High homologous nucleotide to GBV-C was amplified from DNA of MT2 and HeLa cells and PBMC of human and chimpanzee
Abstract
Aim: To determine whether the nucleotide sequences homologous to GBV-C genome exist in the DNA from cell lines of human origin and from peripheral blood mononuclear cells (PBMC) of human and chimpanzee.
Methods: DNA of MT2 cell, HeLa cell, and PBMC from six human and one chimpanzee were prepared by using PCR Temperate Preparation Kit. All of the DNA preparations were digested with DNase-free RN ase and then were extracted by phenol-chloroform method. By using these DNA as templates, direct nested-PCR (dPCR) respectively amplified with GBV-C 5'-NCR and NS3 primers were carried out. Nucleotide sequences of the dPCR products were analyzed and positions in chromosomes of the amplification fragments were detected by using primed in situ (PRINS) sequence-specific labeling with fluorescein.
Results: DNA fragments amplified with GBV-C 5'-NCR primers were obtained from the DNA of MT2 and HeLa cells and the DNA in 4 of 6 human PBMC samples. DNA fragments amplified with GBV-C NS3 primers were obtained from MT2 and HeLa cells and the DNA in 5 of 6 human PBMC samples and one chimpanzee PBMC sample. The homology of the nucleotide sequences from these amplification products compared with the reported GBV-C genome sequence was from 73.80 % to 79.15 %. Fluorescence staining spots by using PRINS were shown in the PBMC and their chromosomes with positive dPCR results.
Conclusion: The nucleotide sequences with high homology to GBV-C genome at the 5'-NCR and/or NS3 region exist in the DNA of MT2 and HeLa cells and the PBMC DNA of human and chimpanzee. These sequences locate in the chromosomes of PBMC with positive dPCR results.
Keywords:
Methods: DNA of MT2 cell, HeLa cell, and PBMC from six human and one chimpanzee were prepared by using PCR Temperate Preparation Kit. All of the DNA preparations were digested with DNase-free RN ase and then were extracted by phenol-chloroform method. By using these DNA as templates, direct nested-PCR (dPCR) respectively amplified with GBV-C 5'-NCR and NS3 primers were carried out. Nucleotide sequences of the dPCR products were analyzed and positions in chromosomes of the amplification fragments were detected by using primed in situ (PRINS) sequence-specific labeling with fluorescein.
Results: DNA fragments amplified with GBV-C 5'-NCR primers were obtained from the DNA of MT2 and HeLa cells and the DNA in 4 of 6 human PBMC samples. DNA fragments amplified with GBV-C NS3 primers were obtained from MT2 and HeLa cells and the DNA in 5 of 6 human PBMC samples and one chimpanzee PBMC sample. The homology of the nucleotide sequences from these amplification products compared with the reported GBV-C genome sequence was from 73.80 % to 79.15 %. Fluorescence staining spots by using PRINS were shown in the PBMC and their chromosomes with positive dPCR results.
Conclusion: The nucleotide sequences with high homology to GBV-C genome at the 5'-NCR and/or NS3 region exist in the DNA of MT2 and HeLa cells and the PBMC DNA of human and chimpanzee. These sequences locate in the chromosomes of PBMC with positive dPCR results.