高分辨率熔解曲线分析技术用于乙醇脱氢酶1B和乙醛脱氢酶2基因的快速分型

发布时间：2018-07-11 10:24

本文选题：乙醇脱氢酶 + 乙醛脱氢酶　；参考：《南方医科大学》2012年硕士论文

【摘要】：背景与目的乙醇脱氢酶(alcohol dehydrogenase, ADH)和乙醛脱氢酶(aldehyde dehydrogenase,ALDH)是在人体内乙醇代谢途径的两个关键酶,负责催化人体的乙醇分解代谢,即催化乙醇向乙醛转化以及乙醛向乙酸盐分解的关键酶。ADH的活性增强可以加速乙醇向乙醛转化,而ALDH活性的降低则使乙醛向乙酸盐转化受到限制,同样使乙醛的浓度显著增高。乙醛浓度的增加与酒精相关性疾病的发生发展有一定关系。ADH和ALDH基因多态性具有种族特异性,在不同种族人群中分布是不一样的。据研究显示,亚洲人的酒精依赖性疾病主要与ADH1B和ALDH2基因变异密切相关。 ADH1B基因位于4号染色体长臂2区2带,ADH1B*1基因在第3号外显子发生143GA变异,形成ADH1B*2,导致p1亚基的第47位氨基酸从精氨酸(Arginine)转换为组氨酸(Histidine)从而形成p2,酶活性增高。ALDH2基因位于12号染色体长臂2区4带,ALDH2*1基因在第12号外显子发生1510GA变异,形成ALDH2*2,与此同时,酶的第487位氨基酸由谷氨酸(Glutamic acid)变为赖氨酸(Lysine)。野生型ALDH2*1具有ALDH活性,突变型ALDH2*2没有ALDH酶活性。ADH1B和ALDH2基因的变异会使酶代谢活性改变,导致饮酒在不同种族和个体间酒精代谢发生改变。这些年来,研究发现这两种变异与各个组织器官的癌变,乙醇相关的肝脏病变、迟发型老人痴呆、冠心病、2型糖尿病并发症、血液中胆固醇水平都有不同程度的联系。因此,一种准确快速的基因型分析方法对于有关的临床研究和人群普查等广阔研究领域是十分必要的。近年发展起来的高分辨率熔解曲线分析(High-resolution melting, HRM)技术已被证明是一种具有低成本、高通量、速度快、操作简便、高灵敏度等优点的用于基因突变检测、基因分型和SNP检测的工具。不同核酸分子的片段长短、碱基组成、GC分布等是不同的,因此在加热变性后所有的双链DNA分子都会有自己相应的熔解曲线形状和位置。当PCR扩增目的片段含有突变/SNP时,目的产物经过变性-复性会产生异源双链,异源双链中突变/SNP位点是不匹配的,所以双链DNA在升温过程中先解链,此时荧光染料从局部解链的双链DNA分子上释放出来,根据荧光强度与温度曲线图可以判断是否具有突变/SNP,同时不同的位点和杂合度都会影响熔解曲线的峰形。HRM分析过程中,随着双链DNA分子的扩增,由于荧光染料结合到重新产生的双链DNA分子,荧光信号不断的增强。当进入DNA扩增平台期时,荧光信号同时也进入了平台期。随着温度的不断升高,DNA双链逐渐解开,结合上的饱和染料被释放出来,荧光信号逐渐减弱。直到温度升到95℃,所有的双链DNA分子完全解开,通过检测荧光值,建立荧光值随温度升高的变化过程得到熔解曲线。HRM技术的基本原理就是根据熔解曲线的差异性来对样品进行区分。以往的变异检测技术,限制性内切酶片段长度多态性(PCR-Restriction Fragment Length Polymorphism, PCR-RFLP),单链构象多态性分析(Single-Strand Conformation Polymorphisms, SSCP),两对交叉引物PCR(PCR with Confronting Two-Pair Primers, PCR-CTPP),扩增产物长度多态性(Amplified Product Length Polymorphism, APLP),变性高效液相色谱(Denaturing High Performance Liquid Chromatography, DHPLC)等具有一定的局限性,如耗时长,操作繁琐,准确度低,成本相对较高等。与这些方法相比,HRM分析方法具有一定的优势：成本低,只需饱和染料不需要昂贵的探针；在PCR反应结束后,不需要后续工作而直接进行熔解曲线反应；快速、高通量,一次可以同时检测96或384个样本；闭管操,防止样本DNA遭受污染；经过HRM分析处理后的PCR扩增产物还可以直接进行DNA测序,十分方便快捷。HRM具有较高的敏感性和特异性,目前已广泛应用于生命科学、医学、农学、畜牧业等各个领域的研究工作中。基于HRM上述特点,本研究拟应用HRM技术,建立ADH1B和ALDH2的检测方法,对预测个体患酒精相关疾病的风险和研究酶变异与酒精相关疾病的关系提供方便,同时为中国南方酒精相关疾病患者的遗传咨询、临床诊断提供有用的信息。材料与方法 1.标本：共收集200例全血标本。采用标准的饱和酚／氯仿法提取外周血中的基因组DNA。 