Okayama Naoko, Fujimura Kozue, Nakamura Junji, Suehiro Yutaka, Hamanaka Yuichiro, Hinoda Yuji
Department of Clinical Laboratory Science, Yamaguchi University School of Medicine, 1-1-1, Minami-Kogushi, Ube, Yamaguchi 755-8505, Japan.
Clin Chem Lab Med. 2004 Jan;42(1):13-6. doi: 10.1515/CCLM.2004.004.
Tetra-primer amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) is a new efficient method for single-nucleotide polymorphism (SNP) genotyping. To determine the optimal conditions for ARMS-PCR we attempted to genotype ten SNPs. DNA was extracted from the peripheral blood of 168 unrelated healthy Japanese volunteers. Two problems inhibited uniform efficiency of the amplification of three bands. The first problem was the lower amplification efficiency of the shorter and allele-specific products compared with the largest product. This phenomenon was overcome by increasing the relative concentration of the inner primers. The second problem was non-specific amplification of the shorter products. To reduce the amplification of these non-specific bands, adjusting any one of the following PCR conditions was effective: i) reducing the ratio of the inner primer concentration relative to that of the outer primers; ii) increasing the annealing temperature for the initial 5-10 cycles; iii) hot start PCR. With these procedures all ten of the SNPs were successfully genotyped. Our present data may be useful in the further application of tetra-primer ARMS-PCR to SNP genotyping.
四引物扩增阻滞突变系统-聚合酶链反应（ARMS-PCR）是一种用于单核苷酸多态性（SNP）基因分型的新型高效方法。为确定ARMS-PCR的最佳条件，我们尝试对10个SNP进行基因分型。从168名无亲缘关系的健康日本志愿者的外周血中提取DNA。有两个问题影响了三条带扩增效率的一致性。第一个问题是与最大产物相比，较短的等位基因特异性产物扩增效率较低。通过增加内引物的相对浓度克服了这一现象。第二个问题是较短产物的非特异性扩增。为减少这些非特异性条带的扩增，调整以下任何一个PCR条件均有效：i）降低内引物浓度与外引物浓度的比例；ii）提高最初5-10个循环的退火温度；iii）热启动PCR。通过这些方法，所有10个SNP均成功进行了基因分型。我们目前的数据可能有助于四引物ARMS-PCR在SNP基因分型中的进一步应用。
