Okayama Naoko, Fujimura Kozue, Suehiro Yutaka, Hamanaka Yuichiro, Fujiwara Motoki, Matsubara Tomoyo, Maekawa Tsuyoshi, Hazama Shoichi, Oka Masaaki, Nohara Hiroaki, Kayano Kozo, Okita Kiwamu, Hinoda Yuji
Division of Clinical Laboratory, Yamaguchi University Hospital, Yamaguchi University School of Medicine, Yamaguchi, Japan.
J Clin Lab Anal. 2002;16(1):56-8. doi: 10.1002/jcla.2075.
A nonsynonymous single nucleotide polymorphism (Asp299Gly) in the Toll-like receptor-4 (TLR-4) gene affects the responsiveness to lipopolysaccharide in humans. To analyze this important polymorphism more efficiently, we developed a simple polymerase chain reaction (PCR) restriction length fragment polymorphism (RFLP) assay and examined the Asp299Gly allele frequency in a Japanese population. The PCR primer was designed with 1- or 2-bp mismatches, creating the recognition sequence for restriction enzyme BsaBI or BstXI, allowing RFLP analysis of the digested products. Genotyping was carried out with this assay for 275 DNA specimens from 107 healthy volunteers and 168 patients with various diseases, including ulcerative colitis (n = 86). The Asp299Gly allele of the TLR-4 gene was not detected in any of the specimens, suggesting that it is very rare in Japanese.
Toll样受体4（TLR-4）基因中的一个非同义单核苷酸多态性（Asp299Gly）影响人类对脂多糖的反应性。为了更有效地分析这一重要多态性，我们开发了一种简单的聚合酶链反应（PCR）-限制性片段长度多态性（RFLP）检测方法，并检测了日本人群中Asp299Gly等位基因频率。PCR引物设计有1或2个碱基错配，产生限制性酶BsaBI或BstXI的识别序列，从而允许对消化产物进行RFLP分析。使用该检测方法对来自107名健康志愿者和168名患有各种疾病（包括溃疡性结肠炎，n = 86）的患者的275个DNA样本进行基因分型。在任何样本中均未检测到TLR-4基因的Asp299Gly等位基因，这表明该等位基因在日本人中非常罕见。
