Takara la taq hot start polymerase

A single-molecule PCR approach was used to identify specific breakpoints or ends of mtDNA^{8 (link)}. Briefly, single mtDNA molecules were amplified in 42 cycles of PCR using TaKaRa LA Taq Hot Start polymerase (Clontech; for large products when detecting breakpoints) or Ranger DNA polymerase (Bioline; for short products when detecting ends). Total template DNA was diluted to a grade at which only a part of multiple identical reactions resulted in amplification products (ideally less than 50%). Under these conditions, most of the positive reactions are likely to originate from a single mtDNA molecule. Amplification products representing single linker-ligated mtDNA ends were directly sequenced using an mtDNA-specific primer. Deletion breakpoints were mapped by re-amplifying single-deletion amplicons using diverse primer pairs located within the amplified region and direct sequencing of re-amplified products.

Total DNA from left ventricular muscle was isolated through column purification with QIAamp DNA Mini Kit (QIAGEN N.V., Venlo, Netherlands) as described by the manufacturer. Each sample was eluted in 200 μl elution buffer provided with the kit and stored without freezing at 4°C. LR-PCR was used in order to detect mtDNA deletions. For that purpose, almost the entire mtDNA was amplified between primers musMT2482F (5’-GTTCAACGATTAAAGTCCTACGTG-3’) and musMT1005R (5’-CCAGTATGCTTACCTTGTTACGAC-3’) (first number, 5’-end of the primer; F-forward or R-reverse) by the use of TaKaRa LA Taq Hot Start polymerase (Clontech). The LR-PCR was performed under the following conditions: 95°C for 2.5 min, 30 cycles of 92°C for 20 s and 66.8°C for 5:30 min, and 72°C for 10 min. The PCR products were loaded on a 1% agarose gel with Quick-Load 1 kb Extend DNA Ladder (New England Biolabs, NEB).