[7] Direct sequencing of polymerase chain reaction products from low melting temperature agarose

This chapter describes a method in which polymerase chain reaction (PCR) products purified in low melting temperature agarose can be sequenced directly using commercially available sequencing kits with little or no variation from the directions of the manufacturer.

Abstract

This chapter describes a method in which polymerase chain reaction (PCR) products purified in low melting temperature agarose can be sequenced directly using commercially available sequencing kits with little or no variation from the directions of the manufacturer. Therefore, the PCR product can be prepared for sequencing in a single step and sequenced under the same conditions as a normal double-stranded template using techniques already available in most laboratories. PCR primers may be used without end labeling, and the PCR reaction products need not be processed prior to low-melting-temperature agarose gel electrophoresis. The chapter discusses a number of different parameters that have the potential to affect the quality of sequence obtained. Templates of any size may be used to obtain reliable sequence information using either the original PCR primers or internal primers as sequencing primers. In rare cases in which this technique fails to yield good sequences, it was noted that several parameters may be changed to improve the results.