Lasergene megalign program

PCR primer pairs specific for each gene of the eight-gene hemO cluster35 (link) were designed based on alignment of gene cluster sequences of six A. baumannii strains [strains ACICU21 (link), SDF22 (link), MDR-ZJ0625 (link), TYTH-127 (link), TCDC-AB0711526 (link), and AB005718 (link)], available as of May, 2013. The sequence alignment was performed by using Lasergene MegAlign program from DNASTAR, Inc. (Madison, WI). Primers were chosen from regions of highly conserved sequences and are shown in Supplementary Table S5 online. The PCR amplification of the hemO gene cluster was performed for the clinical isolates using genomic DNA as a template. Individual PCR reactions involved a 50 μl reaction mixture containing 1 × 5 PRIME MasterMix, 200 nM of forward and reverse primers, and 1 μl of genomic DNA template. Amplification was carried out with 5 min at 95°C; 36 cycles of 30 s at 95°C, 30 s at 55°C, and 1 min at 72°C; and 10 min at 72°C. The resulting PCR products were visualized and imaged through agarose gel electrophoresis analysis.

PCR products from both control and HCC-induced groups were sequenced. All loci were directly sequenced in both forward and reverse directions using the same primers used for PCR amplification by dye terminator cycle sequencing technique. Sequence alignment of the resulted sequences with the corresponding functional p53 gene sequence available in GenBank to locate sequence variants in DNA and protein sequences were done by the CLUSTAL W method using the Lasergene MegAlign program (version 15.1, DNASTAR Inc.).