DNA typing for SLA-DRB1 by PCR using sequence-specific primers (PCR-SSP)
1997
Kawakami, K. (Society for Techno-Innovation of Agriculture, Forestry and Fisheries, Tokyo (Japan)) | Takeda, K. | Onishi, A. | Nakajima, E. | Komatsu, M.
We reported a method based on PCR amplification with sequence-specific primers (PCR-SSP) fro SLA-typing, and new method for sizing of amplified fragments on the basis of difference of fluorescent dyes using automated DNA fragment analyzer. PCR-SSP method, sequence specific primers amplified only the complementary target allele. Identification of the allele is based on the presence or absence of amplified products observed after electrophoresis. We amplified of the first domain in the second exon of swine DRB 1 region of several pig breeds by PCR and determined the nucleotide sequences. We have designed several sets of primers with or without fluorescent label which will positively identify the sharing of sequence motifs between alleles. The products amplified with non-labeled primer were detected by agarose gel electrophoresis, and those with labeled primer were visualized by automated fluorescence detection on the ABI DNA Sequencer and analyzed with Genescan Analysis software. DNA samples from 30 DRB 1 clones and cDNA from 3 individuals have been typed by the PCR-SSP technique. PCR-SSP results obtained using non-labeled and labeled primer correlated well with each other. No false-positive or negative typing results were obtained. We have very promising preliminary data using this typing strategy for SLA-DRB 1 alleles. PCR-SSP typing method wee used for many of the primer pairs in the SLA-DRB region. Due to the sharing of sequence motifs between alleles, this typing will not be truly allele-specific but rather be group-specific
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