Fluorescence detection in automated DNA sequence analysis

  title={Fluorescence detection in automated DNA sequence analysis},
  author={Lloyd M. Smith and Jane Z. Sanders and Robert J. Kaiser and Peter Hughes and Chris Dodd and Charles R. Connell and Cheryl R. Heiner and Stephen B. H. Kent and Leroy E. Hood},
We have developed a method for the partial automation of DNA sequence analysis. Fluorescence detection of the DNA fragments is accomplished by means of a fluorophore covalently attached to the oligonucleotide primer used in enzymatic DNA sequence analysis. A different coloured fluorophore is used for each of the reactions specific for the bases A, C, G and T. The reaction mixtures are combined and co-electrophoresed down a single polyacrylamide gel tube, the separated fluorescent bands of DNA… 
Rapid DNA sequencing based upon single molecule detection.
The ability to sequence large fragments of DNA will significantly reduce the amount of subcloning and the number of overlapping sequences required to assemble megabase segments of sequence information.
Colorimetric-detected DNA sequencing.
  • S. Beck
  • Chemistry, Medicine
    Analytical biochemistry
  • 1987
A sensitive, colorimetric method for visualizing the band pattern of DNA sequencing reaction is described, which is so sensitive that the sequencing reactions can be performed under the same conditions and concentrations as those for the radioactive detection.
A system for rapid DNA sequencing with fluorescent chain-terminating dideoxynucleotides.
A DNA sequencing system based on the use of a novel set of four chain-terminating dideoxynucleotides, each carrying a different chemically tuned succinylfluorescein dye distinguished by its fluorescent emission, and the sequence analysis of M13 phage DNA made with this system is described.
Separation and analysis of DNA sequence reaction products by capillary gel electrophoresis.
The potential of capillary gel electrophoresis with laser induced fluorescence detection as a tool for DNA sequence determination with synthetic oligonucleotides and single-stranded phage DNA as templates is demonstrated.
DNA sequence analysis is one of the most important techniques of modern molecular biology. It permits the rapid and complete determination of the precise chemical structure of the genetic material.
Sequencing of antisense DNA analogues by capillary gel electrophoresis with laser-induced fluorescence detection.
A linear relationship independent of experimental conditions between the relative electrophoretic migration time and the oligonucleotide base number was observed and is shown to be consistent with a simplified version of this model and can be used to facilitate the sequence determination.
A ligase-mediated gene detection technique.
An assay for the presence of given DNA sequences has been developed, based on the ability of two oligonucleotides to anneal immediately adjacent to each other on a complementary target DNA molecule, which permits the rapid and standardized identification of single-copy gene sequences in genomic DNA.
High-speed DNA sequencing: an approach based upon fluorescence detection of single molecules.
A laser based technique for the rapid sequencing of large fragments of DNA based upon the detection of single, fluorescently tagged nucleotides cleaved from a single DNA fragment which is orders of magnitude faster than existing methods.
High-throughput fluorescent DNA sequence analysis: Methods and automation
This strategy combines rapid preparation of template DNA using a modification of the polymerase chain reaction (PCR), automation of the DNA sequencing reactions using a robotic laboratory workstation, and subsequent analysis of the fluorescent-labeled reaction products on a commercial automated fluorescent sequencer.
Capillary gel electrophoresis for DNA sequencing. Laser-induced fluorescence detection with the sheath flow cuvette.
Capillary polyacrylamide gel electrophoresis separation of dideoxycytidine chain-terminated DNA fragments is reported, with preliminary mass detection limit of 10(-20) mol of fluorescein-labeled DNA fragments.


DNA sequencing with chain-terminating inhibitors.
A new method for determining nucleotide sequences in DNA is described, which makes use of the 2',3'-dideoxy and arabinon nucleoside analogues of the normal deoxynucleoside triphosphates, which act as specific chain-terminating inhibitors of DNA polymerase.
DNA sequence analysis by primed synthesis.
  • A. Smith
  • Medicine, Biology
    Methods in enzymology
  • 1980
This chapter discusses the techniques for DNA sequence analysis by primed synthesis, which makes use of the ability of DNA polymerases to synthesize a complementary radioactive copy of a single-stranded DNA template using restriction enzyme generated DNA fragments as primers.
The synthesis of oligonucleotides containing an aliphatic amino group at the 5' terminus: synthesis of fluorescent DNA primers for use in DNA sequence analysis.
A rapid and versatile method has been developed for the synthesis of oligonucleotides which contain an aliphatic amino group at their 5' terminus. This amino group reacts specifically with a variety
Sequencing end-labeled DNA with base-specific chemical cleavages.
The chapter presents techniques for producing discrete DNA fragments, end-labeling DNA, segregating end- labeled fragments, extracting DNA from gels, and the protocols for partially cleaving it at specific bases using the chemical reactions.
Non-radioactive hybridization probes prepared by the chemical labelling of DNA and RNA with a novel reagent, photobiotin.
The sensitivity of detection of target RNA in dot-blots and Northern blots was equivalent to that obtained with 32p-labelled DNA probes, and photobiotin was also used for the labelling of proteins with biotin.
Solid-phase methods for sequencing of nucleic acids I. Simultaneous sequencing of different oligodeoxyribonucleotides using a new, mechanically stable anion-exchange paper.
A solid-phase method for simultaneous sequencing of large numbers of oligodeoxyribonucleotides has been developed using a new, mechanically stable anion-exchange paper. The excellent mechanical
Oligonucleotide synthesis : a practical approach
An introduction to modern methods of DNA synthesis / Michael J. Gait -- Preparation of protected deoxyribonucleotides / Roger A. Jones -- Solid phase synthesis of oligodeoxyribonucleotides by the