Optimal conditions to use Pfu exo(-) DNA polymerase for highly efficient ligation-mediated polymerase chain reaction protocols

Ligation-Mediated Polymerase Chain Reaction (LMPCR) is the most sensitive s equencing technique available to map single-stranded DNA breaks at the nucl eotide level of resolution using genomic DNA. LMPCR has been adapted to map DNA damage and reveal DNA-protein interactions inside living cells. Howeve r, the sequence context (GC content), the global break frequency and the cu rrent combination of DNA polymerases used in LMPCR affect the quality of th e results. In this study, we developed and optimized an LMPCR protocol adap ted for Pyrococcus furiosus exo(-) DNA polymerase (Pfu exo(-)). The relativ e efficiency of Pfu exo(-) was compared to T7-modified DNA polymerase (Sequ enase 2.0) at the primer extension step and to Thermus aquaticus DNA polyme rase (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 molecul es 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. S ubstitution of NaCl for KCl in the PCR reaction buffer of Taq strikingly im proved the efficiency of the DNA polymerase. Pfu exo(-) was clearly more ef ficient than Taq to specifically amplify extremely GC-rich genomic DNA sequ ences. Our results show that a combination of Pfu exo(-) at the primer exte nsion step and Taq at the POR amplification step is ideal for in vivo DNA a nalysis and DNA damage mapping using LMPCR.