脑胶质瘤病人遗传易感基因单核苷酸多态性的研究

目的筛查与脑胶质瘤发病相关基因的单核苷酸多态性，进一步寻找胶质瘤易感基因。方法收集入院脑胶质瘤病人的血液。应用PCR扩增的方法对肿瘤相关的基因的特定片段进行扩增。利用变性高效液相色谱技术进行筛查。双向DNA直接测序，在GENBANK进行对比找到突变的碱基，并进行分析。结果变性高效液相色谱筛查后表明，在HLA-DMA基因中检测到了已知杂和性突变，并发现新的突变，HLA-DMA4EC杂合性缺失突变。结论变性高效液相色谱技术在筛查基因的SNPs方面显示了其快速、高通量和准确的特点。HLA-DMA基因第4号外显子第111位碱基缺失突变是一个新的SNP，可能是胶质母细胞瘤的易感突变之一。     

变性高效液相色谱法检测基因单核苷酸多态性

我们采用变性高效液相色谱 (ｄＨＰＬＣ)技术检测单核苷酸多态性 (ＳＮＰ)。根据Ｃａｒｇｉｌｌ提供的思路是 :聚合酶链反应(ＰＣＲ)扩增出包含多态性位点的片段 ,变性后缓慢复性 ,当样品为杂合子时 ,将出现 4种组分 :2种同源双链和 2种异源杂合双链 ,所以在最佳分离条件下 ,可能出现 4个吸收峰。人儿茶酚胺甲基转移酶基因密码子 136和 15 8处分别存在ＣＴＣ→ＣＴＧ和ＧＴＧ→ＡＴＧ的多态性 ;5 羟色胺受体 2ａ基因 74位和 10 2位碱基处分别存在Ｃ→Ａ及Ｔ→Ｃ的多态性。我们用ｄＨＰＬＣ与酶切 2种方法分别对上述 2个基因的 4个多态性位…     

变性高效液相色谱法检测单核苷酸多态性的研究进展

单核苷酸多态(single nucleotide polymorphisms,SNPs)是指某一人群的正常个体基因组内特定核苷酸位置上存在不同碱基,且其最低的基因频率>1%,即在基因组核苷酸水平上单碱基突变引起的DNA序列多态性[1-2]。通常说的SNPs包括单个碱基的转换、颠换,以及单个碱基的缺失和插入。在人类DNA多态性中,SNPs大约占90%。人类基因组中每千个核苷酸就有一个以上的SNPs,因此整个人类基因组30亿对碱基中共有300万以上的SNPs。其中约有25~40万个为基因编码区的突变(coding region SNPs,cSNPs),cSNPs中又有20%~30%可引起蛋白质编码序列改变并导…     

单核苷酸多态性与2型糖尿病易感基因相关性的研究进展

近年来,随着经济发展和生活方式的改变,2型糖尿病(T2DM)发病率在我国呈现逐渐升高的趋势。T2DM是一种遗传因素和环境因素共同作用而形成的多基因遗传的复杂性疾病,并以胰岛素分泌受损和(或)胰岛素抵抗为主要的临床特点,其遗传方式属于常染色体多基因隐性遗传,由于异常基因的遗传使后代具有糖尿病易感性。随着单核苷酸多态性(SNP)分析技术的发展以及全基因组关联研究结果的相继报道,至今已发现许多常见基     

江苏地区2型糖尿病易感基因单核苷酸多态性与妊娠期糖尿病关系的研究

目的探讨江苏地区2型糖尿病易感基因CDKAL1、CDKN2A-CDKN2B、TCF7L2及HHEX-IDE的单核苷酸多态性（SNP）与妊娠期糖尿病（GDM）的关系。方法采用病例对照法及多重SNaPshot SNP分型技术检测185例无GDM倾向的健康孕妇（对照组）及176例GDM孕妇（GDM组）CDKAL1、CDKN2A-CDKN2B、TCF7L2及HHEX-IDE的SNP位点基因分型,并对所有SNP位点进行连续不平衡分析。结果比较GDM组和对照组孕妇的等位基因频率,CDKAL1（rs7754840）的等位基因C为GDM的一个危险因素。CC型携带者患GDM的风险是未携带者的1.73倍[P〈0.01,OR=1.73,95%CI：1.28~2.33]。CDKN2A-CDKN2B（rs10811661）、TCF7L2（rs7903146）及HHEX-IDE（rs1111875）检测位点的基因分布差异无统计学差异（P〉0.05）。CDKAL1（rs7754840）、CDKN2A-CDKN2B（rs10811661）、TCF7L2（rs7903146）及HHEX-IDE（rs1111875）的基因型频率均符合遗传平衡法则。结论 CDKAL1（rs7754840）与江苏地区GDM易感密切相关。     

