Wu D Y, Ugozzoli L, Pal B K, Wallace R B
Department of Molecular Biochemistry, Beckman Research Institute of the City of Hope, Duarte, CA 91010.
Proc Natl Acad Sci U S A. 1989 Apr;86(8):2757-60. doi: 10.1073/pnas.86.8.2757.
A rapid nonradioactive approach to the diagnosis of sickle cell anemia is described based on an allele-specific polymerase chain reaction (ASPCR). This method allows direct detection of the normal or the sickle cell beta-globin allele in genomic DNA without additional steps of probe hybridization, ligation, or restriction enzyme cleavage. Two allele-specific oligonucleotide primers, one specific for the sickle cell allele and one specific for the normal allele, together with another primer complementary to both alleles were used in the polymerase chain reaction with genomic DNA templates. The allele-specific primers differed from each other in their terminal 3' nucleotide. Under the proper annealing temperature and polymerase chain reaction conditions, these primers only directed amplification on their complementary allele. In a single blind study of DNA samples from 12 individuals, this method correctly and unambiguously allowed for the determination of the genotypes with no false negatives or positives. If ASPCR is able to discriminate all allelic variation (both transition and transversion mutations), this method has the potential to be a powerful approach for genetic disease diagnosis, carrier screening, HLA typing, human gene mapping, forensics, and paternity testing.
描述了一种基于等位基因特异性聚合酶链反应（ASPCR）的快速非放射性诊断镰状细胞贫血的方法。该方法可直接检测基因组DNA中的正常或镰状细胞β-珠蛋白等位基因，无需额外的探针杂交、连接或限制性酶切步骤。在与基因组DNA模板进行的聚合酶链反应中，使用了两种等位基因特异性寡核苷酸引物，一种针对镰状细胞等位基因，一种针对正常等位基因，以及另一种与两种等位基因都互补的引物。等位基因特异性引物在其3'末端核苷酸上彼此不同。在适当的退火温度和聚合酶链反应条件下，这些引物仅在其互补等位基因上引导扩增。在对12名个体的DNA样本进行的单盲研究中，该方法正确且明确地确定了基因型，无假阴性或假阳性。如果ASPCR能够区分所有等位基因变异（转换和颠换突变），则该方法有可能成为遗传疾病诊断、携带者筛查、HLA分型、人类基因图谱绘制、法医学和亲子鉴定的有力方法。
