Consecutive cycles of precise, unidirectional 14-bp deletions using a BseRI/BsgI trimming plasmid.
A straightforward method for generating precise, consecutive, unidirectional 14-bp deletions into cloned DNA, adopted from the trimming principle developed by Szybalski and his colleagues, is presented. The method utilizes pTRIM14, a plasmid constructed with the class-IIS restriction enzyme recognition sites for BseRI and BsgI arranged in the form of a cassette, just upstream from the cloned DNA. Class-IIS restriction enzymes cleave DNA downstream of their recognition sites. pTRIM14, containing the cloned DNA, is processed through a trimming cycle that involves sequential restriction digestions with BsgI and then BseRI, followed by treatment with Mung Bean nuclease and then with ligase. One trimming cycle results in a net 14-bp deletion. We demonstrate precise, consecutive deletions at very high efficiency.
Ariazi
E A
EA
University of Wisconsin Madison 53792, USA.
Gould
M N
MN
D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods; D004274:DNA, Recombinant / Q000235:genetics / Q000378:metabolism*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D017422:Sequence Analysis, DNA; D017384:Sequence Deletion
D004274:DNA, Recombinant; C082523:endodeoxyribonuclease BseRI; C100361:endodeoxyribonuclease BsgI; D015252:Deoxyribonucleases, Type II Site-Specific