Insert selection by BamHI methyltransferase protection in P1 phage-based cloning.
A P1-based cloning system has been tested which depends upon (i) in vitro selection for vectors containing inserts mediated by methyltransferase (MTase) protection, and (ii) in vivo vector arm Cre-mediated recombination following electroporation. Specifically, chromosomal DNA was digested with BglII, dephosphorylated, methylated with BamHI MTase and ligated into the BamHI site of the vector, thereby destroying that site. Subsequent BamHI digestion acted as the in vitro selection, eliminating vector religation products prior to electroporation into cells expressing the Cre recombinase. Electroporation with linearized vector gave approx. 10(6) transformants per microgram vector, depending on vector concentration. Cloning of BglII fragments of gamma DNA using the in vitro selection system led to 1.3-4-fold fewer transformants per microgram vector. Plasmids recovered from these clones were all found to contain a gamma BglII fragment and the representation of fragments in the clones was independent of the length of the fragments. Both in vitro selection and electroporation are applicable to library construction using size-selected human DNA, with size selection either before or after ligation and BamHi digestion.
Bobovnikova
Y
Y
Department of Microbiology, Mount Sinai School of Medicine, New York, NY 10029, USA.
Kim
S J
SJ
Wetmur
J G
JG
D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D001426:Bacterial Proteins / Q000378:metabolism*; D017101:Bacteriophage P1 / Q000235:genetics*; D010582:Bacteriophage lambda / Q000235:genetics; D016466:CHO Cells; D003001:Cloning, Molecular / Q000379:methods*; D006224:Cricetinae; D004254:DNA Nucleotidyltransferases / Q000378:metabolism; D004270:DNA, Circular / Q000378:metabolism; D004274:DNA, Recombinant / Q000235:genetics* / Q000378:metabolism; D004279:DNA, Viral / Q000235:genetics / Q000378:metabolism; D015257:DNA-Cytosine Methylases / Q000378:metabolism*; D015245:Deoxyribonuclease BamHI / Q000378:metabolism*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D018274:Electroporation; D015723:Gene Library; D005822:Genetic Vectors / Q000235:genetics*; D006801:Humans; D019426:Integrases; D008745:Methylation; D011995:Recombination, Genetic; D012641:Selection, Genetic; D014764:Viral Proteins
D001426:Bacterial Proteins; D004270:DNA, Circular; D004274:DNA, Recombinant; D004279:DNA, Viral; D014764:Viral Proteins; C042525:DNA modification methylase BamHI; D015257:DNA-Cytosine Methylases; C045073:Cre recombinase; D004254:DNA Nucleotidyltransferases; D019426:Integrases; D015245:Deoxyribonuclease BamHI; C024404:BglII endonuclease; D015252:Deoxyribonucleases, Type II Site-Specific