Differential Evolution of Antiretroviral Restriction Factors in Pteropid Bats as Revealed by APOBEC3 Gene Complexity

Hayward, Joshua A; Tachedjian, Mary; Cui, Jie; Cheng, Adam Z; Johnson, Adam; Baker, Michelle L; Harris, Reuben S; Wang, Lin-Fa; Tachedjian, Gilda

All PCR amplification assays were performed using the Roche FastStart High Fidelity PCR system (Cat # 04738292001) utilizing an annealing temperature gradient of 54-64 C in 2 C increments. Each reaction was made up to a total of 20 ll, containing 1 unit of polymerase, 2 ng of total cDNA, and 8 pmol of each primer. All other parameters for PCR amplification were performed according to the manufacturer's recommended protocol. PCR reaction products were separated by agarose gel electrophoresis, using a 1% (w/v) agarose gel run at 120 V for 25 min. All DNA bands were excised from the gel and purified using the Qiagen QIAquick Gel Extraction kit (Cat # 28706) according to the manufacturer's protocol. The gel extracted PCR products were cloned into the pCR2.1 plasmid vector using the Invitrogen TOPO TA Cloning kit (Cat # 450641) according to the manufacturer's protocol and transformed into Escherichia coli TOP10 (Invitrogen). The nucleotide sequence of clones were determined by Sanger sequencing using the M13F (5 0 -GTAAAACGACGGCCAG-3 0 ) and M13R (5 0 -CAGGAAACAGCTATGAC-3 0 ) sequencing primers.