complex and genotyping them is quite challenging. Using these three sequencing platforms, we were able to meet all requirements for G group-level high resolution and high volume HLA typing. PMID:26423536

  12. Analysis of separate isolates of Bordetella pertussis repeated DNA sequences.

    PubMed

    McPheat, W L; Hanson, J H; Livey, I; Robertson, J S

    1989-06-01

    Two independent isolates of a Bordetella pertussis repeated DNA unit were sequenced and shown to be an insertion sequence element with five nucleotide differences between the two copies. The sequences were 1053 bp in length with near-perfect terminal inverted repeats of 28 bp, had three open reading frames, and were each flanked by short direct repeats. The two insertion sequences showed considerable homology to two other B. pertussis repeated DNA sequences reported recently: IS481 and a 530 bp repeated DNA unit. The B. pertussis insertion sequence would appear to comprise a group of closely related sequences differing mainly in flanking direct repeats and the terminal inverted repeats. The two isolates reported here, which were from the adenylate cyclase and agglutinogen 2 regions of the genome, were numbered IS48lvl and IS48lv2 respectively. PMID:2559151

  13. Conserved Sequences at the Origin of Adenovirus DNA Replication

    PubMed Central

    Stillman, Bruce W.; Topp, William C.; Engler, Jeffrey A.

    1982-01-01

    The origin of adenovirus DNA replication lies within an inverted sequence repetition at either end of the linear, double-stranded viral DNA. Initiation of DNA replication is primed by a deoxynucleoside that is covalently linked to a protein, which remains bound to the newly synthesized DNA. We demonstrate that virion-derived DNA-protein complexes from five human adenovirus serological subgroups (A to E) can act as a template for both the initiation and the elongation of DNA replication in vitro, using nuclear extracts from adenovirus type 2 (Ad2)-infected HeLa cells. The heterologous template DNA-protein complexes were not as active as the homologous Ad2 DNA, most probably due to inefficient initiation by Ad2 replication factors. In an attempt to identify common features which may permit this replication, we have also sequenced the inverted terminal repeated DNA from human adenovirus serotypes Ad4 (group E), Ad9 and Ad10 (group D), and Ad31 (group A), and we have compared these to previously determined sequences from Ad2 and Ad5 (group C), Ad7 (group B), and Ad12 and Ad18 (group A) DNA. In all cases, the sequence around the origin of DNA replication can be divided into two structural domains: a proximal A · T-rich region which is partially conserved among these serotypes, and a distal G · C-rich region which is less well conserved. The G · C-rich region contains sequences similar to sequences present in papovavirus replication origins. The two domains may reflect a dual mechanism for initiation of DNA replication: adenovirus-specific protein priming of replication, and subsequent utilization of this primer by host replication factors for completion of DNA synthesis. Images PMID:7143575

  14. Multiplexed Sequence Encoding: A Framework for DNA Communication

    PubMed Central

    Zakeri, Bijan; Carr, Peter A.; Lu, Timothy K.

    2016-01-01

    Synthetic DNA has great propensity for efficiently and stably storing non-biological information. With DNA writing and reading technologies rapidly advancing, new applications for synthetic DNA are emerging in data storage and communication. Traditionally, DNA communication has focused on the encoding and transfer of complete sets of information. Here, we explore the use of DNA for the communication of short messages that are fragmented across multiple distinct DNA molecules. We identified three pivotal points in a communication—data encoding, data transfer & data extraction—and developed novel tools to enable communication via molecules of DNA. To address data encoding, we designed DNA-based individualized keyboards (iKeys) to convert plaintext into DNA, while reducing the occurrence of DNA homopolymers to improve synthesis and sequencing processes. To address data transfer, we implemented a secret-sharing system—Multiplexed Sequence Encoding (MuSE)—that conceals messages between multiple distinct DNA molecules, requiring a combination key to reveal messages. To address data extraction, we achieved the first instance of chromatogram patterning through multiplexed sequencing, thereby enabling a new method for data extraction. We envision these approaches will enable more widespread communication of information via DNA. PMID:27050646

  15. Multiplexed Sequence Encoding: A Framework for DNA Communication.

    PubMed

    Zakeri, Bijan; Carr, Peter A; Lu, Timothy K

    2016-01-01

    Synthetic DNA has great propensity for efficiently and stably storing non-biological information. With DNA writing and reading technologies rapidly advancing, new applications for synthetic DNA are emerging in data storage and communication. Traditionally, DNA communication has focused on the encoding and transfer of complete sets of information. Here, we explore the use of DNA for the communication of short messages that are fragmented across multiple distinct DNA molecules. We identified three pivotal points in a communication-data encoding, data transfer & data extraction-and developed novel tools to enable communication via molecules of DNA. To address data encoding, we designed DNA-based individualized keyboards (iKeys) to convert plaintext into DNA, while reducing the occurrence of DNA homopolymers to improve synthesis and sequencing processes. To address data transfer, we implemented a secret-sharing system-Multiplexed Sequence Encoding (MuSE)-that conceals messages between multiple distinct DNA molecules, requiring a combination key to reveal messages. To address data extraction, we achieved the first instance of chromatogram patterning through multiplexed sequencing, thereby enabling a new method for data extraction. We envision these approaches will enable more widespread communication of information via DNA. PMID:27050646

