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Rapid and reliable protocol for direct sequencing of material amplified by the polymerase chain reaction
N Kusukawa1, T Uemori, K Asada
1Takara Shuzo Co., Ltd., Biotechnology Research Laboratories, Shiga, Japan.
Biotechniques
|July 1, 1990
Abstract:
A simple and reliable method is described for direct sequencing of material generated by the polymerase chain reaction. The protocol is based on the purification of the amplified double-stranded product by polyethylene glycol precipitation, annealing of primer with template by a "snap-cooling" procedure and sequencing by the dideoxy chain termination method with the use of Klenow fragment or Taq polymerase. The limit of the size of PCR products that can be sequenced is also discussed.