Fidelity of DNA polymerases in DNA amplification.

Abstract:

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 (Taq) 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 approximately 3.4 x 10(-5) for modified T7, 1.3 x 10(-4) for Klenow fragment, and 2.1 x 10(-4) for Taq polymerases after a 10(6)-fold amplification. The error rate for T4 DNA polymerase was not more than 3 x 10(-6) error per base duplication. The predominant mutations were sequenced and found to be transitions of G.C to A.T for T4 and modified T7 DNA polymerases, and A.T to G.C for Taq polymerase. Klenow fragment induced both possible transitions and deletions of 2 and 4 base pairs.

Polymerases:

Topics:

Fidelity

Status:

new topics/pols set partial results complete validated

Results:

Polymerase Reference Property Result Context
Klenow fragment Keohavong P1989 Nucleotide Substitution Rate 0.00013 errors/bp Technique: DGGE
T4 Keohavong P1989 Nucleotide Substitution Rate 3E-06 errors/bp Technique: DGGE
Sequenase Keohavong P1989 Nucleotide Substitution Rate 3.4E-05 errors/bp Technique: DGGE
Taq pol I Keohavong P1989 Nucleotide Substitution Rate 0.00021 errors/bp Technique: DGGE

Entry validated by:

Log in to edit reference All References

Using Polbase tables:

Sorting:

Tables may be sorted by clicking on any of the column titles. A second click reverses the sort order. <Ctrl> + click on the column titles to sort by more than one column (e.g. family then name).

Filtering:

It is also possible to filter the table by typing into the search box above the table. This will instantly hide lines from the table that do not contain your search text.