DEVELOPING HIGH RESOLUTION RFLPS FOR HUMAN GENETIC ANALYSIS

大多数人类染色体的遗传图谱已经生成 使用限制性片段长度多态性（RFLP）。 在5000人中， 在已知的RFLP中，只有1,500个多态性 在编码基因中。 这代表了50- 100，000个基因中的一小部分 被认为存在于整个基因组中。 为了 描述生物学上重要的人类基因的多态性，我们有 采用了标准方法和新方法来识别 遗传标记 这些方法包括通过DNA鉴定RFLP 杂交，单链构象表征 基因非编码部分的多态性（SSCP），以及 微卫星位点在已知基因中或附近。 多态性一直是 在色氨酸羟化酶基因（TPH）中描述，色氨酸羟化酶基因的γ 2亚基 GABA受体（GABARG 2），一种调节 载脂蛋白，酪氨酸激酶基因（RLK），转酮酶，血清素 受体（HTR 1A）和液泡H+-ATP酶基因。 这些基因中的许多 在人类多态性研究中心， 三代家庭的集合。 这些家庭受雇于 开发人类基因组的遗传图谱。 此外，我们已经开始 建立一个高信息量的标记集合， 通过聚合酶链反应。 这些标记是微卫星， 基因组中含有2-5个碱基对的重复序列的区域， 长度，高度多态性。 到目前为止，我们已经合成了引物 65个微卫星位点，并进行了测试，以确认其 多态 这些标记对于连锁作图是极其有价值的 项目，以及分析等位基因共享之间的兄弟姐妹。 一 我们正在广泛研究的一种特殊标记物， 坏死因子（TNF）基因簇。 TNF基因位于 HLA基因座在免疫功能中起着至关重要的作用。 我们还 在CD 4基因中建立了微卫星重复序列。 CD 4和TNF 在暴露于艾滋病毒的患者群体中， 与这些个体的疾病有关。

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.