Open AccessIncomplete primer extension duringin vitroDNA amplification catalyzed byTaqpolymerase; exploitation for DNA sequencing
Author(s) -
David B. Olsen,
Fritz Eckstein
Publication year - 1989
Publication title -
nucleic acids research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 9.008
H-Index - 537
eISSN - 1362-4954
pISSN - 0305-1048
DOI - 10.1093/nar/17.23.9613
Subject(s) - biology , oligonucleotide , taq polymerase , primer (cosmetics) , microbiology and biotechnology , primer extension , agarose gel electrophoresis , primer dimer , gel electrophoresis , multiple displacement amplification , polymerase chain reaction , hot start pcr , dna , polymerase , polyacrylamide gel electrophoresis , agarose , dna polymerase , multiplex polymerase chain reaction , biochemistry , dna extraction , thermus aquaticus , enzyme , base sequence , gene , chemistry , organic chemistry
Polyacrylamide gel electrophoresis of DNA fragments obtained by the polymerase chain reaction using Taq polymerase revealed the presence of multiple fragments shorter than the expected product. These abortive extension products were observed even when analysis by agarose gel electrophoresis showed only a single band. The production of prematurely terminated fragments can be exploited for the sequencing of PCR products if phosphorothioate groups are incorporated base specifically during the reaction in the presence of two oligonucleotide primers, one of which is 5'-32P-labeled. The addition of snake venom phosphodiesterase to the reaction mixture after completion of the amplification cycles digests each fragment from the 3'-end to a phosphorothioate group so that the sequence can be read by polyacrylamide gel electrophoresis.