  16. Quantitative Comparison of Large-Scale DNA Enrichment Sequencing Data.

    PubMed

    Lienhard, Matthias; Chavez, Lukas

    2016-01-01

    DNA enrichment followed by sequencing (DNA-IP seq) is a versatile tool in molecular biology with a wide variety of applications. Computational analysis of differential DNA enrichment between conditions is important for identifying epigenetic alterations in disease compared to healthy controls and for revealing dynamic epigenetic modifications throughout normal and distorted cell differentiation and development. We present a protocol for genome-wide comparative analysis of DNA-IP sequencing data to identify statistically significant differential sequencing coverage between two conditions by considering variation across replicates. The protocol provides a detailed description for the comparative analysis of DNA-IP sequencing data including basic data processing, quality controls, and identification of differential enrichment using the Bioconductor package "MEDIPS". PMID:27008016

  17. Compiling Multicopy Single-Stranded DNA Sequences from Bacterial Genome Sequences

    PubMed Central

    Yoo, Wonseok; Lim, Dongbin

    2016-01-01

    A retron is a bacterial retroelement that encodes an RNA gene and a reverse transcriptase (RT). The former, once transcribed, works as a template primer for reverse transcription by the latter. The resulting DNA is covalently linked to the upstream part of the RNA; this chimera is called multicopy single-stranded DNA (msDNA), which is extrachromosomal DNA found in many bacterial species. Based on the conserved features in the eight known msDNA sequences, we developed a detection method and applied it to scan National Center for Biotechnology Information (NCBI) RefSeq bacterial genome sequences. Among 16,844 bacterial sequences possessing a retron-type RT domain, we identified 48 unique types of msDNA. Currently, the biological role of msDNA is not well understood. Our work will be a useful tool in studying the distribution, evolution, and physiological role of msDNA. PMID:27103888

  18. Characterization of group A Streptococcus strains recovered from Mexican children with pharyngitis by automated DNA sequencing of virulence-related genes: unexpectedly large variation in the gene (sic) encoding a complement-inhibiting protein.

    PubMed

    Mejia, L M; Stockbauer, K E; Pan, X; Cravioto, A; Musser, J M

    1997-12-01

    Sequence variation was studied in several target genes in 54 strains of group A Streptococcus (GAS) cultured from children with pharyngitis in Mexico City. Although 16 distinct emm alleles were identified, only 4 had not been previously described. Virtually all bacteria (31 of 33 [94%] with the streptococcal pyrogenic exotoxin gene (speA) had emm1-related, emm3, or emm6 alleles. The gene (sic) encoding an extracellular GAS protein that inhibits complement function was unusually variable among isolates with the emm1 family of alleles, with a total of seven variants identified. The data suggest that many GAS strains infecting Mexican children are genetically similar to organisms commonly encountered in the United States and western Europe. Sequence variation in the sic gene is useful for rapid differentiation among GAS isolates with the emm1 family of alleles. PMID:9399523

  19. Characterization of group A Streptococcus strains recovered from Mexican children with pharyngitis by automated DNA sequencing of virulence-related genes: unexpectedly large variation in the gene (sic) encoding a complement-inhibiting protein.

    PubMed Central

    Mejia, L M; Stockbauer, K E; Pan, X; Cravioto, A; Musser, J M

    1997-01-01

    Sequence variation was studied in several target genes in 54 strains of group A Streptococcus (GAS) cultured from children with pharyngitis in Mexico City. Although 16 distinct emm alleles were identified, only 4 had not been previously described. Virtually all bacteria (31 of 33 [94%] with the streptococcal pyrogenic exotoxin gene (speA) had emm1-related, emm3, or emm6 alleles. The gene (sic) encoding an extracellular GAS protein that inhibits complement function was unusually variable among isolates with the emm1 family of alleles, with a total of seven variants identified. The data suggest that many GAS strains infecting Mexican children are genetically similar to organisms commonly encountered in the United States and western Europe. Sequence variation in the sic gene is useful for rapid differentiation among GAS isolates with the emm1 family of alleles. PMID:9399523

  20. An Evolution Based Biosensor Receptor DNA Sequence Generation Algorithm

    PubMed Central

    Kim, Eungyeong; Lee, Malrey; Gatton, Thomas M.; Lee, Jaewan; Zang, Yupeng

    2010-01-01

    A biosensor is composed of a bioreceptor, an associated recognition molecule, and a signal transducer that can selectively detect target substances for analysis. DNA based biosensors utilize receptor molecules that allow hybridization with the target analyte. However, most DNA biosensor research uses oligonucleotides as the target analytes and does not address the potential problems of real samples. The identification of recognition molecules suitable for real target analyte samples is an important step towards further development of DNA biosensors. This study examines the characteristics of DNA used as bioreceptors and proposes a hybrid evolution-based DNA sequence generating algorithm, based on DNA computing, to identify suitable DNA bioreceptor recognition molecules for stable hybridization with real target substances. The Traveling Salesman Problem (TSP) approach is applied in the proposed algorithm to evaluate the safety and fitness of the generated DNA sequences. This approach improves efficiency and stability for enhanced and variable-length DNA sequence generation and allows extension to generation of variable-length DNA sequences with diverse receptor recognition requirements. PMID:22315543

  1. Biological nanopore MspA for DNA sequencing

    NASA Astrophysics Data System (ADS)

    Manrao, Elizabeth A.

