Efficient production of single-stranded DNA as long as 2 kb for sequencing of PCR-amplified DNA
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 performed under optimal conditions (double-stranded PCR). Then, a 5-10 μl 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 is approximately 0.4 μM. 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.
Publication Source (Journal or Book title)
Kaltenboeck, B., Spatafora, J., Zhang, X., Kousoulas, K., Blackwell, M., & Storz, J. (1992). Efficient production of single-stranded DNA as long as 2 kb for sequencing of PCR-amplified DNA. BioTechniques, 12 (2) Retrieved from https://repository.lsu.edu/animalsciences_pubs/776