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. modified this method, avoiding the need for prior disruption of the template cells, and incorporating radio-labelled deoxynucleotides, rather than end-labelling the sequencing primer (7). We have further developed this method to be applied to direct sequencing of multiple biotinylated PCR products, without the need for cloning, using agarose gel purification of the amplified DNA. Heating simultaneously melts the agarose gel slice and denatures the DNA and the single biotinylated strand of DNA is captured on solid phase for sequencing.