Direct sequencing of PCR products in agarose gel slices.

Direct DNA sequencing of amplified polymerase chain reaction (PCR) products offers several advantages over cloning of amplified DNA products. It is faster (1 day versus 3—5 days) and in DNA samples containing sequence polymorphisms both the normal and mutated sequence can be detected in the same sequencing reaction. The major problems encountered in direct sequencing of amplified DNA have been contamination by PCR primers and deoxynucleotides, and overcoming fast template renaturation. Techniques which have been used to purify the amplified product include: differential precipitation, electrophoretic purification, absorption of DNA to specific matrices, or differential filtration (i.e. centricon) (1—6). Each of these techniques is capable of improving the quality of sequence data, but all are time consuming and subject to some performance variability. The problem of template reannealing has also been approached by several methods including: rapid cooling following denaturation, use of thermal stable polymerases, increased primer concentration and preparation of a single-stranded DNA templates by: asymmetric PCR, biotinylation of one primer or use of strandspecific nuclease (2,5,7,8). We have devised a strategy for direct sequencing of PCR products using a simple modification of the standard Sequenase® protocol (U.S. Biochemicals, Cleveland, OH).