Enhancement of PCRs by partial restriction digestion of genomic templates.
Genomic DNA preparations derived from mammalian cells can often exhibit poor template activity in PCR, particularly when carried out on target sequences present at low copy number. Using genomic DNA bearing SV40 sequences integrated into host chromosomal DNA at low copy number as a target, we show that template efficiency can be dramatically enhanced after treatment of the genomic template with restriction enzymes for varying periods of time. Also, our results indicate that, while template activity was enhanced by all of the restriction enzymes tested, optimal digestion time varied for each enzyme.
Vázquez
R
R
Biochemistry Division, City College, City University of New York, NY 10031, USA.
Steinberg
M L
ML
D002461:Cell Line, Transformed; D004247:DNA / Q000235:genetics* / Q000378:metabolism; D017931:DNA Primers; D004262:DNA Restriction Enzymes / Q000378:metabolism; D004279:DNA, Viral / Q000235:genetics* / Q000378:metabolism; D005784:Gene Amplification; D005818:Genetic Engineering; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D013539:Simian virus 40; D013698:Templates, Genetic; D013997:Time Factors
D017931:DNA Primers; D004279:DNA, Viral; D004247:DNA; D004262:DNA Restriction Enzymes