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"Nonrandom" DNA sequence analysis in bacteriophage M13 by the dideoxy chain-termination method.

M Poncz·D Solowiejczyk·M Ballantine·E Schwartz·S Surrey
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Abstract

We describe a rapid "nonrandom" DNA sequence analysis procedure that facilitates the nucleotide sequence determination of large contiguous regions of DNA. The method consists of cloning a restriction endonuclease fragment of interest into bacteriophage M13 followed by construction of a series of nuclease BAL-31 deletion mutants originating from a single site in M13 that is close to the DNA insert. Determination of the size of the deletion mutant is accomplished by hybridization to a complementary single-stranded probe derived from M13 containing that total insert followed by nuclease S1 treatment. Single-stranded M13-insert DNAs of progressively smaller sizes are isolated and analyzed by using a site-specific M13 DNA primer and the dideoxy chain-termination method. In this way, analysis of the DNA sequence proceeds from one end of the total insert to the other in a nonrandom fashion due to generation of a controlled overlapping set of deletion mutants.

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Base SequenceChromosome DeletionCloning, MolecularColiphagesDNADNA, RecombinantGlobinsHumansJournal ArticleResearch Support, U.S. Gov't, P.H.S.

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M Poncz, D Solowiejczyk, M Ballantine, E Schwartz, S Surrey. 1982. "Nonrandom" DNA sequence analysis in bacteriophage M13 by the dideoxy chain-termination method.. https://doi.org/10.1073/pnas.79.14.4298

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