Optimal conditions and specific characteristics of Vent exo- DNA polymerase in ligation-mediated polymerase chain reaction protocols.
An optimized procedure for the ligation-mediated polymerase chain reaction (PCR) technique using Thermococcus litoralis exo- DNA polymerase (Vent exo-) was developed. The optimal dosage of Vent exo- at the primer extension and PCR amplification steps as well as the optimal DNA quantity to use were established. We showed that Vent exo- can efficiently create the blunt-ended termini required for subsequent linker ligation. Vent exo- proves to be more efficient than Pyrococcus furiosus exo- (Pfu exo-) for this task. Vent exo- resolves highly GC-rich sequence substantially better than Thermus aquaticus DNA polymerase (Taq) and with a similar efficiency as Pfu exo-. The DNA/DNA polymerase activity ratio is significantly higher for Vent exo- than for Pfu exo-, which is reflected by the sensibility of Vent exo- in efficiently amplifying genomic DNA. Furthermore, the range of efficiency of Vent exo- demonstrates the importance of conducting evaluative testing to identify the optimal dosage of use of this polymerase to obtain successful PCR amplification. Optimal MgSO4 concentrations to use with Vent exo- were established. Our results show that Vent exo- DNA polymerase produces bands of uniform and strong intensity and can efficiently be used for the analysis of DNA in living cells by ligation-mediated PCR.
Vigneault
François
F
Unité de Recherche en Génétique Humaine et Moléculaire, Research Center, Hôpital St-François d'Assise, Centre Hospitalier Universitaire de Québec, QC, Canada.
Drouin
Régen
R
D004247:DNA / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D011088:DNA Ligases / Q000378:metabolism*; D017931:DNA Primers / Q000235:genetics; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D051860:Fragile X Messenger Ribonucleoprotein 1; D020862:GC Rich Sequence / Q000235:genetics; D006801:Humans; D048031:JNK Mitogen-Activated Protein Kinases / Q000235:genetics / Q000378:metabolism; D008214:Lymphocytes / Q000378:metabolism; D008297:Male; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism; D016133:Polymerase Chain Reaction; D020027:Pyrococcus furiosus / Q000201:enzymology; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D019914:Taq Polymerase / Q000378:metabolism; D013696:Temperature; D013698:Templates, Genetic; D019712:Thermococcus / Q000201:enzymology*; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism
D004247:DNA; D011088:DNA Ligases; D017931:DNA Primers; D004259:DNA-Directed DNA Polymerase; D051860:Fragile X Messenger Ribonucleoprotein 1; D048031:JNK Mitogen-Activated Protein Kinases; D009419:Nerve Tissue Proteins; D016601:RNA-Binding Proteins; D019914:Taq Polymerase; D016159:Tumor Suppressor Protein p53; C497683:FMR1 protein, human; C070331:Tli polymerase