2.分子分析方法： (1)针对ADH1B和ALDH2基因以及143GA和1510GA变异位点设计PCR扩增体系及优化PCR条件。通过DNA测序获得ADH1B和ALDH2各种基因型样品。 (2)建立检测ADH1B和ALDH2基因的高分辨率熔解曲线分析技术。针对ADH1B和ALDH2基因以及143GA和1510GA变异位点设计HRM分析的引物,利用经测序已知的基因型样品优化HRM分析条件,从而建立ADH1B和ALDH2基因的高分辨率熔解曲线分型的分析方法。 (3)根据HRM的分析结果,从各种基因型中选取一定数量的样品进行测序分析,以验证评价该方法的准确性。并且选取每种ADH1B和ALDH2基因野生纯合子和突变纯合子各10个样本进行重复实验。同时对ADH1B基因的野生纯合子和突变纯合子样本进行了2倍稀释法,进行5个浓度检测,探讨该方法检测敏感性。 3.统计学分析：对构建的ADH1B和ALDH2基因已知突变分型检测体系进行准确性和稳定性分析。利用部分样本直接测序对比来验证其准确性；采用重复性实验以确定该体系的稳定性与准确性。同时分析东莞地区随机汉族人群的ADH1B和ALDH2的基因频率和基因型频率,并应用χ2检验该群体样品是否符合Hardy-Weinberg平衡。使用的统计学分析软件为SPSS13.0。 4.实验结果的综合分析、总结。结果建立了稳定可靠的双重HRM检测体系。此体系中,所选取的扩增目的DNA序列以及所设计的引物的特异性高。ADH1B和ALDH2基因的野生纯合子G/G,杂合子G/A,突变纯合子A/A三种基因型能够在HRM上进行明显的区分。用盲法分析对该方法进行评价,用此方法分析东莞地区200份汉族人基因组DNA样品,从每组基因型中随机选出部分样品进行测序对照,结果符合率为100%,证实了该方法的准确性很好；进行重复实验,发现三次实验的变异系数(CV)的范围从0.021%到0.062%,证实了该方法的稳定性很好。在2倍稀释试验中,5个浓度均可以准确分型,可以知道6.25ng浓度仍然可以用于此检测体系。 χ2检验结果显示所选群体符合Hardy-Weinberg平衡,ADH1B与ALDH2基因频率与以前所报道的数据基本符合,从另一个方面说明了该方法的准确性。结论饮酒已被认为是危害人类健康的严重危险因素,容易导致多种酒精相关疾病。酒精在体内剂量效应和作用时间,不仅仅与酒量和饮酒频率相关,还与易感基因ADH1B和ALDH2代谢能力有强关联性。ADH1B和ALDH2两种基因的变异与酒精依赖性疾病密切相关,所以对ADH1B和ALDH2两种基因进行快速分型,在中国人群中进行快速筛查,不仅能够对酒精相关疾病风险进行评估,还有助于研究ADH1B和ALDH2基因与其他相关疾病的致病机理。高分辨率熔解曲线分析技术是近几年兴起一种基于核酸的物理性质的基因突变检测技术。这种检测技术没有局限于突变碱基位点与类型,不需使用序列特异性探针,在PCR结束后直接进行高分辨率熔解曲线分析来完成对样品基因型的分析。HRM因其操作简便、快速,成本低,结果准确,高通量,并且闭管操作而备受关注。高分辨率熔解曲线分析技术只需要在普通PCR基础上增加一个饱和染料既可以进行未知突变扫描,也可以对已知突变进行基因分型,还可以分析短片段重复序列。HRM分析的这些优势使它具有极强的可操作性,近年来成为国内外新兴的各研究领域学、方法学研究和应用热点。本课题研究中建立的针对ADH1B和ALDH2基因的高分辨率熔解曲线分析检测方法能够快速、准确地同时对ADH1B和ALDH2基因进行检测,并且实验的重复性和稳定性好、敏感性高、体系可靠。本研究对中国南方人群进行检测,发现所检测的200人中不存在ADH1B*1/*1和ALDH2*2/*2组合的个体,所以推测这种基因型组合在中国南方是很少见的。有关文献报道,ADH1B*1等位基因会增加食道癌,肝癌等癌症发生的风险性。同时很多的研究表明ALDH2*1/*2或ALDH2*2/*2个体由于乙醛的积累导致其患有食道癌等癌症的风险更大,然而同时对这两个基因进行联合研究的很少,因此同时纳入ADH1B和ALDH2两基因的病例-对照研究是下一步进行疾病风险研究的重点工作。本研究为ADH1B和ALDH2基因的检测建立了一种简单、高通量、快速、经济和灵敏的检测方法,有助于酒精相关疾病的遗传学咨询,流行病学调查,并为研究ADH1B和ALDH2基因与其他相关疾病的关系提供了一项常规检测手段。本研究方法的一些处理技巧如改变扩增子长度以及加尾处理具有普遍的代表性与通用性,可为其它基因同时分型的检测方法研究提供借鉴与参考。
[Abstract]:Background and purpose