急性心肌梗死易感基因多态性研究现状

急性心肌梗死（AMI）是一种多基因与环境相互作用而产生的疾病,目前是人类最主要的死亡原因之一^[1]。因此,针对AMI病因的研究始终没有中断过。除了传统的危险因素（包括高血压、高血脂、糖尿病、吸烟等）之外,家族史被认为是一个独立的危险因素。随着近年来人类基因组学和分子生物学技术的发展,各国科学家在大规模病例对照研究中对AMI的易感基因及其多态性进行了深入的调查研究,确定了一系列的AMI易感基因及相关单核苷酸多态性（SNP）位点。候选易感基因主要集中在以下4个方面^[2-4]：（1）肾素-血管紧张素系统;     

聚合酶链反应/变性高效液相色谱技术检测载脂蛋白E基因型

目的 建立快速检测载脂蛋白E(apo E)基因型的变性高效液相色谱技术。 方法 标准酚-氯仿法提取180份健康人外周血基因组DNA,用聚合酶链反应/变性高效液相色谱（PCR/DHPLC）技术测定apo E基因型,分析人群中apo E基因型和等位基因的分布频率。 结果 共检出6种基因型：ε3/ε3占73.89%,ε2/ε3和ε3/ε4各占15.56%和7.22%,ε2/ε2、ε2/ε4和ε4/ε4共占3.34%。3种等位基因中,最常见的是ε3,占85.28%,ε2和ε4各占10.00%和4.72%。 结论 建立了一种基于DHPLC的apo E基因分型方法,该方法具有简便、准确的特点。     

应用变性高效液相色谱检测CD31563位点单核苷酸多态性

目的应用变性高效液相色谱（DHPLC）检测CD31 563位点单核苷酸多态性。方法聚合酶链反应（PCR）扩增健康正常人位于17q23编码CD31基因第8外显子563位点附近的DNA片段，应用DHPLC技术对扩增片段进行多态性分析，将不同峰型的PCR片段进行序列测定，并与参考序列对照分析。结果PCR扩增片段为203bp，经DHPLC分析有3种不同峰型，经与测序结果比较，G／G纯合峰型、A／A纯合峰型、G／A杂合峰型明显不同，3种基因型的个体得到明确区分。74名健康正常人中CD31-563S（AGC）的基因频率为0．514，CD31-563N（AAC）的基因频率为0.486。结论DHPLC可以高效、经济、准确的检测CD31-563位点单核苷酸多态性。     

利用DNA池和变性高效液相色谱分析bcr和 abl基因序列标签位点多态性

为了探讨bcr和abl基因的单核苷酸多态性(SNP)与慢性髓细胞性白血病(CML)的关系，利用DNA池(DNA pooling)结合变性高效液相色谱(dHPLC)技术对bcr和abl基因上的9个序列标签位点(sequence-tagged site，STS)进行序列变异的筛查分析，并通过测序对筛查结果进行验证。研究结果表明，在9个STS片段中检出了4个片段中的多态性位点和3个片段中与参考序列不一致的变异。结论：U07000片段中的SNP在慢性髓性细胞白血病病人和对照人群中的基因频率分布有显著差异。     

儿童白血病易感基因及其多态性的研究进展

白血病约占儿童恶性肿瘤的1/3,其发病是环境因素(物理因素、化学因素和生物因素)和遗传因素(代谢酶基因、DNA修复酶基因和毒物受体基因及其多态性等)交互作用的结果.本文主要综述近年来与儿童白血病发病有关的易感基因及其多态性的研究进展.     

Affordable assays for genotyping single nucleotide polymorphisms in insects

Insect genome projects and DNA sequence databases are providing unprecedented amounts of information about variation at specific nucleotides in protein- and RNA-coding genes. Single nucleotide polymorphisms (SNPs) are abundant in all insect species so far examined and are proving useful in population genetics, linkage mapping and marker-assisted selection. A number of studies has already identified SNPs associated with insecticide resistance, especially mutations conferring reduced target site sensitivity. Unfortunately, most modern, high-throughput, automated SNP detection technologies are expensive or require the use of expensive equipment and are therefore not accessible to laboratories on a limited budget or to our colleagues in developing countries. In this review, we provide a chronological and comprehensive list of all SNP methods. We emphasize and explain those techniques in which genotypes can be identified by eye or that only require agarose gel electrophoresis. We provide examples where these techniques have or are currently being applied to insects.     

