[Application of polymerase chain reaction in amplification of human cytomegalovirus B gene code region]

A nucleic acid amplification procedure, the polymerase chain reaction (PCR), has been used to amplify the human cytomegalovirus (HCMV) B gene code region, and its glycoprotein 52 kd antigenic domain. The primers used in the PCR assay were based on a conserved region of the HCMV B gene sequence. By using primer 1 (5'-AAAGAATTCATGGAATCCAGGATCTG-3', upstream nucleotides 157 to 2877), primer 2 (5'-AAAGAATTCATGAACGTGAAGGAATCG-3', upstream nucleotides 1846 to 2877), and primer 4(5'-ATAAAGCTTAATCAGACGTTCTCTTCTTC-3', downstream nucleotides 157 to 2877 and 1846 to 2877), the HCMV B gene code region sequence and its glycoprotein 52 kd antigenic domain sequence were amplified from the recombinant plasmid pBH1 DNA containing the HCMV B gene. Amplification cycles consisted of denaturation at 92 degrees C for 1 min, annealing at 55 degrees C for 1 min, and extension at 72 degrees C for 1.5 min. The cycles were carried out 30 times in three different water baths, respectively. After the last extension, 10 microliter of each reaction mixture was removed and subjected to electrophoresis on 1.2% agarose gel. Ten microgram of PCR products were obtained from 0.2 microgram of template DNA after the 30 times cycles, and were enough for being used in the restriction site analysis and the construction of clones.