Ethanol dehydrogenase (alcohol dehydrogenase, ADH) and acetaldehyde dehydrogenase (aldehyde dehydrogenase, ALDH) are two key enzymes in the metabolic pathway of ethanol in human body, which are responsible for the catalytic metabolism of ethanol in human body, that is, the activity enhancement of the key enzyme, which catalyzes the conversion of ethanol into acetaldehyde and acetaldehyde to acetic acid, can accelerate ethanol to B. The reduction of aldehydes, while the decrease of ALDH activity, makes the conversion of acetaldehyde to acetic acid limited and also increases the concentration of acetaldehyde significantly. The increase of acetaldehyde concentration is related to the occurrence and development of alcohol related diseases. The polymorphism of.ADH and ALDH genes is racial specificity, and the distribution is different in different ethnic groups. According to the study, it is shown that The alcohol dependence diseases of Asians are closely related to the variation of ADH1B and ALDH2 genes.
The ADH1B gene is located in the 2 band of the 2 region of the long arm of chromosome 4. The ADH1B*1 gene changes 143GA in exon third and forms ADH1B*2, causing the forty-seventh amino acids of the P1 subunit to convert from arginine (Arginine) to histidine (Histidine) to form P2, and the activity of the enzyme increases in the 4 band of the 2 region of the long arm of chromosome 12, and the ALDH2*1 gene is outside twelfth. At the same time, the 487th bit amino acid of the enzyme changed from glutamic acid (Glutamic acid) to lysine (Lysine). The wild type ALDH2*1 had ALDH activity. The mutant ALDH2*2 had no ALDH enzyme activity.ADH1B and ALDH2 gene, which could change the activity of enzyme metabolism, causing alcohol to alcohol in different races and individuals. Metabolism has changed. Over the years, studies have found that these two variants have different degrees of association with cancer of various tissues and organs, alcohol related liver diseases, delayed Alzheimer's disease, coronary heart disease, type 2 diabetes complications, and blood cholesterol levels. Broad research areas such as population census and so on are very necessary.
