Kaltenboeck B, Spatafora J W, Zhang X, Kousoulas K G, Blackwell M, Storz J
Dept. of Veterinary Microbiology and Parasitology, School of Veterinary Medicine, Louisiana State University, Baton Rouge 70803-8416.
Biotechniques. 1992 Feb;12(2):164, 166, 168-71.
A modification of the asymmetric PCR method is described, which reliably facilitates sequencing of PCR-amplified DNA. This procedure produces single-stranded DNA fragments as long as two kilobases that are suitable for dideoxy DNA sequencing. First, a PCR for double-stranded DNA is preformed under optimal conditions (double-stranded PCR). Then, a 5-10-microliters fraction of the double-stranded PCR and a single primer are used to generate single-stranded DNA in a separate PCR (single-stranded PCR). The concentration of the single primer are used to generate single-stranded DNA in a separate PCR (single-stranded PCR). The concentration of the single primer is approximately 0.4 microM. Usually 15 to 25 cycles of single-stranded PCR are optimal to produce single-stranded DNA for four to eight sequencing reactions. The single-stranded DNA is purified by centrifugal ultrafiltration and used directly in dideoxy sequencing. This method was employed to produce high-quality single-stranded DNA templates from a variety of organisms for efficient DNA sequencing of PCR-amplified DNA.
本文描述了一种不对称PCR方法的改进方法，该方法能可靠地促进PCR扩增DNA的测序。此程序可产生长达两千碱基的单链DNA片段，适用于双脱氧DNA测序。首先，在最佳条件下进行双链DNA的PCR（双链PCR）。然后，取5 - 10微升双链PCR产物和一条单引物，用于在单独的PCR中生成单链DNA（单链PCR）。单引物的浓度约为0.4微摩尔。通常，15至25个循环的单链PCR最适合为四至八个测序反应生成单链DNA。单链DNA通过离心超滤纯化，可直接用于双脱氧测序。该方法用于从多种生物体中制备高质量的单链DNA模板，以高效地对PCR扩增的DNA进行测序。