    Unlocking the information hidden in the human genome provides insight into the inner workings of complex biological systems and can be used to greatly improve health-care. In order to allow for widespread sequencing, new technologies are required that provide fast and inexpensive readings of DNA. Nanopore sequencing is a third generation DNA sequencing technology that is currently being developed to fulfill this need. In nanopore sequencing, a voltage is applied across a small pore in an electrolyte solution and the resulting ionic current is recorded. When DNA passes through the channel, the ionic current is partially blocked. If the DNA bases uniquely modulate the ionic current flowing through the channel, the time trace of the current can be related to the sequence of DNA passing through the pore. There are two main challenges to realizing nanopore sequencing: identifying a pore with sensitivity to single nucleotides and controlling the translocation of DNA through the pore so that the small single nucleotide current signatures are distinguishable from background noise. In this dissertation, I explore the use of Mycobacterium smegmatis porin A (MspA) for nanopore sequencing. In order to determine MspA's sensitivity to single nucleotides, DNA strands of various compositions are held in the pore as the resulting ionic current is measured. DNA is immobilized in MspA by attaching it to a large molecule which acts as an anchor. This technique confirms the single nucleotide resolution of the pore and additionally shows that MspA is sensitive to epigenetic modifications and single nucleotide polymorphisms. The forces from the electric field within MspA, the effective charge of nucleotides, and elasticity of DNA are estimated using a Freely Jointed Chain model of single stranded DNA. These results offer insight into the interactions of DNA within the pore. With the nucleotide sensitivity of MspA confirmed, a method is introduced to controllably pass DNA through the pore

  2. Integrated on-line system for DNA sequencing by capillary electrophoresis: From template to called bases

    SciTech Connect

    Ton, H.; Yeung, E.S.

    1997-02-15

    An integrated on-line prototype for coupling a microreactor to capillary electrophoresis for DNA sequencing has been demonstrated. A dye-labeled terminator cycle-sequencing reaction is performed in a fused-silica capillary. Subsequently, the sequencing ladder is directly injected into a size-exclusion chromatographic column operated at nearly 95{degree}C for purification. On-line injection to a capillary for electrophoresis is accomplished at a junction set at nearly 70{degree}C. High temperature at the purification column and injection junction prevents the renaturation of DNA fragments during on-line transfer without affecting the separation. The high solubility of DNA in and the relatively low ionic strength of 1 x TE buffer permit both effective purification and electrokinetic injection of the DNA sample. The system is compatible with highly efficient separations by a replaceable poly(ethylene oxide) polymer solution in uncoated capillary tubes. Future automation and adaptation to a multiple-capillary array system should allow high-speed, high-throughput DNA sequencing from templates to called bases in one step. 32 refs., 5 figs.

  3. An Optimal Seed Based Compression Algorithm for DNA Sequences

    PubMed Central

    Gopalakrishnan, Gopakumar; Karunakaran, Muralikrishnan

    2016-01-01

    This paper proposes a seed based lossless compression algorithm to compress a DNA sequence which uses a substitution method that is similar to the LempelZiv compression scheme. The proposed method exploits the repetition structures that are inherent in DNA sequences by creating an offline dictionary which contains all such repeats along with the details of mismatches. By ensuring that only promising mismatches are allowed, the method achieves a compression ratio that is at par or better than the existing lossless DNA sequence compression algorithms. PMID:27555868

  4. DNA sequence analysis with droplet-based microfluidics

    PubMed Central

    Abate, Adam R.; Hung, Tony; Sperling, Ralph A.; Mary, Pascaline; Rotem, Assaf; Agresti, Jeremy J.; Weiner, Michael A.; Weitz, David A.

    2014-01-01

    Droplet-based microfluidic techniques can form and process micrometer scale droplets at thousands per second. Each droplet can house an individual biochemical reaction, allowing millions of reactions to be performed in minutes with small amounts of total reagent. This versatile approach has been used for engineering enzymes, quantifying concentrations of DNA in solution, and screening protein crystallization conditions. Here, we use it to read the sequences of DNA molecules with a FRET-based assay. Using probes of different sequences, we interrogate a target DNA molecule for polymorphisms. With a larger probe set, additional polymorphisms can be interrogated as well as targets of arbitrary sequence. PMID:24185402

  5. DNA Methyltransferase Accessibility Protocol for Individual Templates by Deep Sequencing

    PubMed Central

    Darst, Russell P.; Nabilsi, Nancy H.; Pardo, Carolina E.; Riva, Alberto; Kladde, Michael P.

    2013-01-01

    A single-molecule probe of chromatin structure can uncover dynamic chromatin states and rare epigenetic variants of biological importance that bulk measures of chromatin structure miss. In bisulfite genomic sequencing, each sequenced clone records the methylation status of multiple sites on an individual molecule of DNA. An exogenous DNA methyltransferase can thus be used to image nucleosomes and other protein–DNA complexes. In this chapter, we describe the adaptation of this technique, termed Methylation Accessibility Protocol for individual templates, to modern high-throughput sequencing, which both simplifies the workflow and extends its utility. PMID:22929770

  6. Profiling DNA Methylomes from Microarray to Genome-Scale Sequencing

    PubMed Central

    Huang, Yi-Wen; Huang, Tim H.-M.; Wang, Li-Shu

    2010-01-01

    DNA cytosine methylation is a central epigenetic modification which plays critical roles in cellular processes including genome regulation, development and disease. Here, we review current and emerging microarray and next-generation sequencing based technologies that enhance our knowledge of DNA methylation profiling. Each methodology has limitations and their unique applications, and combinations of several modalities may help build the entire methylome. With advances on next-generation sequencing technologies, it is now possible to globally map the DNA cytosine methylation at single-base resolution, providing new insights into the regulation and dynamics of DNA methylation in genomes. PMID:20218736