变性高效液相色谱法快速诊断21三体综合征

目的用变性高效液相色谱法(DHPLC)分析三体综合征患者的双链DNA,快速产前诊断21三体综合征。方法根据所设计的引物对21号染色体D21S11、D21S1411和D21S1412共3个短串联重复序列(STR)位点进行PCR扩增,然后在50℃柱温条件下用DHPLC对PCR扩增产物进行检测和分析。结果健康对照者D21S11、D21S1411和D21S1412的DHPLC峰形呈现一个或两个高低相同的波峰,21三体综合征患者的DHPLC峰形呈现两个高低不同的波峰,且其中一个波峰高度接近为另一个波峰的2倍。结论 DHPLC具有灵敏度高、特异性强、简便快捷等,适合广泛应用于21三体综合征的快速诊断。     

用变性高效液相色谱术筛查甲基化CpG结合蛋白2基因变异

目的用变性高效液相色谱(denaturing high-performance liquid chromatography,DHPLC)分析和DNA测序筛查甲基化CpG结合蛋白2(methyl-CpG-binding protein 2,MECP2)基因突变,评价DHPLC检测MECP2基因变异的可用性。方法建立检测MECP2基因变异的DHPLC分析法,对52例孤独症患儿MECP2基因3个外显子分为8个片段进行PCR扩增,扩增产物用DHPLC筛查,并以DNA测序验证。结果在Exon 4-1、4-2、4-3、4-4片段,分别发现有17个、2个、2个和1个样本峰型异常,DNA测序分析发现为6种MECP2基因变异。结论DHPLC技术可用于检测MECP2基因变异,方法敏感、重复性好。     

运用变性高效液相色谱快速检测CTX-M超广谱β内酰胺酶基因型

目的应用变性高效液相色谱（DHPLC）技术对确认产ESBLs的大肠埃希菌和肺炎克雷伯菌临床分离株CTX-M型质粒酶进行基因分型，探讨其灵敏度和特异度，以建立快速检测CTX-M型ESBLs的新方法。方法采用PCR技术从大肠埃希菌和肺炎克雷伯菌临床分离株中扩增出CTX—M型质粒的编码序列，用DHPLC技术对扩增产物进行分析和DNA测序，确定其基因突变的类型，通过比对两者结果后确定其基因型。结果从34株产ESBLs大肠埃希菌和肺炎克雷伯菌临床分离株中扩增出35份CTX-M型ESBLs编码序列（其中1株同时扩增到2份CTX—M型编码序列）。11份与标准菌株CTX—M-3一致；4份与标准菌株CTX—M-9一致；测序确定分别为CTX—M-3型和CTX—M-9型；20份表现为3种形态各异的异常洗脱峰，测序结果表明20份样本与其标准株对比均存在着基因突变，DHPLC中异常峰一致的样本均为同-基因亚型。结论DH—PLC可用于ESBLs基因分型的检测，能快速检测CTX—M的基因型，具有准确性高、简便快捷、经济等特点，有很大的临床应用价值。     

Rapid detection of glucose-6-phosphate dehydrogenase gene mutations by denaturing high-performance liquid chromatography

OBJECTIVES: Glucose-6-phosphate dehydrogenase (G6PD) deficiency is a common inherited disorder worldwide. Different kinds of G6PD mutations may result in variable severity of clinical onset in G6PD-deficient individuals. In this study, a reliable molecular diagnostic method was developed for rapid detection of G6PD gene mutation. DESIGN AND METHODS: Primers were designed to amplify G6PD gene fragments that were subjected to mutation screening using denaturing high-performance liquid chromatography (DHPLC) analysis. Mutations were identified by their distinct elution peak patterns and were confirmed by DNA sequencing. The assay was further validated against 29 samples from individuals with G6PD deficiency. RESULTS: A DHPLC-based assay for G6PD mutation detection was established. The 9 common G6PD mutations in the Taiwanese and Chinese population could be distinguished through the analysis of DNA elution patterns. During the validation test with the 29 G6PD deficiency specimens, two additional rare mutations, T517C and C519G, were unveiled. Overall, the DHPLC-based mutation detection was 100% concordant with the DNA sequencing results. CONCLUSION: Compared to other genotyping techniques, this method requires significantly less technical time to perform and has a greatly increased throughput capacity. Hence, the DHPLC method represents a major technical advance for G6PD genotyping and should benefit G6PD-deficient individuals for proper clinical care.     

