ESTABLISHING A ROBUST MULTIPLEX SNP-SCORING SYSTEM

描述（改编自研究者摘要）：根据新的 人类基因组计划目标（1998-2003），100，000 - 300，000个 人类基因组中的核苷酸多态性（SNP）将在 未来五年为了利用这一宝贵的资源， 需要开发高通量SNP评分技术。尽管许多 用于PCR后分析的高通量方法是可用的， SNP评分的先决条件，即，PCR扩增，一直是一个限制性步骤 并成为高通量SNP评分的瓶颈。这是因为随着 传统的基于单位点的PCR，扩增大量的 SNP是非常费力的，并且对于许多实验室来说可能负担不起。作为 为了打破这一瓶颈，该应用程序建议选择6000个 高质量SNPs。在这些SNP中，将从数据库中选择4，000个 包含在不同人群中具有已知杂合性的SNP。的 在分析了6,000个SNP后，将选择另外2,000个SNP， 有限的信息，他们已知的杂合子在不同的人类 人口。作为副产品，所得到的信息可以作为 有效利用这些多态性的重要指导方针。6000人 高质量的SNPs将被整合到150组中，每组40个标记。 每组中的标志物将以多重方式用PCR扩增。 两轮PCR方案。来自多重扩增的PCR产物将被 可通过各种基因分型方法进行分析， 常用的碎裂工具。对于后者，限制 选择酶消化用于等位基因序列区分。因为更多 60%以上的SNPs可以通过两种4-碱基限制性内切酶进行分析， 在PCR期间具有或不具有单碱基转换的识别位点， 可以使每组中的标记物可通过单一酶分析， 通过两种酶分析所有选择的SNP。实验条件 将制定出多路复用系统，以便分析所有组 通过具有检测目标的灵敏度的单组条件 1 ng基因组DNA中的序列。

DESCRIPTION (Adapted from the Investigator's Abstract): According to the New Goals for the Human Genome Project (1998-2003), 100,000-300,000 single nucleotide polymorphisms (SNPs) in the human genome will be discovered during the next five years. To utilize this valuable resource, robust and high-throughput SNP scoring technology needs to be developed. Although many high-throughput methods for post-PCR analysis are available, a primary prerequisite for SNP scoring, i.e., PCR amplification, has been a limiting step and presents a bottleneck for high-throughput SNP scoring. This is because with the conventional single-locus-based PCR, amplification of a large number of SNPs is very laborious and may not be affordable for many laboratories. As a step toward breaking this bottleneck, this application proposes to select 6,000 high-quality SNPs. Of these SNPs, 4,000 will be chosen from the databases containing SNPs with known heterozygosities in different human populations. The other 2,000 will be chosen after analyzing 6,000 SNPs without or with very limited information about their known heterozygosities in different human populations. As a by-product, the resulting information may serve as an important guideline for efficiently utilizing these polymorphisms. The 6,000 high-quality SNPs will be incorporated into 150 groups with 40 markers in each. Markers in each group will be robustly amplified in a multiplex way with a two-round PCR protocol. PCR products from the multiplex amplification will be analyzable by various genotyping approaches ranging from DNA-arrayed chips to commonly available fragmentation instruments. For the latter, restriction enzyme digestion is chosen for allelic sequence discrimination. Because greater than 60 percent SNPs can be analyzed by two restriction enzymes of 4-base recognition sites with or without single-base conversion during PCR, it is possible to have the markers in each group analyzable by a single enzyme and to analyze all selected SNPs by as few as two enzymes. The experimental conditions for the multiplex system will be worked out so that all groups will be analyzed by a single set of conditions with a sensitivity of detecting the target sequences in 1 ng genomic DNA.