  7. Profiling DNA methylomes from microarray to genome-scale sequencing.

    PubMed

    Huang, Yi-Wei; Huang, Tim H-M; Wang, Li-Shu

    2010-04-01

    DNA cytosine methylation is a central epigenetic modification which plays critical roles in cellular processes including genome regulation, development and disease. Here, we review current and emerging microarray and next-generation sequencing based technologies that enhance our knowledge of DNA methylation profiling. Each methodology has limitations and their unique applications, and combinations of several modalities may help build the entire methylome. With advances on next-generation sequencing technologies, it is now possible to globally map the DNA cytosine methylation at single-base resolution, providing new insights into the regulation and dynamics of DNA methylation in genomes. PMID:20218736

  8. Current-voltage characteristics of double-strand DNA sequences

    NASA Astrophysics Data System (ADS)

    Bezerril, L. M.; Moreira, D. A.; Albuquerque, E. L.; Fulco, U. L.; de Oliveira, E. L.; de Sousa, J. S.

    2009-09-01

    We use a tight-binding formulation to investigate the transmissivity and the current-voltage (I-V) characteristics of sequences of double-strand DNA molecules. In order to reveal the relevance of the underlying correlations in the nucleotides distribution, we compare the results for the genomic DNA sequence with those of artificial sequences (the long-range correlated Fibonacci and Rudin-Shapiro one) and a random sequence, which is a kind of prototype of a short-range correlated system. The random sequence is presented here with the same first neighbors pair correlations of the human DNA sequence. We found that the long-range character of the correlations is important to the transmissivity spectra, although the I-V curves seem to be mostly influenced by the short-range correlations.

  9. Configuring the Orion Guidance, Navigation, and Control Flight Software for Automated Sequencing

    NASA Technical Reports Server (NTRS)

    Odegard, Ryan G.; Siliwinski, Tomasz K.; King, Ellis T.; Hart, Jeremy J.

    2010-01-01

    The Orion Crew Exploration Vehicle is being designed with greater automation capabilities than any other crewed spacecraft in NASA s history. The Guidance, Navigation, and Control (GN&C) flight software architecture is designed to provide a flexible and evolvable framework that accommodates increasing levels of automation over time. Within the GN&C flight software, a data-driven approach is used to configure software. This approach allows data reconfiguration and updates to automated sequences without requiring recompilation of the software. Because of the great dependency of the automation and the flight software on the configuration data, the data management is a vital component of the processes for software certification, mission design, and flight operations. To enable the automated sequencing and data configuration of the GN&C subsystem on Orion, a desktop database configuration tool has been developed. The database tool allows the specification of the GN&C activity sequences, the automated transitions in the software, and the corresponding parameter reconfigurations. These aspects of the GN&C automation on Orion are all coordinated via data management, and the database tool provides the ability to test the automation capabilities during the development of the GN&C software. In addition to providing the infrastructure to manage the GN&C automation, the database tool has been designed with capabilities to import and export artifacts for simulation analysis and documentation purposes. Furthermore, the database configuration tool, currently used to manage simulation data, is envisioned to evolve into a mission planning tool for generating and testing GN&C software sequences and configurations. A key enabler of the GN&C automation design, the database tool allows both the creation and maintenance of the data artifacts, as well as serving the critical role of helping to manage, visualize, and understand the data-driven parameters both during software development

  10. Identification of genes in anonymous DNA sequences. Final report: Report period, 15 April 1993--15 April 1994

    SciTech Connect

    Fields, C.A.

    1994-09-01

    This Report concludes the DOE Human Genome Program project, ``Identification of Genes in Anonymous DNA Sequence.`` The central goals of this project have been (1) understanding the problem of identifying genes in anonymous sequences, and (2) development of tools, primarily the automated identification system gm, for identifying genes. The activities supported under the previous award are summarized here to provide a single complete report on the activities supported as part of the project from its inception to its completion.

  11. Sequence specific generation of a DNA panhandle permits PCR amplification of unknown flanking DNA.

    PubMed Central

    Jones, D H; Winistorfer, S C

    1992-01-01

    We present a novel method for the PCR amplification of unknown DNA that flanks a known segment directly from human genomic DNA. PCR requires that primer annealing sites be present on each end of the DNA segment that is to be amplified. In this method, known DNA is placed on the uncharacterized side of the sequence of interest via DNA polymerase mediated generation of a PCR template that is shaped like a pan with a handle. Generation of this template permits specific amplification of the unknown sequence. Taq (DNA) polymerase was used to form the original template and to generate the PCR product. 2.2 kb of the beta-globin gene, and 657 bp of the 5' flanking region of the cystic fibrosis transmembrane conductance regulator gene, were amplified directly from human genomic DNA using primers that initially flank only one side of the region amplified. This method will provide a powerful tool for acquiring DNA sequence information. Images PMID:1371352

