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Nucleic Acids Res. 1981 July 10; 9(13): 3015–3027.
PMCID: PMC327328

Shotgun DNA sequencing using cloned DNase I-generated fragments.

Abstract

A method for DNA sequencing has been developed that utilises libraries of cloned randomly-fragmented DNA. The DNA to be sequenced is first subjected to limit attach by a non-specific endonuclease (DNase I in the presence of Mn++), fractionated by size and cloned in a single-stranded phage vector. Clones are then picked at random and used to provide a template for sequencing by the dideoxynucleotide chain termination method. This technique was used to sequence completely a 4257 bp EcoRI fragment of bovine mitochondrial DNA. The cloned fragments were evenly distributed with respect to the EcoRI fragment, and completion of the entire sequence required the construction of only a single library. In general, once a clone library has been prepared, the speed of this approach (greater than 1000 nucleotides of randomly selected sequence per day) is limited mainly by the rate at which the data can be processed. Because the clones are selected randomly, however, the average amount of new sequence information per clone is substantially diminished as the sequence near completion.

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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. [PubMed]
  • Schreier PH, Cortese R. A fast and simple method for sequencing DNA cloned in the single-stranded bacteriophage M13. J Mol Biol. 1979 Mar 25;129(1):169–172. [PubMed]
  • Anderson S, Gait MJ, Mayol L, Young IG. A short primer for sequencing DNA cloned in the single-stranded phage vector M13mp2. Nucleic Acids Res. 1980 Apr 25;8(8):1731–1743. [PMC free article] [PubMed]
  • 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]
  • Heidecker G, Messing J, Gronenborn B. A versatile primer for DNA sequencing in the M13mp2 cloning system. Gene. 1980 Jun;10(1):69–73. [PubMed]
  • Messing J, Crea R, Seeburg PH. A system for shotgun DNA sequencing. Nucleic Acids Res. 1981 Jan 24;9(2):309–321. [PMC free article] [PubMed]
  • Staden R. A strategy of DNA sequencing employing computer programs. Nucleic Acids Res. 1979 Jun 11;6(7):2601–2610. [PMC free article] [PubMed]
  • Gingeras TR, Milazzo JP, Sciaky D, Roberts RJ. Computer programs for the assembly of DNA sequences. Nucleic Acids Res. 1979 Sep 25;7(2):529–545. [PMC free article] [PubMed]
  • Staden R. A new computer method for the storage and manipulation of DNA gel reading data. Nucleic Acids Res. 1980 Aug 25;8(16):3673–3694. [PMC free article] [PubMed]
  • Messing J, Gronenborn B, Müller-Hill B, Hans Hopschneider P. Filamentous coliphage M13 as a cloning vehicle: insertion of a HindII fragment of the lac regulatory region in M13 replicative form in vitro. Proc Natl Acad Sci U S A. 1977 Sep;74(9):3642–3646. [PubMed]
  • Gronenborn B, Messing J. Methylation of single-stranded DNA in vitro introduces new restriction endonuclease cleavage sites. Nature. 1978 Mar 23;272(5651):375–377. [PubMed]
  • Cordell B, Bell G, Tischer E, DeNoto FM, Ullrich A, Pictet R, Rutter WJ, Goodman HM. Isolation and characterization of a cloned rat insulin gene. Cell. 1979 Oct;18(2):533–543. [PubMed]
  • Rigby PW, Dieckmann M, Rhodes C, Berg P. Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J Mol Biol. 1977 Jun 15;113(1):237–251. [PubMed]
  • Young IG, Anderson S. The genetic code in bovine mitochondria: sequence of genes for the cytochrome oxidase subunit II and two tRNAs. Gene. 1980 Dec;12(3-4):257–265. [PubMed]
  • Maniatis T, Hardison RC, Lacy E, Lauer J, O'Connell C, Quon D, Sim GK, Efstratiadis A. The isolation of structural genes from libraries of eucaryotic DNA. Cell. 1978 Oct;15(2):687–701. [PubMed]
  • Yamamoto KR, Alberts BM, Benzinger R, Lawhorne L, Treiber G. Rapid bacteriophage sedimentation in the presence of polyethylene glycol and its application to large-scale virus purification. Virology. 1970 Mar;40(3):734–744. [PubMed]
  • 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]
  • Sanger F, Coulson AR. The use of thin acrylamide gels for DNA sequencing. FEBS Lett. 1978 Mar 1;87(1):107–110. [PubMed]
  • Melgar E, Goldthwait DA. Deoxyribonucleic acid nucleases. II. The effects of metals on the mechanism of action of deoxyribonuclease I. J Biol Chem. 1968 Sep 10;243(17):4409–4416. [PubMed]
  • Weiss B, Jacquemin-Sablon A, Live TR, Fareed GC, Richardson CC. Enzymatic breakage and joining of deoxyribonucleic acid. VI. Further purification and properties of polynucleotide ligase from Escherichia coli infected with bacteriophage T4. J Biol Chem. 1968 Sep 10;243(17):4543–4555. [PubMed]
  • Ehrlich SD, Bertazzoni U, Bernardi G. The specificity of pancreatic deoxyribonuclease. Eur J Biochem. 1973 Dec 3;40(1):143–147. [PubMed]
  • Bernardi A, Gaillard C, Bernardi G. The specificity of five DNAases as studied by the analysis of 5'-terminal doublets. Eur J Biochem. 1975 Apr 1;52(3):451–457. [PubMed]
  • Sutcliffe JG. pBR322 restriction map derived from the DNA sequence: accurate DNA size markers up to 4361 nucleotide pairs long. Nucleic Acids Res. 1978 Aug;5(8):2721–2728. [PMC free article] [PubMed]
  • Lutter LC. Deoxyribonuclease I produces staggered cuts in the DNA of chromatin. J Mol Biol. 1977 Nov 25;117(1):53–69. [PubMed]
  • Heffron F, So M, McCarthy BJ. In vitro mutagenesis of a circular DNA molecule by using synthetic restriction sites. Proc Natl Acad Sci U S A. 1978 Dec;75(12):6012–6016. [PubMed]
  • Berkner KL, Folk WR. Polynucleotide kinase exchange reaction: quantitave assay for restriction endonuclease-generated 5'-phosphoroyl termini in DNA. J Biol Chem. 1977 May 25;252(10):3176–3184. [PubMed]
  • Westergaard O, Brutlag D, Kornberg A. Initiation of deoxyribonucleic acid synthesis. IV. Incorporation of the ribonucleic acid primer into the phage replicative form. J Biol Chem. 1973 Feb 25;248(4):1361–1364. [PubMed]
  • Anderson S, Bankier AT, Barrell BG, de Bruijn MH, Coulson AR, Drouin J, Eperon IC, Nierlich DP, Roe BA, Sanger F, et al. Sequence and organization of the human mitochondrial genome. Nature. 1981 Apr 9;290(5806):457–465. [PubMed]
  • Shenk TE, Carbon J, Berg P. Construction and analysis of viable deletion mutants of simian virus 40. J Virol. 1976 May;18(2):664–671. [PMC free article] [PubMed]
  • Frischauf AM, Garoff H, Lehrach H. A subcloning strategy for DNA sequence analysis. Nucleic Acids Res. 1980 Dec 11;8(23):5541–5549. [PMC free article] [PubMed]
  • Gingeras TR, Roberts RJ. Steps toward computer analysis of nucleotide sequences. Science. 1980 Sep 19;209(4463):1322–1328. [PubMed]

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