Vázquez R, Steinberg M L
Biochemistry Division, City College, City University of New York, NY 10031, USA.
Biotechniques. 1999 Jan;26(1):91-5. doi: 10.2144/99261st02.
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.
从哺乳动物细胞中提取的基因组DNA制剂在PCR中常常表现出较差的模板活性,尤其是在对低拷贝数的靶序列进行PCR时。以整合到宿主染色体DNA上的低拷贝数SV40序列的基因组DNA为靶标,我们发现,用限制酶对基因组模板进行不同时间的处理后,模板效率可显著提高。此外,我们的结果表明,虽然所有测试的限制酶都提高了模板活性,但每种酶的最佳消化时间各不相同。