  12. Plasmonic Nanopores for Trapping, Controlling Displacement, and Sequencing of DNA

    PubMed Central

    2015-01-01

    With the aim of developing a DNA sequencing methodology, we theoretically examine the feasibility of using nanoplasmonics to control the translocation of a DNA molecule through a solid-state nanopore and to read off sequence information using surface-enhanced Raman spectroscopy. Using molecular dynamics simulations, we show that high-intensity optical hot spots produced by a metallic nanostructure can arrest DNA translocation through a solid-state nanopore, thus providing a physical knob for controlling the DNA speed. Switching the plasmonic field on and off can displace the DNA molecule in discrete steps, sequentially exposing neighboring fragments of a DNA molecule to the pore as well as to the plasmonic hot spot. Surface-enhanced Raman scattering from the exposed DNA fragments contains information about their nucleotide composition, possibly allowing the identification of the nucleotide sequence of a DNA molecule transported through the hot spot. The principles of plasmonic nanopore sequencing can be extended to detection of DNA modifications and RNA characterization. PMID:26401685

  13. Plasmonic Nanopores for Trapping, Controlling Displacement, and Sequencing of DNA.

    PubMed

    Belkin, Maxim; Chao, Shu-Han; Jonsson, Magnus P; Dekker, Cees; Aksimentiev, Aleksei

    2015-11-24

    With the aim of developing a DNA sequencing methodology, we theoretically examine the feasibility of using nanoplasmonics to control the translocation of a DNA molecule through a solid-state nanopore and to read off sequence information using surface-enhanced Raman spectroscopy. Using molecular dynamics simulations, we show that high-intensity optical hot spots produced by a metallic nanostructure can arrest DNA translocation through a solid-state nanopore, thus providing a physical knob for controlling the DNA speed. Switching the plasmonic field on and off can displace the DNA molecule in discrete steps, sequentially exposing neighboring fragments of a DNA molecule to the pore as well as to the plasmonic hot spot. Surface-enhanced Raman scattering from the exposed DNA fragments contains information about their nucleotide composition, possibly allowing the identification of the nucleotide sequence of a DNA molecule transported through the hot spot. The principles of plasmonic nanopore sequencing can be extended to detection of DNA modifications and RNA characterization. PMID:26401685

  14. Semiconductor-based DNA sequencing of histone modification states

    PubMed Central

    Cheng, Christine S.; Rai, Kunal; Garber, Manuel; Hollinger, Andrew; Robbins, Dana; Anderson, Scott; Macbeth, Alyssa; Tzou, Austin; Carneiro, Mauricio O.; Raychowdhury, Raktima; Russ, Carsten; Hacohen, Nir; Gershenwald, Jeffrey E.; Lennon, Niall; Nusbaum, Chad; Chin, Lynda; Regev, Aviv; Amit, Ido

    2013-01-01

    The recent development of a semiconductor-based, non-optical DNA sequencing technology promises scalable, low-cost and rapid sequence data production. The technology has previously been applied mainly to genomic sequencing and targeted re-sequencing. Here we demonstrate the utility of Ion Torrent semiconductor-based sequencing for sensitive, efficient and rapid chromatin immunoprecipitation followed by sequencing (ChIP-seq) through the application of sample preparation methods that are optimized for ChIP-seq on the Ion Torrent platform. We leverage this method for epigenetic profiling of tumour tissues. PMID:24157732

  15. Semiconductor-based DNA sequencing of histone modification states.

    PubMed

    Cheng, Christine S; Rai, Kunal; Garber, Manuel; Hollinger, Andrew; Robbins, Dana; Anderson, Scott; Macbeth, Alyssa; Tzou, Austin; Carneiro, Mauricio O; Raychowdhury, Raktima; Russ, Carsten; Hacohen, Nir; Gershenwald, Jeffrey E; Lennon, Niall; Nusbaum, Chad; Chin, Lynda; Regev, Aviv; Amit, Ido

    2013-01-01

    The recent development of a semiconductor-based, non-optical DNA sequencing technology promises scalable, low-cost and rapid sequence data production. The technology has previously been applied mainly to genomic sequencing and targeted re-sequencing. Here we demonstrate the utility of Ion Torrent semiconductor-based sequencing for sensitive, efficient and rapid chromatin immunoprecipitation followed by sequencing (ChIP-seq) through the application of sample preparation methods that are optimized for ChIP-seq on the Ion Torrent platform. We leverage this method for epigenetic profiling of tumour tissues. PMID:24157732

  16. ATRF Houses the Latest DNA Sequencing Technologies | Poster

    Cancer.gov

    By Ashley DeVine, Staff Writer By the end of October, the Advanced Technology Research Facility (ATRF) will be one of the few facilities in the world to house all of the latest DNA sequencing technologies.

  17. Microchannel DNA Sequencing by End-Labelled Free Solution Electrophoresis

    SciTech Connect

    Barron, A.

    2005-09-29

    The further development of End-Labeled Free-Solution Electrophoresis will greatly simplify DNA separation and sequencing on microfluidic devices. The development and optimization of drag-tags is critical to the success of this research.

