A directed nucleotide-sequencing approach for single-stranded vectors based on recloning intermediates of a progressive DNA synthesis reaction.
A simple method for site-directed nucleotide sequencing is presented that uses a novel procedure for generating nested 'deletions' within inserts of single-stranded clones. In this method, single-stranded template, sequencing primer, and the Klenow fragment of Escherichia coli DNA polymerase I are used to initiate progressive DNA synthesis of the entire insert of the clone. By time-dependent sampling and pooling of intermediates from the synthesis reaction a series of nested double-stranded DNA subfragments of the insert can be created. Nested subclones are then produced by S1-endonuclease treatment and oriented subcloning methods. First, smaller quantities of template DNA can be used, equivalent to a fraction of a small DNA sequencing prep. Second, it works with single-stranded M13 phage DNA rather than requiring the preparation of double-stranded replicative form DNA as in ExoIII-based methods. Third, the 'deletions' it generates can span areas of simple nucleotide sequence or secondary structure that often halt digestion in the single-stranded exonuclease-based method. Last, the method is adaptable to a larger variety of insert cloning sites than the ExoIII-based method. The main disadvantage of the method is that, due to the lower efficiency of subcloning larger DNA fragments, subclone inserts larger than 3 kb are generated only infrequently.
Burton
F H
FH
Department of Microbiology and Immunology, University of North Carolina, Chapel Hill 27514.
Loeb
D D
DD
McGraw
R A
RA
Edgell
M H
MH
Hutchison
C A
CA
D001435:Bacteriophages / Q000235:genetics; D001483:Base Sequence; D002872:Chromosome Deletion; D003001:Cloning, Molecular; D004256:DNA Polymerase I / Q000235:genetics; D004261:DNA Replication; D004277:DNA, Single-Stranded / Q000235:genetics*; D005822:Genetic Vectors; D009711:Nucleotides / Q000235:genetics
D004277:DNA, Single-Stranded; D009711:Nucleotides; D004256:DNA Polymerase I