Tan J, Xie T, Xu Q, Xu D, Wang X
Renal Transplant Center, Shanghai First People's Hospital.
Chin Med J (Engl). 1996 Sep;109(9):720-3.
To establish a rapid genotyping for HLA-DR alleles by polymerase chain reaction with sequence-specific primers (PCR-SSP) for clinical application.
The subjects of study included 69 recipients, 43 unrelated donors and 5 cell lines. Genomic DNA was prepared from peripheral blood leukocytes by a salting-out method. Thirty primers were designed according to the HLA-DRB nucleotide sequences, and synthesized on a 391 DNA synthesizer. Twenty separate PCR reactions were performed for each sample. The amplification was accomplished by 34 cycles consisting of denaturation at 94 degrees C for 30 seconds, annealing at 60 degrees C for 50 seconds and extension at 72 degrees C for 40 seconds. The specificity of matching was determined by standard DNAs and Southern hybridization using DIG labeling probes.
All 112 samples and 5 cell lines were able to be typed by PCR-SSP. No false positive or false negative typing results were obtained. The reproducibility was 100%. The size of the specific product was in concordance with the size of the designed primers. The overall time for genotyping was 4 hours. The typing results were confirmed by Southern hybridization.
Genotyping for HLA-DR by PCR-SSP is a rapid and accurate matching technique suited for clinical application.
通过序列特异性引物聚合酶链反应（PCR-SSP）建立一种用于临床应用的HLA-DR等位基因快速基因分型方法。
研究对象包括69名受者、43名无关供者和5个细胞系。采用盐析法从外周血白细胞中提取基因组DNA。根据HLA-DRB核苷酸序列设计30条引物，并在391 DNA合成仪上合成。每个样本进行20个独立的PCR反应。扩增通过34个循环完成，包括94℃变性30秒、60℃退火50秒和72℃延伸40秒。通过标准DNA和使用地高辛标记探针的Southern杂交确定匹配的特异性。
所有112个样本和5个细胞系均能用PCR-SSP进行分型。未获得假阳性或假阴性分型结果。重复性为100%。特异性产物的大小与设计引物的大小一致。基因分型的总时间为4小时。分型结果通过Southern杂交得到证实。
PCR-SSP法进行HLA-DR基因分型是一种适用于临床应用的快速、准确的匹配技术。