High-resolution melting (HRM) technology developed in recent years has been proved to be a tool for gene mutation detection, genotyping and SNP detection, such as low cost, high throughput, fast speed, simple operation and high sensitivity. The length, base composition and GC distribution of different nucleic acid molecules are the tools of gene mutation detection, genotyping and SNP detection. It is different that all double stranded DNA molecules have their own corresponding melting curve shape and position after heating denaturation. When the target fragment of PCR amplification contains mutant /SNP, the target product passes the denaturation refolding to produce the heterogenous double chain, and the mutation of the /SNP loci in the heterologous double chain is not matched, so the double chain DNA first dissolves the chain during the heating process. At this time, the fluorescent dye is released from the double strand DNA molecule of the local chain, and the mutation /SNP can be judged according to the fluorescence intensity and temperature curve. At the same time, the different sites and heterozygosity affect the peak shape.HRM analysis of the melting curve. With the amplification of the double stranded DNA molecules, the fluorescent dyes are combined to regenerate. Double stranded DNA molecules, the fluorescence signal is constantly enhanced. When the DNA amplification platform is entered, the fluorescence signal also enters the platform phase. As the temperature increases, the DNA double chain gradually dissolves, the combined saturated dye is released and the fluorescence signal gradually decreases. Until the temperature rises to 95 degrees, all the double stranded DNA molecules are completely unopened and passed through The basic principle of the fusion curve.HRM technology is to detect the fluorescence value and establish the change process of the fluorescence value with the temperature rising. The basic principle of the fusion curve is to distinguish the sample according to the difference of the melting curve.
The previous mutation detection techniques, PCR-Restriction Fragment Length Polymorphism (PCR-RFLP), single strand conformation polymorphism analysis (Single-Strand Conformation Polymorphisms, SSCP), two pairs of cross primer PCR (PCR with), amplified product length polymorphism (Fragment). Amplified Product Length Polymorphism, APLP), denatured high-performance liquid chromatography (Denaturing High Performance Liquid Chromatography, DHPLC) has some limitations, such as long time consuming, tedious operation, low accuracy and higher cost. Compared with these methods, the HRM analysis method has a certain advantage: low cost and only saturation. The dye does not need an expensive probe; after the PCR reaction ends, it does not need to follow the work to directly react to the melting curve; fast, high throughput, 96 or 384 samples can be detected at the same time; closed tube exercises to prevent sample DNA from being polluted; and after HRM analysis, the PCR enlargement can also be directly carried out by DNA sequencing, very convenient Fast.HRM has high sensitivity and specificity, and has been widely used in the research work of life science, medicine, agriculture and animal husbandry. Based on the above characteristics of HRM, this study intends to use HRM technology to establish the detection methods of ADH1B and ALDH2 to predict the risk of individual alcohol related diseases and to study the enzyme variation and alcohol. It provides convenience for the relationship of related diseases, and provides useful information for genetic counseling and clinical diagnosis of patients with alcohol related diseases in southern China.
Materials and methods
1. samples: a total of 200 blood samples were collected. Genomic DNA. from peripheral blood was extracted by standard saturated phenol / chloroform method.
2. method of molecular analysis:
(1) to design PCR amplification system and optimize PCR conditions for ADH1B and ALDH2 genes and 143GA and 1510GA mutation sites. All kinds of ADH1B and ALDH2 genotypes were obtained by DNA sequencing.
(2) to establish high resolution fusion curve analysis technique for detecting ADH1B and ALDH2 genes. According to the primers of HRM analysis for ADH1B and ALDH2 genes and 143GA and 1510GA mutation sites, the analytical conditions of HRM analysis were optimized by sequencing known genotype samples, and the analysis method of high resolution melting curve classification of ADH1B and ALDH2 based causes was established.
(3) according to the results of HRM analysis, a certain number of samples were selected from various genotypes to verify the accuracy of the method, and 10 samples of each ADH1B and ALDH2 gene Nobu Juriko and the mutant homozygote were repeated. Meanwhile, the wild homozygote and mutant homozygote samples of the ADH1B gene were sampled. 2 times dilution method was used, and 5 concentrations were detected to explore the sensitivity of the method.
