"Nonrandom" DNA sequence analysis in bacteriophage M13 by the dideoxy chain-termination method.
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.
D001483:Base Sequence; D002872:Chromosome Deletion; D003001:Cloning, Molecular / Q000379:methods*; D003090:Coliphages / Q000235:genetics*; D004247:DNA; D004274:DNA, Recombinant; D005914:Globins / Q000235:genetics; D006801:Humans
D004274:DNA, Recombinant; D005914:Globins; D004247:DNA