Improvement of PCR amplified DNA sequencing with the aid of detergents

Abstract

The problem of sequencing short double stranded DNA like products of the polymerase chain reaction (PCR) is the tendency of the templates to reanneal. The use of dimethyl sulphoxide (DMSO) can hep to overcome this problem1. Using the T7 sequencing96 kit (Pharmacia), we have found that addition of 0.5 % of nonidet P-40 (NP-40) or the combination of 0.5 % MM0 with 0.5 % Tween 20 to the sequencing reaction mix2 enhances the intensity of signals obtained and also reduces the frequency of unspecific bands appearing in certain positions. Double stranded DNA spanning 115bp of the gag region of HlV^ben3 (1875-1989) was amplified by the polymerase chain reaction4 (PCR). Amplified DNA was purified by electrophoresis on 2 % agarose gels. 0.25 pmole of the electroeluted DNA and 20 pmoles of one PCR primer were denaturated by boiling for 3 min in the presence of detergent and immediately snap-cooled on dry ice to minimize reannealing. For sequencing of the DNA 10 /tCi of 35SdATP, T7 DNA polymerase, and labeling mix were added to the annealing mixture. The resulting mixture was divided into four equal parts in tubes each containing another 'short'-termination mix. The sequencing reaction was kept at 37°C for five minutes. Then 2/i of solution containing 0.25 mM dNTP, 50 mM NaCl and detergent (see below) were added, and the reaction was left for five minutes at 37 °C. The detergent concentration was maintained throughout all additions to the reaction mixtures. Several detergents were tested alone and in combination; DMSO 10%,