Nucleic Acids Research, 2001, Vol. 29, No. 16 e83
© 2001 Oxford University Press
Optimal conditions to use Pfu exo– DNA polymerase for highly efficient ligation-mediated polymerase chain reaction protocols
1Unité de Recherche en Génétique Humaine et Moléculaire, Centre de Recherche, Hôpital Saint-François d’Assise, Centre Hospitalier Universitaire de Québec, 10 rue de l’Espinay, Québec, QC G1L 3L5, Canada, 2Division of Pathology, Department of Medical Biology, Faculty of Medicine, Laval University, Québec, Canada and 3Laboratory of Cancer Etiology and Chemoprevention, Faculty of Pharmacy, Laval University, Québec, Canada
Ligation-Mediated Polymerase Chain Reaction (LMPCR) is the most sensitive sequencing technique available to map single-stranded DNA breaks at the nucleotide level of resolution using genomic DNA. LMPCR has been adapted to map DNA damage and reveal DNA–protein interactions inside living cells. However, the sequence context (GC content), the global break frequency and the current combination of DNA polymerases used in LMPCR affect the quality of the results. In this study, we developed and optimized an LMPCR protocol adapted for Pyrococcus furiosus exo– DNA polymerase (Pfu exo–). The relative efficiency of Pfu exo– was compared to T7-modified DNA polymerase (Sequenase 2.0) at the primer extension step and to Thermus aquaticus DNA polymerase (Taq) at the PCR amplification step of LMPCR. At all break frequencies tested, Pfu exo– proved to be more efficient than Sequenase 2.0. During both primer extension and PCR amplification steps, the ratio of DNA molecules per unit of DNA polymerase was the main determinant of the efficiency of Pfu exo–, while the efficiency of Taq was less affected by this ratio. Substitution of NaCl for KCl in the PCR reaction buffer of Taq strikingly improved the efficiency of the DNA polymerase. Pfu exo– was clearly more efficient than Taq to specifically amplify extremely GC-rich genomic DNA sequences. Our results show that a combination of Pfu exo– at the primer extension step and Taq at the PCR amplification step is ideal for in vivo DNA analysis and DNA damage mapping using LMPCR.
* To whom correspondence should be addressed at: Unité de Recherche en Génétique Humaine et Moléculaire, Centre de Recherche, Hôpital Saint-François d’Assise, CHUQ, 10 rue de l’Espinay, Quebec, QC G1L 3L5, Canada. Tel: +1 418 525 4402; Fax: +1 418 525 4195; Email: regen.drouin{at}crsfa.ulaval.ca
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