LaForge K S, Shick V, Spangler R, Proudnikov D, Yuferov V, Lysov Y, Mirzabekov A, Kreek M J
Laboratory of the Biology of Addictive Diseases, The Rockefeller University, New York, New York 10021, USA.
Am J Med Genet. 2000 Oct 9;96(5):604-15. doi: 10.1002/1096-8628(20001009)96:5<604::aid-ajmg5>3.0.co;2-f.
The human mu opioid receptor (MOR) plays a central role in mediating the effects of opioids, both endogenous and exogenous. Epidemiological studies have shown that addiction in general, and especially opiate addiction, has a heritable component. Clinical and laboratory studies suggest that the MOR gene may contribute to the heritable component of vulnerability to develop opiate addiction. Naturally occurring single nucleotide polymorphisms (SNPs) have been identified in the MOR gene by conventional methods. Two coding region SNPs, the A118G and C17T substitutions, occur at high allelic frequencies (10.5% and 6.6%, respectively, in our previous studies). These common SNPs cause amino acid changes in the receptor, and may have implications for differences in individual responses to opioids, as well as decreased or increased vulnerability to opiate addiction. The A118G substitution encodes a variant receptor with binding and signal transduction differences in response to beta-endorphin in cellular assays. Recent innovations in microchip technology offer new potential methods for SNP detection. We report here on the development of two separate approaches using custom oligonucleotide gelpad microarrays for detection of these two common SNPs of the MOR gene in human DNA samples. First, PCR-amplified genomic DNA samples were used to produce target sequences, which were labeled with fluorescent dye and hybridized to custom microchips. Oligonucleotides on these reusable microchips were designed to query nucleotide substitutions at positions 17 and 118 of the MOR gene. Thirty-six human DNA samples were assayed both on these custom microchips and by conventional automated gel sequencing, with highly concordant identification of both heterozygous and homozygous substitutions. A second approach was developed for the C17T SNP utilizing single nucleotide extension on custom microchips. These custom gelpad microchips have potential for the rapid and inexpensive detection of specific SNPs for genetic and genomic studies.
人类μ阿片受体（MOR）在介导内源性和外源性阿片类药物的作用中起着核心作用。流行病学研究表明，一般成瘾，尤其是阿片类成瘾，具有遗传成分。临床和实验室研究表明，MOR基因可能是导致阿片类成瘾易感性遗传成分的原因。通过传统方法已在MOR基因中鉴定出自然发生的单核苷酸多态性（SNP）。两个编码区SNP，即A118G和C17T替换，以高等位基因频率出现（在我们之前的研究中分别为10.5%和6.6%）。这些常见的SNP会导致受体中的氨基酸变化，可能对个体对阿片类药物反应的差异以及阿片类成瘾易感性的降低或增加产生影响。A118G替换编码一种变体受体，在细胞试验中对β-内啡肽的结合和信号转导存在差异。微芯片技术的最新创新为SNP检测提供了新的潜在方法。我们在此报告使用定制寡核苷酸凝胶垫微阵列检测人类DNA样本中MOR基因这两个常见SNP的两种不同方法的开发。首先，使用PCR扩增的基因组DNA样本产生靶序列，用荧光染料标记并与定制微芯片杂交。这些可重复使用微芯片上的寡核苷酸被设计用于查询MOR基因第17和118位的核苷酸替换。对36个人类DNA样本在这些定制微芯片上以及通过传统的自动凝胶测序进行检测，对杂合和纯合替换的鉴定高度一致。针对C17T SNP开发了第二种方法，利用定制微芯片上的单核苷酸延伸。这些定制凝胶垫微芯片具有快速、廉价地检测特定SNP用于遗传和基因组研究的潜力。