  18. DNA sequencing using polymerase substrate-binding kinetics

    PubMed Central

    Previte, Michael John Robert; Zhou, Chunhong; Kellinger, Matthew; Pantoja, Rigo; Chen, Cheng-Yao; Shi, Jin; Wang, BeiBei; Kia, Amirali; Etchin, Sergey; Vieceli, John; Nikoomanzar, Ali; Bomati, Erin; Gloeckner, Christian; Ronaghi, Mostafa; He, Molly Min

    2015-01-01

    Next-generation sequencing (NGS) has transformed genomic research by decreasing the cost of sequencing. However, whole-genome sequencing is still costly and complex for diagnostics purposes. In the clinical space, targeted sequencing has the advantage of allowing researchers to focus on specific genes of interest. Routine clinical use of targeted NGS mandates inexpensive instruments, fast turnaround time and an integrated and robust workflow. Here we demonstrate a version of the Sequencing by Synthesis (SBS) chemistry that potentially can become a preferred targeted sequencing method in the clinical space. This sequencing chemistry uses natural nucleotides and is based on real-time recording of the differential polymerase/DNA-binding kinetics in the presence of correct or mismatch nucleotides. This ensemble SBS chemistry has been implemented on an existing Illumina sequencing platform with integrated cluster amplification. We discuss the advantages of this sequencing chemistry for targeted sequencing as well as its limitations for other applications. PMID:25612848

  19. DNA sequencing using polymerase substrate-binding kinetics.

    PubMed

    Previte, Michael John Robert; Zhou, Chunhong; Kellinger, Matthew; Pantoja, Rigo; Chen, Cheng-Yao; Shi, Jin; Wang, BeiBei; Kia, Amirali; Etchin, Sergey; Vieceli, John; Nikoomanzar, Ali; Bomati, Erin; Gloeckner, Christian; Ronaghi, Mostafa; He, Molly Min

    2015-01-01

    Next-generation sequencing (NGS) has transformed genomic research by decreasing the cost of sequencing. However, whole-genome sequencing is still costly and complex for diagnostics purposes. In the clinical space, targeted sequencing has the advantage of allowing researchers to focus on specific genes of interest. Routine clinical use of targeted NGS mandates inexpensive instruments, fast turnaround time and an integrated and robust workflow. Here we demonstrate a version of the Sequencing by Synthesis (SBS) chemistry that potentially can become a preferred targeted sequencing method in the clinical space. This sequencing chemistry uses natural nucleotides and is based on real-time recording of the differential polymerase/DNA-binding kinetics in the presence of correct or mismatch nucleotides. This ensemble SBS chemistry has been implemented on an existing Illumina sequencing platform with integrated cluster amplification. We discuss the advantages of this sequencing chemistry for targeted sequencing as well as its limitations for other applications. PMID:25612848

  20. Levenshtein error-correcting barcodes for multiplexed DNA sequencing

    PubMed Central

    2013-01-01

    Background High-throughput sequencing technologies are improving in quality, capacity and costs, providing versatile applications in DNA and RNA research. For small genomes or fraction of larger genomes, DNA samples can be mixed and loaded together on the same sequencing track. This so-called multiplexing approach relies on a specific DNA tag or barcode that is attached to the sequencing or amplification primer and hence appears at the beginning of the sequence in every read. After sequencing, each sample read is identified on the basis of the respective barcode sequence. Alterations of DNA barcodes during synthesis, primer ligation, DNA amplification, or sequencing may lead to incorrect sample identification unless the error is revealed and corrected. This can be accomplished by implementing error correcting algorithms and codes. This barcoding strategy increases the total number of correctly identified samples, thus improving overall sequencing efficiency. Two popular sets of error-correcting codes are Hamming codes and Levenshtein codes. Result Levenshtein codes operate only on words of known length. Since a DNA sequence with an embedded barcode is essentially one continuous long word, application of the classical Levenshtein algorithm is problematic. In this paper we demonstrate the decreased error correction capability of Levenshtein codes in a DNA context and suggest an adaptation of Levenshtein codes that is proven of efficiently correcting nucleotide errors in DNA sequences. In our adaption we take the DNA context into account and redefine the word length whenever an insertion or deletion is revealed. In simulations we show the superior error correction capability of the new method compared to traditional Levenshtein and Hamming based codes in the presence of multiple errors. Conclusion We present an adaptation of Levenshtein codes to DNA contexts capable of correction of a pre-defined number of insertion, deletion, and substitution mutations. Our improved

  1. Local alignment of two-base encoded DNA sequence

    PubMed Central

    Homer, Nils; Merriman, Barry; Nelson, Stanley F

    2009-01-01

    Background DNA sequence comparison is based on optimal local alignment of two sequences using a similarity score. However, some new DNA sequencing technologies do not directly measure the base sequence, but rather an encoded form, such as the two-base encoding considered here. In order to compare such data to a reference sequence, the data must be decoded into sequence. The decoding is deterministic, but the possibility of measurement errors requires searching among all possible error modes and resulting alignments to achieve an optimal balance of fewer errors versus greater sequence similarity. Results We present an extension of the standard dynamic programming method for local alignment, which simultaneously decodes the data and performs the alignment, maximizing a similarity score based on a weighted combination of errors and edits, and allowing an affine gap penalty. We also present simulations that demonstrate the performance characteristics of our two base encoded alignment method and contrast those with standard DNA sequence alignment under the same conditions. Conclusion The new local alignment algorithm for two-base encoded data has substantial power to properly detect and correct measurement errors while identifying underlying sequence variants, and facilitating genome re-sequencing efforts based on this form of sequence data. PMID:19508732

  2. Discovering simple DNA sequences by the algorithmic significance method.

    PubMed

    Milosavljević, A; Jurka, J

    1993-08-01

    A new method, 'algorithmic significance', is proposed as a tool for discovery of patterns in DNA sequences. The main idea is that patterns can be discovered by finding ways to encode the observed data concisely. In this sense, the method can be viewed as a formal version of the Occam's Razor principle. In this paper the method is applied to discover significantly simple DNA sequences. We define DNA sequences to be simple if they contain repeated occurrences of certain 'words' and thus can be encoded in a small number of bits. Such definition includes minisatellites and microsatellites. A standard dynamic programming algorithm for data compression is applied to compute the minimal encoding lengths of sequences in linear time. An electronic mail server for identification of simple sequences based on the proposed method has been installed at the Internet address pythia/anl.gov. PMID:8402207

