Fidelity of DNA polymerases in DNA amplification

Journal Article · Fri Dec 01 00:00:00 EST 1989 · Proceedings of the National Academy of Sciences of the United States of America; (USA)

DOI:https://doi.org/10.1073/pnas.86.23.9253· OSTI ID:7138311

Keohavong, P; Thilly, W G ^[1]

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1. Massachusetts Institute of Technology, Cambridge (USA)

Denaturing gradient gel electrophoresis (DGGE) was used to separate and isolate the products of DNA amplification by polymerase chain reaction (PCR). The strategy permitted direct enumeration and identification of point mutations created by T4, modified T7, Klenow fragment of polymerase I, and Thermus aquaticus (Tag) DNA polymerases. Incorrectly synthesized sequences were separated from the wild type by DGGE as mutant/wild-type heteroduplexes and the heteroduplex fraction was used to calculate the average error rate (mutations per base duplication). The error rate induced in the 104-base-pair low-temperature melting domain of exon 3 of the human hypoxanthine/guanine phosphoribosyltransferase (HPRT) gene was {approx} 3.4 {times} 10{sup {minus}5} for modified T7, 1.3 {times} 10{sup {minus}4} for Klenow fragment, and 2.1 {times} 10{sup {minus}4} for Taq polymerases after a 10{sup 6}-fold amplification. The error rate for T4 DNA polymerase was not more than 3 {times} 10{sup {minus}6} error per base duplication. The predominant mutations were sequenced and found to be transitions of G{center dot}C to A{center dot}T for T4 and modified T7 DNA polymerases, and A{center dot}T to G{center dot}C for Taq polymerase. Klenow fragment induced both possible transitions and deletions of 2 and 4 base pairs.

• DOE Contract Number: FG02-86ER60448
• OSTI ID: 7138311
• Journal Information: Proceedings of the National Academy of Sciences of the United States of America; (USA), Vol. 86:23; ISSN 0027-8424
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
DNA POLYMERASES
BIOCHEMISTRY
GENE MUTATIONS
DNA SEQUENCING
HYPOXANTHINE PHOSPHORIBOSYLTRANSFERASE
GENE AMPLIFICATION
BACTERIOPHAGES
ELECTROPHORESIS
TEMPERATURE EFFECTS
CHEMISTRY
ENZYMES
GLYCOSYL TRANSFERASES
MICROORGANISMS
MUTATIONS
NUCLEOTIDYLTRANSFERASES
PARASITES
PENTOSYL TRANSFERASES
PHOSPHORUS-GROUP TRANSFERASES
POLYMERASES
STRUCTURAL CHEMICAL ANALYSIS
TRANSFERASES
VIRUSES
550200* - Biochemistry