Application of Single Molecule Detection to Dna Sequencing

Goodwin, Peter M.; Cai, Hong; Jett, James H.; Ishaug-Riley, Susan L.; Machara, Nicholas P.; Semin, David; Orden, Alan Van; Keller, Richard A.

doi:10.1080/07328319708002914

Cited by 41 publications

(29 citation statements)

References 8 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Others have sought to develop sequencing methods that rely on detecting single molecules (e.g., via exonuclease digestion [72,[85][86][87][88] or by detecting the nature or existence of PCR products ampliWed therefrom [67][68][69][70][71]); recently, the incorporation of nucleotides has been observed on single DNA strands by Xuorescence microscopy [89]. Future progress depends on the ability to observe primer extension with ever lower nucleotide concentrations.…”

Section: Discussionmentioning

confidence: 99%

Single-nucleotide polymorphism detection using nanomolar nucleotides and single-molecule fluorescence

Twist

Winson

Rowland

et al. 2004

Analytical Biochemistry

View full text Add to dashboard Cite

Section: Discussionmentioning

confidence: 99%

Single-nucleotide polymorphism detection using nanomolar nucleotides and single-molecule fluorescence

Twist

Winson

Rowland

et al. 2004

Analytical Biochemistry

View full text Add to dashboard Cite

“…There have been many proposals to develop new sequencing technologies based on single molecule measurements, generally either by observing the interaction of particular proteins with DNA (6,(11)(12)(13) or by using ultra high-resolution scanned probe microscopy (14). Although none of these methods has been demonstrated experimentally, they are interesting because they promise high sensitivity, low cost, and in some cases a high degree of parallelization (15).…”

mentioning

confidence: 99%

“…Scanned probe microscopes have not yet been able to demonstrate simultaneously the resolution and chemical specificity needed to resolve individual bases (14). Other proposals turn to nature for inspiration and seek to combine optical techniques with enzymes that have been fine-tuned by evolution to operate as machines that assemble and disassemble DNA with inherent single-base resolution (6,11,12). Although there have been single molecule studies of DNA polymerase (16,17), RNA polymerase (18,19), and exonuclease (20,21), measuring the activity of these enzymes with single-base resolution has been an elusive goal.…”

mentioning

confidence: 99%

Sequence information can be obtained from single DNA molecules

Braslavsky

Hébert

Kartalov

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

427

245

View full text Add to dashboard Cite

The completion of the human genome draft has taken several years and is only the beginning of a period in which large amounts of DNA and RNA sequence information will be required from many individuals and species. Conventional sequencing technology has limitations in cost, speed, and sensitivity, with the result that the demand for sequence information far outstrips current capacity. There have been several proposals to address these issues by developing the ability to sequence single DNA molecules, but none have been experimentally demonstrated. Here we report the use of DNA polymerase to obtain sequence information from single DNA molecules by using fluorescence microscopy. We monitored repeated incorporation of fluorescently labeled nucleotides into individual DNA strands with single base resolution, allowing the determination of sequence fingerprints up to 5 bp in length. These experiments show that one can study the activity of DNA polymerase at the single molecule level with single base resolution and a high degree of parallelization, thus providing the foundation for a practical single molecule sequencing technology.T he Sanger method of DNA sequencing (1) and subsequent developments in automation (2) and computation (3) revolutionized the world of biological sciences and eventually led to the sequencing of the consensus human genome (4, 5). The successes of this and other genome projects have only whetted the appetite of the scientific community, and many applications of DNA sequencing have been proposed that will require cheaper, faster, or more sensitive sequencing technology than conventional methods currently provide. After the determination of the consensus human genome, there is a desire to sequence many individual human genomes to provide highresolution genotypes that can be used to determine the complex relationships among disease, pharmaceutical efficacy, and genetic variability (6-8). Similarly, aggressive technological innovation is required for the field of comparative genomics to reach its full potential (4). Finally, mRNA sequencing is valuable to determine exon splicing patterns (9) and as a tool to discover gene function from context-specific expression data (10).There have been many proposals to develop new sequencing technologies based on single molecule measurements, generally either by observing the interaction of particular proteins with DNA (6, 11-13) or by using ultra high-resolution scanned probe microscopy (14). Although none of these methods has been demonstrated experimentally, they are interesting because they promise high sensitivity, low cost, and in some cases a high degree of parallelization (15). Unlike conventional technology, their speed and read length would not be inherently limited by the resolving power of electrophoretic separation. Single molecule sensitivity might permit direct sequencing of mRNA from rare cell populations or perhaps even individual cells.A major obstacle in the development of single molecule sequencing schemes is that DNA has an extraordinarily ...

show abstract

“…With respect to analytical purposes as well as for single molecule DNA-sequencing it is of great interest to achieve a most efficient detection of all molecules within a sample volume [34][35][36][37][38]. In this case a sample survey is not sufficient, but a complete counting of all molecules within a sample volume is the ultimate goal.…”

Section: Introductionmentioning

confidence: 99%

Highly Efficient Single Molecule Detection in Different Micro and submicrometer Channels with cw-excitation

Dörre¹,

Stephan²,

Eigen

2001

Single Mol.

View full text Add to dashboard Cite

In this article we describe a detailed comparison between single molecule detection in three different types of microchannels. The overall detection efficiencies and the signal-to-noise ratios are compared. A new data evaluation technique that is used is explained in detail. The average number of photons per molecule is calculated from the measurements and the detection properties of each channel with respect to the diffusional characteristics of the dye molecules are investigated. From the detection efficiencies advantages and drawbacks of the individual methods are elucidated as well as possible applications of the techniques are illustrated.

show abstract

Application of Single Molecule Detection to Dna Sequencing

Cited by 41 publications

References 8 publications

Single-nucleotide polymorphism detection using nanomolar nucleotides and single-molecule fluorescence

Single-nucleotide polymorphism detection using nanomolar nucleotides and single-molecule fluorescence

Sequence information can be obtained from single DNA molecules

Highly Efficient Single Molecule Detection in Different Micro and submicrometer Channels with cw-excitation

Contact Info

Product

Resources

About