High Throughput Detection & Genotyping For SNPs

高吞吐量SNP识别。LNG已经在包括酒精中毒在内的50多个神经遗传候选基因中发现了100多个单核苷酸多态(SNPs)。在LNG发现的SNP中，大约有20%会导致非同义氨基酸的变化，这些变化可能会改变编码蛋白质的功能或基因表达的变化。检测到的序列变异的意义是深远的：例如，我们发现的一种罕见的5-羟色胺转运体变异被证明是有功能的，并被发现导致严重的病理：阿斯伯格综合征、治疗难治性强迫症和神经性厌食症，在两个分离的家庭中。例如，常见的HTR2C Ser23Cys和HTR2AAsn452His等位基因已被证明具有功能性，并与精神分裂症患者的氯氮平反应有关。根据酒精、治疗反应、在全基因组或候选连锁或关联研究中的鉴定以及基因组序列数据的可用性，选择用于SNP筛查的基因。由于该项目的目标是确定SNPs在复杂遗传疾病中的作用，我们将重点放在候选基因的蛋白质编码部分以及蛋白质编码区内和两侧可能的调节区上。我们使用变性高效液相色谱(DHPLC)来筛选由477个基因组DNA组成的DNA小组，该小组可以检测到150-450BP大小的DNA双链中90%以上的序列变异，从而丰富了临床和种族多样性。筛选小组的规模和多样性至关重要。例如，仅在两个家系中发现了罕见的5-羟色胺运输错义替换，但具有该替换的个体存在严重的行为问题。除了一例外，他们患有阿斯伯格综合症、严重的治疗抵抗型强迫症或神经性厌食症。在深入研究HTTLPT 5-羟色胺启动子的多态性中，我们发现了一种新的、常见的、改变功能的SNP等位基因，并利用这一信息在两个群体中将5-羟色胺转运体基因与强迫症联系起来。构建筛选小组是为了准确估计我们主要连锁数据集中每个新SNP的等位基因频率。筛查数据集由土著美国人、非裔美国人、高加索人和亚洲人组成，丰富了包括酒精中毒、强迫症、精神分裂症、饮食障碍和躁郁症在内的各种精神疾病。使用基于凝胶的、半自动的或基于毛细管的、全自动的DNA测序方法对从初级筛选方法获得的序列变体进行表征和确认。对使用准确、高通量和低成本方法进行SNP检测的持续需求促使我们开发其他方法。我们的实验方法利用了双链DNA分子之间的热熔化差异，这些双链DNA分子携带完全序列匹配或单核苷酸不匹配，就像在特定SNP的杂合子个体中发现的那样。对SNP杂合子个体的DNA混合物中的链进行变性和重新退火会包含最多50%的重新退火的DNA作为完全匹配的序列，而至少50%的重新退火的DNA将具有单个碱基不匹配。单碱基错配的DNA双链的热熔融特性比完全匹配的双链DNA更不稳定。用Perkin Elmer 7700序列检测器监测双链DNA熔融过程中荧光信号的变化。利用这台特殊的仪器，可以进行热变性实验，并同时分析96个样本的DNA熔化数据。我们将原始的荧光读数转换成一阶导数图，生成每个DNA双链的熔化曲线。利用这些熔融图谱，我们能够检测到大小为100-150个碱基的双链DNA中的单碱基错配。这种方法非常适合大规模检测新的序列变体。高通量基因分型。高通量基因分型程序的核心问题是准确性、灵活性和成本。由于其分析设计的灵活性、低错误率和同时进行96到384个分析的潜力，我们选择了使用在LNG中发现的SNPs或其他实验室描述的SNPs的5‘核酸酶分析方法来确定我们研究人群的等位基因频率，我们使用这种方法进行了大约1,000次个体分析。基因分型完成率为95%，从重复样本计算的错误率不到0.5%。目前，每次化验的试剂成本为0.20-0.30美元。我们已经过渡到SNPlex，这是一种利用DNA连接的高通量、多重基因分型方法，在ABI毛细管测序仪上进行检测，并使用机器人进行DNA准备。我们用这种方法进行了大约6,000种分析，其中包括一个400个座位的基因组控制面板，有效地在病例/对照研究中检测种族分层。

HIGH THROUGHPUT SNP IDENTIFICATION. LNG has discovered over 100 single nucleotide polymorphisms (SNPs) in more than 50 neuro-genetic candidate genes for addictive behaviors, including alcoholism. Approximately 20% of the SNPs discovered by LNG result in non-synonymous amino acid changes that may alter function of the encoded protein or alterations in expression of the gene. The implications of detected serwuence variation have been profound: for example a rare serotonin transporter variant we found was shown to be functional and discovered to lead to severe pathology: Asperger's syndrome, treatment resistant OCD, and anorexia nervosa, in two families in which it is segregating. For example, the common HTR2C Ser23Cys and HTR2A Asn452His alleles have been shown to be functional and linked to the clozapine responsiveness of schizophrenics. Genes for SNP screening were selected on the basis of postulated roles in alcohol, treatment response, identification in whole genome or candidate linkage or association study, and the availability of genomic sequence data. Because a goal of the project is to determine the role of SNPs in complex genetic disorders, we focused our screening in efforts on protein coding portions of the candidate gene and possible regulatory regions within and flanking the protein coding regions. We used denaturing high performance liquid chromatography (dHPLC), which detects greater than 90% of sequence variants in DNA duplexes of 150-450 bp in size, to screen a DNA panel composed of 477 genomic DNAs enriched for clinical and ethnic diversity. Size and diversity of the screening panel are critical. For example, a rare serotonin transport missense substitution was found in only two families, however individuals with