High speed DNA sequencing by capillary electrophoresis

Luckey, John A.; Drossman, Howard; Kostichka, Anthony J.; Mead, David A.; D’Cunha, Jonathan; Norris, Tracy B.; Smith, Lloyd M.

doi:10.1093/nar/18.15.4417

Cited by 235 publications

(107 citation statements)

References 6 publications

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“…In a recent study the investigators concluded that dominant populations can be detected by using T-RFLP analysis as a tool for precise quantification of the PCR product pool along with the capability for potential PCR bias detection (27). The limitations of electrophoresis technology for accurately and precisely determining sizes of fragments within specific size ranges are well documented (15,22,24,25,28,31,47). The velocity at which a DNA fragment moves through a sieving matrix, such as agarose or polyacrylamide, is not linearly correlated with size.…”

Section: Discussionmentioning

confidence: 99%

Fidelity of Select Restriction Endonucleases in Determining Microbial Diversity by Terminal-Restriction Fragment Length Polymorphism

Engebretson

Moyer

2003

Appl Environ Microbiol

111

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An evaluation of 18 DNA restriction endonucleases for use in terminal-restriction fragment length polymorphism (T-RFLP) analysis was performed by using richness and density indices in conjunction with computer simulations for 4,603 bacterial small-subunit rRNA gene sequences. T-RFLP analysis has become a commonly used method for screening environmental samples for precursory identification and community comparison studies due to its precision and high-throughput capability. The accuracy of T-RFLP analysis for describing a community has not yet been thoroughly evaluated. In this study, we attempted to classify restriction endonucleases based upon the ability to resolve unique terminal-restriction fragments (T-RFs) or operational taxonomic units (OTUs) from a database of gene sequences. Furthermore, we assessed the predictive accuracy of T-RFLP at fixed values of community richness (n ‫؍‬ 1, 5, 10, 50, and 100). Classification of restriction endonuclease fidelity was performed by measuring richness and density for the entire database of T-RFs. Further analysis of T-RFLP accuracy for determining richness was performed by iterative, random sampling from the derived database of T-RFs. It became apparent that two constraints were influential for measuring the fidelity of a given restriction endonuclease: (i) the ability to resolve unique sequence variants and (ii) the number of unique T-RFs that fell within a measurable size range. The latter constraint was found to be more significant for estimating restriction endonuclease fidelity. Of the 18 restriction endonucleases examined, BstUI, DdeI, Sau96I, and MspI had the highest frequency of resolving single populations in model communities. All restriction endonucleases used in this study detected <70% of the OTUs at richness values greater than 50 OTUs per modeled community. Based on the results of our in silico experiments, the most efficacious uses of T-RFLP for microbial diversity studies are those that address situations where there is low to intermediate species richness (e.g., colonization, early successional stages, biofilm formation).

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Section: Discussionmentioning

confidence: 99%

Fidelity of Select Restriction Endonucleases in Determining Microbial Diversity by Terminal-Restriction Fragment Length Polymorphism

Engebretson

Moyer

2003

Appl Environ Microbiol

111

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“…Even so, the classic Sanger method has undergone various modifications and refinements in the intervening years, most recently driven by the requirements of the Human Genome Project to facilitate automation and to increase throughput. Perhaps most significantly, contemporary methods use four different base-specific fluorescent dyes (Smith et al 1986) instead of radioactive labels, and cumbersome gel electrophoresis has been replaced by automated capillary electrophoresis (Luckey et al 1990). These developments have dramatically increased the efficiency of Sanger sequencing, which is now widely considered the gold standard for clinical diagnostic use.…”

Section: Introductionmentioning

confidence: 99%

Review of massively parallel DNA sequencing technologies

Moorthie

Mattocks

Wright

2011

HUGO J

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Since the development of technologies that can determine the base-pair sequence of DNA, the ability to sequence genes has contributed much to science and medicine. However, it has remained a relatively costly and laborious process, hindering its use as a routine biomedical tool. Recent times are seeing rapid developments in this field, both in the availability of novel sequencing platforms, as well as supporting technologies involved in processes such as targeting and data analysis. This is leading to significant reductions in the cost of sequencing a human genome and the potential for its use as a routine biomedical tool. This review is a snapshot of this rapidly moving field examining the current state of the art, forthcoming developments and some of the issues still to be resolved prior to the use of new sequencing technologies in routine clinical diagnosis.

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“…A subsequent report from Smith's laboratory described the use of capillary gel electrophoresis for the separation of the reaction products of the four spectral channel, single lane sequencing system [20]. Resolution was between a factor of 1.5 to 2 times superior to slab-gel data.…”

Section: Introductionmentioning

confidence: 99%

“…Smith and coworkers described the use of one-and four-spectral channel on-column fluorescence detectors; both the single-and four-spectral channel detectors produced detection limits of about 100 zmol of labeled fragment [19,20]; Karger's group has reported similar detection limits for their single spectral channel detector [12]. This group has reported a post-column single spectral channel fluorescence detector in the gel electrophoresis separation of the products of a single chain terminating reaction; detection limits were 10 zmol [21].…”

Section: Introductionmentioning

confidence: 99%