[10] Automated fluorescent DNA sequencing of polymerase chain reaction products

This chapter describes two useful methods for the direct sequencing of the products of symmetric polymerase chain reaction (PCR), including the purification of double-stranded template by polyethylene glycol (PEG) precipitation and enzymatic conversion to single-stranded template. It also provides solutions to some of the common problems of directly sequencing PCR products. The methods described provide some useful approaches for the DNA sequencing of templates produced by PCR. These procedures have been employed successfully for the large-scale DNA sequencing of cosmid fragments subcloned in plasmid or M13 vectors and for the sequence analysis of complementary DNAs (cDNAs) cloned in bacteriophage λ vectors. The method describing direct sequencing from PEG-precipitated PCR product has been used successfully for the analysis of Caenorhabditis elegans genomic and cDNA sequences. For every combination of amplification primer pair and target DNA, there is an optimal method for PCR amplification; the ability to sequence the products of any PCR experiment directly will also vary. A coupled PCR/DNA sequencing method that works well for one experimental system may work quite poorly with others.