RRC ID 33461
Author Ahmed A.
Title A vector for sequencing long (40-kb) DNA fragments.
Journal Gene
Abstract An improved vector (pAA113M) has been constructed for sequencing long (40-kb) DNA fragments. The DNA fragment is cloned in the tet gene of the cosmid and subdivided into numerous overlapping segments by IS1-promoted deletions. Plasmids bearing these deletions are fractionated by gel electrophoresis, and shortened further from the opposite end by treatment with specific restriction endonucleases. Thus, a series of short overlapping segments, spread across the entire length of the fragment, become fused to IS1. Each segment can then be sequenced by the dideoxy method using an IS1 primer. The plasmids can replicate either from their normal origin or, in the presence of a filamentous helper phage, from the M13 origin. Hence, each segment can be sequenced as either double-stranded DNA or single-stranded DNA. Sequences of IS1-promoted and restriction enzyme-generated deletions contain overlaps that can be used to assemble the complete 40-kb sequence.
Volume 75(2)
Pages 315-21
Published 1989-2-20
DOI 10.1016/0378-1119(89)90277-1
PII 0378-1119(89)90277-1
PMID 2541052
MeSH Base Sequence* Chromosome Deletion Cloning, Molecular DNA / genetics DNA Transposable Elements DNA, Single-Stranded / genetics Escherichia coli / genetics Genetic Techniques Genetic Vectors* Plasmids* Restriction Mapping
IF 2.984
Times Cited 4
Prokaryotes E. coli pAA113M