  3. Nuclear and mitochondrial DNA sequences from two Denisovan individuals

    PubMed Central

    Sawyer, Susanna; Renaud, Gabriel; Viola, Bence; Hublin, Jean-Jacques; Gansauge, Marie-Theres; Shunkov, Michael V.; Derevianko, Anatoly P.; Prüfer, Kay; Pääbo, Svante

    2015-01-01

    Denisovans, a sister group of Neandertals, have been described on the basis of a nuclear genome sequence from a finger phalanx (Denisova 3) found in Denisova Cave in the Altai Mountains. The only other Denisovan specimen described to date is a molar (Denisova 4) found at the same site. This tooth carries a mtDNA sequence similar to that of Denisova 3. Here we present nuclear DNA sequences from Denisova 4 and a morphological description, as well as mitochondrial and nuclear DNA sequence data, from another molar (Denisova 8) found in Denisova Cave in 2010. This new molar is similar to Denisova 4 in being very large and lacking traits typical of Neandertals and modern humans. Nuclear DNA sequences from the two molars form a clade with Denisova 3. The mtDNA of Denisova 8 is more diverged and has accumulated fewer substitutions than the mtDNAs of the other two specimens, suggesting Denisovans were present in the region over an extended period. The nuclear DNA sequence diversity among the three Denisovans is comparable to that among six Neandertals, but lower than that among present-day humans. PMID:26630009

  4. Nuclear and mitochondrial DNA sequences from two Denisovan individuals.

    PubMed

    Sawyer, Susanna; Renaud, Gabriel; Viola, Bence; Hublin, Jean-Jacques; Gansauge, Marie-Theres; Shunkov, Michael V; Derevianko, Anatoly P; Prüfer, Kay; Kelso, Janet; Pääbo, Svante

    2015-12-22

    Denisovans, a sister group of Neandertals, have been described on the basis of a nuclear genome sequence from a finger phalanx (Denisova 3) found in Denisova Cave in the Altai Mountains. The only other Denisovan specimen described to date is a molar (Denisova 4) found at the same site. This tooth carries a mtDNA sequence similar to that of Denisova 3. Here we present nuclear DNA sequences from Denisova 4 and a morphological description, as well as mitochondrial and nuclear DNA sequence data, from another molar (Denisova 8) found in Denisova Cave in 2010. This new molar is similar to Denisova 4 in being very large and lacking traits typical of Neandertals and modern humans. Nuclear DNA sequences from the two molars form a clade with Denisova 3. The mtDNA of Denisova 8 is more diverged and has accumulated fewer substitutions than the mtDNAs of the other two specimens, suggesting Denisovans were present in the region over an extended period. The nuclear DNA sequence diversity among the three Denisovans is comparable to that among six Neandertals, but lower than that among present-day humans. PMID:26630009

  5. Effects of sequence on DNA wrapping around histones

    NASA Astrophysics Data System (ADS)

    Ortiz, Vanessa

    2011-03-01

    A central question in biophysics is whether the sequence of a DNA strand affects its mechanical properties. In epigenetics, these are thought to influence nucleosome positioning and gene expression. Theoretical and experimental attempts to answer this question have been hindered by an inability to directly resolve DNA structure and dynamics at the base-pair level. In our previous studies we used a detailed model of DNA to measure the effects of sequence on the stability of naked DNA under bending. Sequence was shown to influence DNA's ability to form kinks, which arise when certain motifs slide past others to form non-native contacts. Here, we have now included histone-DNA interactions to see if the results obtained for naked DNA are transferable to the problem of nucleosome positioning. Different DNA sequences interacting with the histone protein complex are studied, and their equilibrium and mechanical properties are compared among themselves and with the naked case. NLM training grant to the Computation and Informatics in Biology and Medicine Training Program (NLM T15LM007359).

  6. Efficient DNA sequencing on microtiter plates using dried reagents and Bst DNA polymerase.

    PubMed

    Earley, J J; Kuivaniemi, H; Prockop, D J; Tromp, G

    1993-01-01

    Sequenase, Taq DNA polymerase and Bst DNA polymerase were tested for sequencing of DNA on microtiter plates using dried down reagents. Several parameters were investigated to expedite the drying process while minimizing damage to the enzyme. Sequenase did not tolerate drying very well, and frequently generated sequences with weak signals and many sites of premature termination. With Taq DNA polymerase it was possible to obtain sequences of good quality. However, there was considerable variation of results between experiments and between batches of microtiter plates. Bst DNA polymerase generated sequences of excellent quality. It was stable for more than a week in dried-down state at -20 degrees C and at least overnight at room temperature. The method described here using Bst DNA polymerase is well suited for laboratory robots and workstations that typically employ 96-well microtiter plates. PMID:8173079

  7. Analysis of the DNA Sequencing Quality and Efficiency of the Apollo100 Robotic Microcycler in a Core Facility Setting

    PubMed Central

    Logsdon, M. E.; Trounstine, M. C.; Zianni, M. R.

