DEVELOPING HIGH RESOLUTION RFLPS FOR HUMAN GENETIC ANALYSIS

大多数人类染色体的遗传图谱已经绘制完成 使用限制性片段长度多态(RFLP)。在5000人中 已经描述的已知RFLP，只有1500个多态 在编码基因方面。这只是50-100,000个基因中的一小部分 它们被认为是在整个基因组中被编码的。为了 描述生物上重要的人类基因的多态，我们有 采用了标准方法和新方法来确定 遗传标记。这些方法包括用DNA鉴定RFLP 单链构象的杂交、表征 基因非编码部分的多态(SSCP)，以及使用 已知基因及其附近的微卫星定位。多态现象一直是 在色氨酸羟基酶基因(TPH)中描述的是 GABA受体(GABARG2)，一种调节 载脂蛋白，酪氨酸激酶基因(RLK)，转酮醇酶，5-羟色胺 受体(HTR1a)和液泡H+-ATPase基因。这些基因中的许多 人类多态中心的标志物已被分型 三代家族的集合。这些家庭受雇于 开发一张人类基因组的遗传图谱。此外，我们已经开始 建立一组可打字的高信息量标记 通过聚合酶链式反应。这些标记是微卫星， 基因组中包含2-5个碱基对的重复序列的区域 长度，这是高度多态的。到目前为止，我们已经合成了引物 65个微卫星座位，并对它们进行了测试，以确认它们的 多态。这些标记对连锁作图非常有价值 项目，以及兄弟姐妹之间等位基因共享的分析。一 我们正在广泛研究的特定标记物是在肿瘤中 肿瘤坏死因子基因簇。肿瘤坏死因子基因位于 在免疫功能中起着至关重要的作用。我们还有 在CD4基因中建立了一个微卫星重复序列。外周血中的CD4和肿瘤坏死因子 在暴露于艾滋病毒的人群中进行基因多态分型 与这些人的疾病有关。

The genetic maps for most of the human chromosomes have been generated using restriction fragment length polymorphisms (RFLPs). Of the 5,000 known RFLPs that have been described, there are only 1,500 polymorphisms in coding genes. This represents a small fraction of the 50-100,000 genes that are believed to be encoded in the entire genome. In order to characterize polymorphisms in biologically important human genes, we have employed both standard approaches, as well as new methods to identify genetic markers. These methods include identification of RFLPs by DNA hybridization, characterization of single-stranded conformation polymorphisms (SSCPs) in non-coding portions of genes, and the use of microsatel-lite loci in and near known genes. Polymorphisms have been described in the tryptophan hydroxylase gene (TPH), the gamma 2 subunit of the GABA receptor (GABARG2), a transcription factor gene that regulates apolipoproteins, a tyrosine kinase gene (RLK), transketolase, a serotonin receptor (HTR1A), and the vacuolar H+-ATPase gene. Many of these genetic markers have been typed in the Centre D'Etude du Polymorphisme Humain collection of three generation families. These families are employed in developing a genetic map of the human genome. In addition, we have begun to establish a collection of highly informative markers that can be typed by the polymerase chain reaction. These markers are microsatellites, regions of the genome that contain repetitive sequences 2-5 base pairs in length, that are highly polymorphic. To date, we have synthesized primers for 65 microsatellite loci and have tested them to confirm their polymorphism. These markers are extremely valuable for linkage mapping projects, as well as analysis of allele sharing amongst siblings. One particular marker that we are extensively studying is in the tumor necrosis factor (TNF) gene cluster. The TNF genes are located within the HLA locus and play a crucial role in immune function. We have also established a microsatellite repeat in the CD4 gene. Both the CD4 and TNF polymorphisms are being typed in groups of patients exposed to HIV to look for associations with disease in these individuals.