ESTABLISHING A ROBUST MULTIPLEX SNP-SCORING SYSTEM

描述(改编自《调查者摘要》)：根据The New 人类基因组计划的目标(1998-2003年)，100,000-300,000个 人类基因组中的核苷酸多态(SNPs)将在 接下来的五年。要利用这一宝贵的资源，强大和 需要开发高通量的SNP评分技术。虽然很多人 高通量的后PCR分析方法是可用的，主要的 SNP评分的先决条件，即聚合酶链式反应扩增，一直是一个限制步骤 并提出了高吞吐量SNP评分的瓶颈。这是因为随着 常规的单基因座为基础的聚合酶链式反应，扩增出大量的 SNPs非常费力，对许多实验室来说可能负担不起。作为一名 为了打破这一瓶颈，该应用程序建议选择6,000 高质量的SNPs。在这些SNP中，将从数据库中选择4000个 在不同的人群中含有已知杂合性的SNPs。这个 其他2,000个将在分析了6,000个SNP后选择，无论是非常 关于它们在不同人类中的已知杂合性的有限信息 人口。作为副产品，产生的信息可能会作为 有效利用这些多态的重要指导原则。6,000人 优质SNPs将被分成150组，每组40个标记。 每组中的标记将以多路复用的方式进行强有力的扩增 两轮聚合酶链式反应。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，从DNA阵列芯片到 常用的碎裂仪器。对于后者，限制 等位基因序列的识别采用酶消化法。因为更大的 两种4碱基限制性内切酶可分析60%以上的SNPs 在聚合酶链式反应中有或没有单碱基转换的识别位点，它是 有可能使每组中的标记可由单个酶分析，并 只需两种酶就可以分析所有选定的SNPs。实验条件 因为将制定多路传输系统，以便对所有组进行分析 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.