PRODUCTION OF THERMOSTABLE DNA POLYMERASE SUITABLE FOR WHOLE-BLOOD POLYMERASE CHAIN REACTION - Biochemistry and Biotechnology: Research and Development - page 4

DNA polymerase, blood of healthy adults was supplied into reaction mixture at various
concentrations (1-20%, v/v). For amplification of FV gene segment containing nucleotide
1691, synthetic primers (F 5'-TGCCCAGTGCTTAACAAGACCA-3' и R 5'-TGTTATCACA
CTGGTGCTAA-3') were applied [11]. The obtained data were analyzed using agarose gel
electrophoresis (Figure 1).
Figure 1. Electrophoregram of amplified FV gene segment containing nucleotide 1691. M, DNA
molecular size markers; K+, PCR product of FV gene segment using 10 ng of purified DNA; 1-20%,
whole blood concentration in the sample.
It is evident from electrophoregram data that the PCR product has the size approximately
equal to 267 bp corresponding to theoretical estimates. It may be concluded therefore that
SSO7D-Klen Taq DNA polymerase displays high enzyme activity even in the presence of
elevated concentrations of whole blood (up to 20%, v/v). Presence of single point mutation
was checked upon treatment of amplified samples with restriction endonuclease Mnl I. It was
shown earlier that restriction of wild-type allele generates products 67, 37 and 163 bp long
whereas the enzymatic cleavage of mutant allele yields fragments of 67 and 200 bp size.
Analysis of derived fragments was performed by agarose gel electrophoresis (Figure 2).
Figure 2. Electrophoregram of restriction fragment length polymorphism analysis of amplified FV gene
segment containing nucleotide 1691. Line M is DNA molecular size markers; line 1 is the untreated
segment; lines 2 and 3 are Mnl I restriction fragments of FV gene segments from two individuals.
The electrophoregram shows that samples from both individuals contain DNA fragments
sized 37, 67 and 163 bp matching wild-type allele of the gene encoding FV. As a result such
patients are homozygous for wild-type allele of FV. Summing up, it was demonstrated that