High Throughput Detection & Genotyping For SNPs

高通量SNP鉴定。LNG在50多个神经遗传候选基因中发现了100多个单核苷酸多态性（SNP），这些基因与成瘾行为有关，包括酗酒。通过LNG发现的大约20%的SNP导致非同义氨基酸变化，这可能改变编码蛋白质的功能或基因表达的改变。检测到的血清素变异的意义是深远的：例如，我们发现的一种罕见的血清素转运蛋白变异被证明是功能性的，并发现导致严重的病理学：在两个家庭中，它是分离的，包括巴氏综合征，治疗抵抗性强迫症和神经性厌食症。例如，常见的HTR 2C Ser 23 Cys和HTR 2A Asn 452 His等位基因已被证明是功能性的，并与精神分裂症患者的氯氮平反应性相关。基于在酒精、治疗反应、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中的假定作用来选择用于SNP筛选的基因。由于该项目的目标是确定SNP在复杂遗传疾病中的作用，因此我们将筛选工作集中在候选基因的蛋白质编码部分以及蛋白质编码区域内和侧翼的可能调控区域上。我们使用变性高效液相色谱法（dHPLC），检测大于90%的150-450 bp大小的DNA双链体中的序列变异，筛选由477个基因组DNA组成的DNA面板丰富的临床和种族多样性。筛选小组的规模和多样性至关重要。例如，一种罕见的5-羟色胺转运错义替代只在两个家庭中发现，然而，与替代的个人有严重的行为问题。除了一个病例外，他们患有神经性厌食症，严重的治疗抵抗性强迫症或神经性厌食症。在深入研究的HTTLPT 5-羟色胺启动子多态性中，我们发现了一个新的、常见的SNP等位基因，该等位基因改变了功能，并利用这一信息将两个人群中的5-羟色胺转运蛋白基因与强迫症联系起来。构建筛选组是为了实现对我们的一级连锁数据集中每个新SNP的等位基因频率的准确估计。筛选数据集由美国土著人、非洲裔美国人、高加索人和亚洲人人群组成，并富含各种精神疾病，包括酒精中毒、强迫症、精神分裂症、进食障碍和双相情感障碍。使用基于凝胶的半自动或基于毛细管的全自动DNA测序方法对从初步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分子之间的热熔解差异，所述双链DNA分子携带完整序列匹配或单核苷酸错配，如在特定SNP的杂合个体中发现的。来自SNP杂合个体的DNA混合物中链的变性和再退火将含有至多50%的再退火DNA作为完全匹配的序列，而至少50%的再退火DNA将具有单碱基错配。具有单碱基错配的DNA双链体的热解链特性固有地比完全匹配的双链DNA更不稳定。使用Perkin Elmer 7700序列检测器监测双链DNA解链过程中荧光信号的变化。有了这个特殊的仪器，它可以进行热变性实验和分析的DNA解链数据在96个样品在同一时间。我们将原始荧光读数转换为一阶导数图，该图产生每个DNA双链体的解链曲线。利用这些解链图谱，我们能够检测100-150 bp大小的双链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异。高通量基因分型。高通量基因分型程序的中心问题是准确性、灵活性和成本。由于其分析设计灵活性、低错误率和同时进行96至384次分析的潜力，我们选择了使用LNG中发现的SNP或其他实验室描述的SNP的5'核酸酶分析，用于我们研究人群中的等位基因频率测定，并且我们使用该方法进行了约1，000次个体分析。基因分型完成率>95%，重复样本计算的错误率小于0.5%。试剂成本目前为每次检测0.20 - 0.30美元。我们已经过渡到SNRP-PCR，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制备。我们用这种方法进行了大约6,000次检测，包括一个400个基因座的基因组对照组，用于有效检测病例/对照研究中的种族分层。

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.