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Proc Natl Acad Sci U S A. 1983 Jul; 80(13): 3963–3965.
doi: 10.1073/pnas.80.13.3963
PMCID: PMC394179
PMID: 6575390

Buffer gradient gels and 35S label as an aid to rapid DNA sequence determination.

M D Biggin, T J Gibson, and G F Hong
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Abstract

Two methods for increasing the length of DNA sequence data that can be read off a polyacrylamide gel are described. We have developed a rapid way to pour a buffer concentration gradient gel that, by altering the vertical band separation on an autoradiograph, allows more sequence to be obtained from a gel. We also show that the use of deoxyadenosine 5'-(alpha-[35S]thio)triphosphate as the label incorporated in dideoxynucleotide sequence reactions increases the sharpness of the bands on an autoradiograph and so increases the resolution achieved.

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Selected References

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  • Sanger F, Nicklen S, Coulson AR. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. [PMC free article] [PubMed] [Google Scholar]
  • Maxam AM, Gilbert W. A new method for sequencing DNA. Proc Natl Acad Sci U S A. 1977 Feb;74(2):560–564. [PMC free article] [PubMed] [Google Scholar]
  • Sanger F, Coulson AR. The use of thin acrylamide gels for DNA sequencing. FEBS Lett. 1978 Mar 1;87(1):107–110. [PubMed] [Google Scholar]
  • Vosberg HP, Eckstein F. Incorporation of phosphorothioate groups into fd and phi X174 DNA. Biochemistry. 1977 Aug 9;16(16):3633–3640. [PubMed] [Google Scholar]
  • Brody RS, Frey PA. Unambiguous determination of the stereochemistry of nucleotidyl transfer catalyzed by DNA polymerase I from Escherichia coli. Biochemistry. 1981 Mar 3;20(5):1245–1252. [PubMed] [Google Scholar]
  • Garoff H, Ansorge W. Improvements of DNA sequencing gels. Anal Biochem. 1981 Aug;115(2):450–457. [PubMed] [Google Scholar]
  • Sanger F, Coulson AR, Barrell BG, Smith AJ, Roe BA. Cloning in single-stranded bacteriophage as an aid to rapid DNA sequencing. J Mol Biol. 1980 Oct 25;143(2):161–178. [PubMed] [Google Scholar]
  • Duckworth ML, Gait MJ, Goelet P, Hong GF, Singh M, Titmas RC. Rapid synthesis of oligodeoxyribonucleotides VI. Efficient, mechanised synthesis of heptadecadeoxyribonucleotides by an improved solid phase phosphotriester route. Nucleic Acids Res. 1981 Apr 10;9(7):1691–1706. [PMC free article] [PubMed] [Google Scholar]
  • Messing J, Vieira J. A new pair of M13 vectors for selecting either DNA strand of double-digest restriction fragments. Gene. 1982 Oct;19(3):269–276. [PubMed] [Google Scholar]
  • Deininger PL. Random subcloning of sonicated DNA: application to shotgun DNA sequence analysis. Anal Biochem. 1983 Feb 15;129(1):216–223. [PubMed] [Google Scholar]
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