the substitution have severe behavioral problems. Except in one case, they have Asperger's Syndrome, severe treatment resistant OCD, or anorexia nervosa. Within the intensively studied HTTLPT serotonin promoter polymorphism we discovered a new, common, SNP allele that alters function and used this information to link the serotonin transporter gene to OCD in two populations. The screening panel was constructed in order to achieve an accurate estimation of allele frequencies for each novel SNP within our primary linkage data sets. The screening dataset is composed of native American, African American, Caucasian and Asian populations and is enriched for various psychiatric diseases including alcoholism, OCD, schizophrenia, eating disorders, and bipolar disorder. Sequence variants obtained from the primary screening method were characterized and confirmed using gel-based, semi-automated, or capillary-based, fully automated, DNA sequencing methods. The continuing requirement for SNP detection using accurate, high throughput, and lower cost methods motivated us to develop other methods. Our experimental approach took advantage of thermal melting differences between double stranded DNA molecules that carry either a complete sequence match or a single nucleotide mismatch, as found in heterozygous individuals for a particular SNP. Denaturation and re-annealing of the strands in a DNA mixture from an individual that is heterozygous for a SNP will contain at most 50% of the re-annealed DNAs as perfectly matched sequences, while at least 50% of the re-annealed DNAs will have a single base mismatch. The thermal melting characteristics of DNA duplexes with a single base mismatch are inherently less stable than perfectly matched double stranded DNAs. A Perkin Elmer 7700 Sequence Detector was used to monitor the change in fluorescence signal over the course of double strand DNA melting. With this particular instrument, it was possible to perform thermal denaturation experiments & analyze the DNA melting data in 96 samples at one time. We transformed the raw fluorescence readings into first derivative plots that produced a melting profile for each DNA duplex. With thesse melting profiles, we were able to detect single-base mismatches in double stranded DNAs 100-150 bp in size. This approach is highly suited for large-scale detection of new sequence variants. HIGH THROUGHPUT GENOTYPING. Central issues in high throughput genotyping procedures are accuracy, flexibility, and cost. Because of its assay design flexibility, low error rate, and potential for performing 96 to 384 assays simultaneously, we selected the 5' nuclease assay using SNPs either discovered in the LNG or SNPs that have been described by other laboratories for allele frequency determinations in our study populations, and we have some 1,000 invidual assays with this method. Genotyping completion rates are >95% and error rates that have been calculated from replicate samples are less than 0.5%. Reagent costs are currently $0.20-$0.30 per assay. We have transitioned to SNPlex, a higher-throughput, multiplex genotyping method utilizing DNA ligation, with detection on an ABI capillary sequencer and DNA preparation using robotics. We have some 6,000 assays with this metho, including a 400-locus genomic control panel effective for detecting ethnic stratification in case/control studies.