    2011-01-01

    Sanger, or dideoxynucleotide sequencing, is an important tool for biomolecular research. An important trend in DNA sequencing is to find new and innovative ways to provide high-quality, reliable sequences in a more efficient manner, using automated capillary electrophoresis. The Apollo100 combines Sanger cycle sequencing and solid-phase reversible immobilization for product purification in a single instrument with robotic liquid handling and microfluidic (Microscale On-chip Valve) chips that have onboard thermal cycling and pneumatic mixing. Experiments were performed to determine how the DNA sequencing results from the Apollo100 compared with conventional, manual methods used in a core facility setting. Through rigorous experimentation of multiple baseline runs and a dilution series of template concentration, the Apollo100 generated sequencing that exceeded 900 bases with a quality score of 20 or above. When comparing actual client samples of amplicons, plasmids, and cosmids, Apollo100 sequencing results did not differ significantly from those reactions prepared manually. In addition, bacterial genomic DNA was sequenced successfully, directly with the Apollo100, although results were of lower quality than the standard manual method. As a result of the microscale capabilities, the Apollo100 offers valuable savings with respect to the quantity of reagents consumed compared with current manual sequencing methods, thereby continuing the demand for smaller template and reagent requirements. In conclusion, the Apollo100 can generate high-quality DNA sequences for common templates equivalent to those produced using manual sequencing methods and increases efficiency through reduced labor and reagents. PMID:21738437

  8. The DNA damage checkpoint allows recombination between divergent DNA sequences in budding yeast

    PubMed Central

    George, Carolyn M.; Lyndaker, Amy M.; Alani, Eric

    2011-01-01

    In the early steps of homologous recombination, single-stranded DNA (ssDNA) from a broken chromosome invades homologous sequence located in a sister or homolog donor. In genomes that contain numerous repetitive DNA elements or gene paralogs, recombination can potentially occur between non-allelic/divergent (homeologous) sequences that share sequence identity. Such recombination events can lead to lethal chromosomal deletions or rearrangements. However, homeologous recombination events can be suppressed through rejection mechanisms that involve recognition of DNA mismatches in heteroduplex DNA by mismatch repair factors, followed by active unwinding of the heteroduplex DNA by helicases. Because factors required for heteroduplex rejection are hypothesized to be targets and/or effectors of the DNA damage response (DDR), a cell cycle control mechanism that ensures timely and efficient repair, we tested whether the DDR, and more specifically, the RAD9 gene, had a role in regulating rejection. We performed these studies using a DNA repair assay that measures repair by single-strand annealing (SSA) of a double-strand break (DSB) using homeologous DNA templates. We found that repair of homeologous DNA sequences, but not identical sequences, induced a RAD9- dependent cell cycle delay in the G2 stage of the cell cycle. Repair through a divergent DNA template occurred more frequently in RAD9 compared to rad9Δ strains. However, repair in rad9Δ mutants could be restored to wild-type levels if a G2 delay was induced by nocodazole. These results suggest that cell cycle arrest induced by the Rad9-dependent DDR allows repair between divergent DNA sequences despite the potential for creating deleterious genome rearrangements, and illustrates the importance of additional cellular mechanisms that act to suppress recombination between divergent DNA sequences. PMID:21978436

  9. HLA typing by direct DNA sequencing.

    PubMed

    Smith, Linda K

    2012-01-01

    Sequencing-based typing is a high resolution method for the identification of HLA polymorphisms. The majority of HLA Class I alleles can be discriminated by their exon 2 and 3 sequence, and for Class II alleles, exon 2 is generally sufficient. There are polymorphic positions in other exons which may require additional sequencing to exclude certain alleles with differences outside exon 2 and 3, depending on the clinical requirement and relevant accredition guidelines. The process involves selective amplification of target alleles by PCR, agarose gel electrophoresis of the PCR products to assess the quantity and quality, followed by purification of PCR amplicons to remove excess primer and dNTPs. Cycle sequencing reactions using Applied Biosystems™ BigDye(®) Terminator Ready Reaction v1.1 or v3.1 Kit are performed, then purification of sequence reactions before electrophoresing using Applied Biosystems™ 3730 or 3730XL Genetic Analyser (or similar). Data is processed by specialised software packages, which compare the sample sequence to the sequences of all possible theoretical allele combinations to assign an accurate genotype. Examination of all nucleotides, both at conserved and polymorphic positions enables the direct identification of new alleles, which may not be possible with techniques such as SSP and SSO typing. PMID:22665229

  10. An integer programming approach to DNA sequence assembly.

    PubMed

    Chang, Youngjung; Sahinidis, Nikolaos V

    2011-08-10

    De novo sequence assembly is a ubiquitous combinatorial problem in all DNA sequencing technologies. In the presence of errors in the experimental data, the assembly problem is computationally challenging, and its solution may not lead to a unique reconstruct. The enumeration of all alternative solutions is important in drawing a reliable conclusion on the target sequence, and is often overlooked in the heuristic approaches that are currently available. In this paper, we develop an integer programming formulation and global optimization solution strategy to solve the sequence assembly problem with errors in the data. We also propose an efficient technique to identify all alternative reconstructs. When applied to examples of sequencing-by-hybridization, our approach dramatically increases the length of DNA sequences that can be handled with global optimality certificate to over 10,000, which is more than 10 times longer than previously reported. For some problem instances, alternative solutions exhibited a wide range of different ability in reproducing the target DNA sequence. Therefore, it is important to utilize the methodology proposed in this paper in order to obtain all alternative solutions to reliably infer the true reconstruct. These alternative solutions can be used to refine the obtained results and guide the design of further experiments to correctly reconstruct the target DNA sequence. PMID:21864794