A simple and rapid method of direct sequencing using Dynabeads

Abstract

Highly specific regions of genomic DNA can now be produced in large quantities by enzymatic amplification using the polymerase chain reaction (PCR) and a thermostable Taq polymerase (Saiki et al. 1988). This has encouraged the development of direct sequencing of the amplified DNA. Apart from the obvious advantage of the avoidance of cloning of DNA, direct sequencing enables both alleles to be analysed simultaneously. The latter is particularly advantageous in heterozygous states where the mutant allele cannot be differentiated from the normal. However, direct dideoxy sequencing of PCR‐amplified DNA is still not consistently satisfactory. The difficulty arises from the nature of the PCR‐amplified DNA which consists of short, linear double‐stranded DNA. The two strands of the DNA can rapidly reanneal during DNA sequencing, blocking or displacing the sequencing primer from the template strand, thereby decreasing the amount of specific termination products formed in the sequencing reactions.