Efficient amplification of the HLA-DQA1 gene in single genomes using a semi-nested polymerase chain reaction.
A semi-nested polymerase chain reaction (PCR) was introduced to amplification of the HLA-DQA1 gene, which is a single-copy gene, in a single genome. The limitation of template DNA for genotyping is 1 ng of genomic DNA, or more in the case of ordinary PCR. When reamplification with the same primers was performed, primer dimer was generated and the sensitivity was not improved. We designed a semi-nested primer for the second round of PCR, using the semi-nested PCR, more than 3 pg of template DNA could be amplified and typed. Furthermore, this method was applied to amplify DQA1 gene in single human sperm having haploid DNA, and followed by typing with sequence-specific oligonucleotide (SSO) probes. The semi-nested PCR technique was found to enhance the sensitivity of the amplification reaction and allowed the successful typing of the HLA-DQA1 gene. This is helpful for genotyping from samples with extremely small amounts of DNA, such as forensic or ancient DNA samples.
Uchihi
R
R
Department of Legal Medicine, Nagoya University School of Medicine, Japan.
Yamamoto
T
T
Kojima
T
T
Tamaki
K
K
Katsumata
Y
Y
D001483:Base Sequence; D005784:Gene Amplification; D005802:Genes, MHC Class II; D015894:Genome, Human; D006683:HLA-DQ Antigens / Q000235:genetics*; D059848:HLA-DQ alpha-Chains; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods
D006683:HLA-DQ Antigens; D059848:HLA-DQ alpha-Chains; C069050:HLA-DQA1 antigen