3. statistical analysis: the accuracy and stability of the established ADH1B and ALDH2 gene mutation detection system were analyzed. The accuracy of the system was verified by the direct sequencing of some samples. The stability and accuracy of the system were determined by repeated experiments. Meanwhile, the ADH1B and ALDH2 of the random Han population in Dongguan region were analyzed. The gene frequencies and genotype frequencies were tested and chi square test was used to test whether the population samples were in line with the Hardy-Weinberg balance. The statistical analysis software used was SPSS13.0.. 2
4. the comprehensive analysis of the results of the experiment is summed up.
Result
A stable and reliable dual HRM detection system was established. In this system, the selected amplified target DNA sequence, the specific high.ADH1B of the primers and the wild homozygote G/G of the ALDH2 gene, the heterozygote G/A, and the mutant homozygote A/A three genotypes can be distinctly distinguished on HRM.
The method was evaluated by blind analysis. By using this method, the genomic DNA samples of 200 Han people in Dongguan area were analyzed, and some samples were randomly selected from each group to be sequenced and sequenced. The result showed that the accuracy of the method was 100%, and the accuracy of the method was good. The range of variation coefficient (CV) of three experiments was found to be from 0. .021% to 0.062% proved that the method has a good stability. In the 2 times dilution test, 5 concentrations can be accurately typed. It is possible to know that the concentration of 6.25ng can still be used in this detection system.
The results of the chi 2 test showed that the selected population conformed to the Hardy-Weinberg balance, and the ADH1B and ALDH2 gene frequencies were basically consistent with the previously reported data, and the accuracy of the method was explained in another aspect.
conclusion
Alcohol consumption has been considered to be a serious risk factor for human health. It is easy to lead to a variety of alcohol related diseases. Alcohol in the body dose effect and time, not only with alcohol and drinking frequency, but also associated with the susceptibility gene ADH1B and ALDH2 metabolic ability of the two genes of.ADH1B and ALDH2 and alcohol dependent diseases It is closely related that rapid typing of the two genes of ADH1B and ALDH2 and rapid screening in Chinese population can not only evaluate the risk of alcohol related diseases, but also help to study the pathogenesis of ADH1B and ALDH2 genes and other related diseases.
High resolution fusion curve analysis technique is a technique for detecting gene mutation based on the physical properties of nucleic acid in recent years. This detection technique is not limited to the mutation base site and type, and does not need a sequence specific probe. After the end of PCR, the high resolution fusion curve is carried out directly to complete the sample genotypes. The analysis of.HRM has attracted much attention because of its simple operation, rapid, low cost, accurate results, high throughput, and closed tube operation. High resolution fusion curve analysis technology only needs to add a saturated dye on the basis of ordinary PCR to carry out the unknown mutation scan, the gene classification of the known mutation, and the analysis of the short segment weight. The advantages of complex sequence.HRM analysis make it highly maneuverable. In recent years, it has become a new research field in both domestic and foreign countries, and has been a hot topic in methodology research and application.
The high resolution fusion curve analysis and detection method for ADH1B and ALDH2 genes established in this study can quickly and accurately detect the ADH1B and ALDH2 genes at the same time, and the repeatability and stability of the experiment are good, the sensitivity is high, and the system is reliable.
This study examined people in southern China and found that there were no individuals of ADH1B*1/*1 and ALDH2*2/*2 combinations in 200 people. Therefore, it was inferred that this genotypic combination was rare in southern China. The relevant literature reported that ADH1B*1 alleles would increase the risk of cancer in the esophagus and liver cancer. ALDH2*1/*2 or ALDH2*2/*2 individuals have a greater risk of cancer such as esophagus cancer due to the accumulation of acetaldehyde. However, there are few joint studies on these two genes, so the case control study, which is incorporated into the ADH1B and ALDH2 two genes simultaneously, is the key task for the next step of disease risk research.
This study establishes a simple, high throughput, rapid, economical and sensitive detection method for the detection of ADH1B and ALDH2 genes, which can help the genetic counseling and epidemiological investigation of alcohol related diseases, and provide a routine test for the study of the relationship between the ADH1B and ALDH2 genes and other related diseases.
Some techniques such as changing the length of the amplicons and adding tail treatment have universal representation and generality, which can be used for reference and reference for the study of the detection methods of other genotyping.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R346

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