Improved double-stranded DNA sequencing using the linear polymerase chain reaction.

One of the problems associated with double-3tranded DNA sequencing is that most of the [5'-3P]-labelied oligonuoleotide primer i3 not extended by the DNA polymerase. Hence, most of the labelled primer is wasted and gels have to be autoradiographed for several days. In the method described in this paper, almost all of the labelled primer is extended by the DNA polymerase and thus a shorter autoradiograph exposure is needed. This method Is a modification of the linear polymerase chain reaction (LPCR) where only one o ligonucleotide 13 present (1). It U363 Taq DNA polymerase and, since polymerisation is at 72°C, results in a low level of sequencing artifacts. Multiple LPCR cycle3 also removes sequencing artifacts. The use of lower than normal dNTP concentrations i3 necessary to achieve