A plasmid-based lacZα gene assay for DNA polymerase fidelity measurement.

Abstract:

A significantly improved DNA polymerase fidelity assay, based on a gapped plasmid containing the lacZα reporter gene in a single-stranded region, is described. Nicking at two sites flanking lacZα, and removing the excised strand by thermocycling in the presence of complementary competitor DNA, is used to generate the gap. Simple methods are presented for preparing the single-stranded competitor. The gapped plasmid can be purified, in high amounts and in a very pure state, using benzoylated-naphthoylated DEAE-cellulose, resulting in a low background mutation frequency (~1 × 10(-4)). Two key parameters, the number of detectable sites and the expression frequency, necessary for measuring polymerase error rates have been determined. DNA polymerase fidelity is measured by gap filling in vitro, followed by transformation into Escherichia coli and scoring of blue/white colonies and converting the ratio to error rate. Several DNA polymerases have been used to fully validate this straightforward and highly sensitive system.

Polymerases:

Topics:

Fidelity

Status:

new topics/pols set partial results complete validated

Results:

Polymerase Reference Property Result Context
Pfu Keith BJ2013 Overall Error Rate 1.6E-06 errors/bp Technique: Blue/white screening (pSJ2)
Pfu Keith BJ2013 Overall Error Rate 3.5E-06 errors/bp Technique: Blue/white screening (pSJ3)
Pfu D215AE143A Keith BJ2013 Overall Error Rate 6.3E-06 errors/bp Technique: Blue/white screening (pSJ2)
Pfu D215AE143A Keith BJ2013 Overall Error Rate 6.7E-06 errors/bp
Taq pol I Keith BJ2013 Overall Error Rate 1E-05 errors/bp Technique: Blue/white screening (pSJ2)
Taq pol I Keith BJ2013 Overall Error Rate 1.1E-05 errors/bp
Pfu D215AE143A/D473G Keith BJ2013 Overall Error Rate 1.8E-05 errors/bp Technique: Blue/white screening (pSJ2)
Pfu D215AE143A/D473G Keith BJ2013 Overall Error Rate 2.4E-05 errors/bp

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.