Application of N-terminally truncated DNA polymerase from Thermus thermophilus (delta Tth polymerase) to DNA sequencing and polymerase chain reactions: comparative study of delta Tth and wild-type Tth polymerases

N-Terminally truncated DNA polymerase from Thermus thermophilus (delta Tth polymerase) lacking 5'-3' exonuclease activity was used for DNA sequencing and polymerase chain reaction (PCR). In contrast to the high background of the sequencing ladder observed with the wild-type Tth polymerase,...

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Bibliographic Details
Published inDNA research Vol. 3; no. 2; p. 87
Main Authors Arakawa, T, Jongsareejit, B, Tatsumi, Y, Tanaka, K, Ikeda, K, Komatsubara, H, Inoue, H, Kawakami, B, Oka, M, Emi, S, Yomo, T, Shima, Y, Negoro, S, Urabe, I
Format Journal Article
LanguageEnglish
Published England 30.04.1996
Subjects
Automation
DNA-Directed DNA Polymerase - chemistry
DNA-Directed DNA Polymerase - metabolism
Mutagenesis
Mutation
Polymerase Chain Reaction - methods
Sequence Analysis, DNA - methods
Thermus thermophilus - enzymology
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Summary:N-Terminally truncated DNA polymerase from Thermus thermophilus (delta Tth polymerase) lacking 5'-3' exonuclease activity was used for DNA sequencing and polymerase chain reaction (PCR). In contrast to the high background of the sequencing ladder observed with the wild-type Tth polymerase, delta Tth polymerase gave readable sequencing patterns which extend up to more than 500 bases from the primer site on cycle sequencing and automated sequencing. The delta Tth polymerase was used for the standard and mutagenic PCR, and net amplification of the DNA and the mutations accumulated during PCR were analyzed. Under mutagenic PCR, the mutation rates were 7.0 x 10(-4) (Tth) and 8.3 x 10(-4) (delta Tth) per nucleotide per cycle of amplification, which were 4-9 times higher than the rates under standard PCR.
ISSN:1340-2838