变性高效液相色谱对CTX-M型超广谱β内酰胺酶基因分型研究

目的 探讨湖南省CTX-M型超广谱B内酰胺酶(ESBLs)基因型分布情况和变性高效液相色谱(DHPLC)方法检测CTX-M型ESBLs基因型的准确性.方法 用多重PCR扩增标准菌株和ESBLs表型阳性的临床菌株blaCTX-M基因,扩增产物经DHPLC分析得到标准菌株和临床菌株色谱峰图,通过比对标准色谱峰图对临床菌株进行基因分型,同时采用单纯随机抽样法选择25株多重PCR扩增阳性菌株进行特异PCR扩增,其产物冉进行基因测序来评估DHPLC法的准确性.结果 142株产ESBLs的肠杆菌科细菌经多重PCR扩增证实109株携带blaCTX-M基因,检出率为76.8%(109/142).109株扩增阳性的菌株经DHPLC分析后检出4种不同的blaCTX-M基因型:33株携带CTX-M-3、19株携带CTX-M-15、5株携带CTX-M-9、52株携带CTX-M-14.25株基因测序结果与DHPLC基因分型结果作比较显示:24株DHPLC的基因分型结果与基因测序结果完全吻合,但有1株DHPLC基因分型为CTX-M-15,而测序为CTX-M-82.结论 DHPLC可对耐药进行基因快速基因分型,具有准确、快速和经济等优点.     

Mutational analysis of CYP2C8 in hypertensive patients using denaturing high performance liquid chromatography

What is known and Objective: CYP2C8 is involved in the cytochrome P450 (CYP) epoxygenase pathway. Arachidonic acid metabolites such as epoxyeicosatrienenoic acids and hydroxyeicosatetrenoic acids, produced may have a role in hypertension. We aimed to develop a medium through‐put method for screening samples of known and new mutations of CYP2C8 using denaturing high performance liquid chromatography (DHPLC). Methods: DNA samples from 200 subjects (hypertensive patients and healthy controls) were screened for SNPs in CYP2C8 using DHPLC. Genotypes and allelic frequencies of CYP2C8 between the healthy controls and patients with hypertension were compared. Results and Discussions: Six variants were detected and two were new; T deletion at 5063 and substitution of C to T at 33468 in exon 8. Differences in variant frequencies were detected between the controls and hypertensive patients. The controls have significantly higher prevalence of C35322C compared to the patients. The functional significance of the SNP at 35322 requires further study. Having homozygous C35322C could be a protective factor for hypertension. What is new and Conclusion: Denaturing high performance liquid chromatography is useful for population screening to identify new and existing SNPs. A higher frequency of the C35322T SNP was observed among hypertensive patients than control subjects. This potentially important observation requires confirmation and the clinical significance assessed.     

Screening for adenine and hypoxanthine phosphoribosyltransferase deficiencies in human erythrocytes by high-performance liquid chromatography

A screening method using high-performance liquid chromatography (HPLC) for the simultaneous detection of deficiencies of adenine phosphoribosyltransferase (APRT) and hypoxanthine phosphoribosyltransferase (HPRT) activities in human erythrocytes is described. Both enzyme reactions of APRT and HPRT in lysates treated with a charcoal-dextran were simultaneously carried out in the same reaction tube and the enzyme activities were determined by measuring the increases in absorbance at 260 nm of adenosine and inosine converted from adenosine-5'-monophosphate and inosine-5'-monophosphate with alkaline phosphatase. Adenosine and inosine were separated from adenine and hypoxanthine by a reversed-phase column.

The method could detect 1% of normal APRT activity and 0.3% of normal HPRT activity. The within-run coefficients of variation for APRT and HPRT activities were 3.2 and 3.4%, respectively.     

变性高效液相色谱技术在β地中海贫血分型诊断中的应用

目的 探讨变性高效液相色谱技术(DHPLC)在快速诊断β地中海贫血及分型中的应用价值.方法 采用外周血红细胞平均体积(MCV)、红细胞分布宽度(RDW)、红细胞脆性和Hb电泳4项指标相结合的方法筛选地中海贫血可疑标本226份.运用PCR反向斑点杂交技术(PCR-RDB)和DHPLC对226份可疑标本进行基因分型确诊.结果 226份可疑静脉血标本中,经PCR-RDB和DHPLC确诊的β地中海贫血为69份,两种方法检测缺失和突变的基因型别完全一致,占地中海贫血筛查总人数的30.5%.其中CD41/CD42(-TCTT)移码突变37例(54%);IVS-Ⅱ-654(C→T)插入序列突变12例(17%);TATA-28(A→G)转录突变10例(15%);CD17(A→T)无义突变5例(7%);CD71/CD72(+A)移码突变5例(7%).结论 DHPLC可快速、高效和准确地对β地中海贫血进行基因分型诊断.