Analysis of DNA polymerase activity in vitro using non-radioactive primer extension assay in an automated DNA sequencer

Although different DNA polymerases have distinct functions and substrate affinities, their general mechanism of action is similar. Thus, they can all be studied using the same technical principle, the primer extension assay employing radioactive tags. Even though fluorescence has been used routinely for many years for DNA sequencing, it has not been used in the in vitro primer extension assay. The use of fluorescence labels has obvious advantages over radioactivity, including safety, speed and ease of manipulation. In the present study, we demonstrated the potential of non-radioactive in vitro primer extension for DNA polymerase studies. By using an M13 tag in the substrate, we can use the same fluorescent M13 primer to study different substrate sequences. This technique allows quantification of the DNA polymerase activity of the Klenow fragment using different templates and under different conditions with similar sensitivity to the radioactive assay.

Nuclear matrix bound DNA polymerase-beta in mouse fibrosarcoma: effect of gamma-radiation.

Nuclear matrices isolated from the mouse fibrosarcoma tumour cells contain the eukaryotic replicative enzyme DNA polymerase-alpha and the presumptive repair enzyme DNA polymerase-beta. Exposure of tumors to various doses of gamma-radiation (1.95 to 6.5 Gy) causes a 2-fold increase in the levels of only DNA polymerase-beta in the nuclear matrix. The increase in the levels of this enzyme is not discernible if the matrices are isolated 24 hr after irradiation. The rise in the levels of the repair enzyme DNA polymerase-beta could be indicative of radiation stress response of the tumour cells and their repair ability.

Comparative evaluation of labelling intensity for detection of enterotoxigenic Escherichia coli.

Twenty four Esch. coli isolates obtained from patients of diarrhoea were tested by DNA hybridization for presence of enterotoxigenic Esch. coli (ETEC). The probe generated for this study was labelled by two different ways using the large Klenow fragment of DNA polymerase-I. It was observed that labelling by sequential harnessing of the exonuclease and polymerase activity of the enzyme was superior to extension of random hexanucleotide primers. This method besides being economic, dispenses with the critical step involved in the thermodynamics of oligoannealing and initiation of DNA synthesis.

In vitro characterization of repair synthesis initiated by T4 endonuclease V on a synthetic DNA substrate.

The size of the repair patch produced by E. coli DNA polymerase (Pol I) following the removal of a pyrimidine dimer from DNA in response to the nicking activity of T4 endonuclease (T4 endo V) was determined. A 48-bp DNA containing a pyrimidine dimer at a defined location was labelled in the damaged strand and incubated with T4 endo V and E. coli endonuclease IV. Subsequently, DNA synthesis by DNA Pol I was carried out in the presence of four dNTPs, ATP and DNA ligase. Analysis of the reaction products on a sequencing gel revealed a ladder of only 4-oligonucleotides, 1-4 nucleotides greater in length than the fragment generated by the combined nicking activities of T4 endo V and E. coli endonuclease IV. Thus we conclude that the in vitro repair patch size of T4 endo V is 4 nucleotides and that in some cases the repaired DNA is not ligated.