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 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.
Kaltenboeck
B
B
Dept. of Veterinary Microbiology and Parasitology, School of Veterinary Medicine, Louisiana State University, Baton Rouge 70803-8416.
Spatafora
J W
JW
Zhang
X
X
Kousoulas
K G
KG
Blackwell
M
M
Storz
J
J
D000818:Animals; D001483:Base Sequence; D004247:DNA / Q000032:analysis; D004277:DNA, Single-Stranded / Q000138:chemical synthesis*; D009710:Nucleotide Mapping / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods*
D004277:DNA, Single-Stranded; D004247:DNA