Direct single stranded sequencing from agarose of polymerase chain reaction products

The polymerase chain reaction (PCR) has made possible the rapid isolation and amplification of specific DNA segments, which can then be used for a wide range of applications (1). Direct sequencing of PCR products has been described, using assymetric PCR, which requires purification of the PCR product, or inclusion of 10 % DMSO in the sequencing reaction (2, 3, 4). However, these methods of sequencing have depended on the production of a pure uncontaminated DNA fragment. Direct sequencing from low melting point agarose has been successfully performed, but still requires a double stranded template (5). Single stranded DNA for sequencing has also been produced from biotinylated PCR products. One PCR primer has a biotin moiety attached at the 5 ' end, and the resultant PCR product can be bound to solid phase using streptavidin-biotin bonding to hold DNA onto streptavidin coated magnetic beads (6). Schofield et al. modifie