本文選題：乙醇脫氫酶 + 乙醛脫氫酶　； 參考：《南方醫(yī)科大學(xué)》2012年碩士論文

【摘要】：背景與目的 乙醇脫氫酶(alcohol dehydrogenase, ADH)和乙醛脫氫酶(aldehyde dehydrogenase,ALDH)是在人體內(nèi)乙醇代謝途徑的兩個(gè)關(guān)鍵酶,負(fù)責(zé)催化人體的乙醇分解代謝,即催化乙醇向乙醛轉(zhuǎn)化以及乙醛向乙酸鹽分解的關(guān)鍵酶。ADH的活性增強(qiáng)可以加速乙醇向乙醛轉(zhuǎn)化,而ALDH活性的降低則使乙醛向乙酸鹽轉(zhuǎn)化受到限制,同樣使乙醛的濃度顯著增高。乙醛濃度的增加與酒精相關(guān)性疾病的發(fā)生發(fā)展有一定關(guān)系。ADH和ALDH基因多態(tài)性具有種族特異性,在不同種族人群中分布是不一樣的。據(jù)研究顯示,亞洲人的酒精依賴性疾病主要與ADH1B和ALDH2基因變異密切相關(guān)。 ADH1B基因位于4號(hào)染色體長(zhǎng)臂2區(qū)2帶,ADH1B*1基因在第3號(hào)外顯子發(fā)生143GA變異,形成ADH1B*2,導(dǎo)致p1亞基的第47位氨基酸從精氨酸(Arginine)轉(zhuǎn)換為組氨酸(Histidine)從而形成p2,酶活性增高。ALDH2基因位于12號(hào)染色體長(zhǎng)臂2區(qū)4帶,ALDH2*1基因在第12號(hào)外顯子發(fā)生1510GA變異,形成ALDH2*2,與此同時(shí),酶的第487位氨基酸由谷氨酸(Glutamic acid)變?yōu)橘嚢彼?Lysine)。野生型ALDH2*1具有ALDH活性,突變型ALDH2*2沒有ALDH酶活性。ADH1B和ALDH2基因的變異會(huì)使酶代謝活性改變,導(dǎo)致飲酒在不同種族和個(gè)體間酒精代謝發(fā)生改變。這些年來,研究發(fā)現(xiàn)這兩種變異與各個(gè)組織器官的癌變,乙醇相關(guān)的肝臟病變、遲發(fā)型老人癡呆、冠心病、2型糖尿病并發(fā)癥、血液中膽固醇水平都有不同程度的聯(lián)系。因此,一種準(zhǔn)確快速的基因型分析方法對(duì)于有關(guān)的臨床研究和人群普查等廣闊研究領(lǐng)域是十分必要的。 近年發(fā)展起來的高分辨率熔解曲線分析(High-resolution melting, HRM)技術(shù)已被證明是一種具有低成本、高通量、速度快、操作簡(jiǎn)便、高靈敏度等優(yōu)點(diǎn)的用于基因突變檢測(cè)、基因分型和SNP檢測(cè)的工具。不同核酸分子的片段長(zhǎng)短、堿基組成、GC分布等是不同的,因此在加熱變性后所有的雙鏈DNA分子都會(huì)有自己相應(yīng)的熔解曲線形狀和位置。當(dāng)PCR擴(kuò)增目的片段含有突變/SNP時(shí),目的產(chǎn)物經(jīng)過變性-復(fù)性會(huì)產(chǎn)生異源雙鏈,異源雙鏈中突變/SNP位點(diǎn)是不匹配的,所以雙鏈DNA在升溫過程中先解鏈,此時(shí)熒光染料從局部解鏈的雙鏈DNA分子上釋放出來,根據(jù)熒光強(qiáng)度與溫度曲線圖可以判斷是否具有突變/SNP,同時(shí)不同的位點(diǎn)和雜合度都會(huì)影響熔解曲線的峰形。HRM分析過程中,隨著雙鏈DNA分子的擴(kuò)增,由于熒光染料結(jié)合到重新產(chǎn)生的雙鏈DNA分子,熒光信號(hào)不斷的增強(qiáng)。當(dāng)進(jìn)入DNA擴(kuò)增平臺(tái)期時(shí),熒光信號(hào)同時(shí)也進(jìn)入了平臺(tái)期。隨著溫度的不斷升高,DNA雙鏈逐漸解開,結(jié)合上的飽和染料被釋放出來,熒光信號(hào)逐漸減弱。直到溫度升到95℃,所有的雙鏈DNA分子完全解開,通過檢測(cè)熒光值,建立熒光值隨溫度升高的變化過程得到熔解曲線。HRM技術(shù)的基本原理就是根據(jù)熔解曲線的差異性來對(duì)樣品進(jìn)行區(qū)分。 以往的變異檢測(cè)技術(shù),限制性內(nèi)切酶片段長(zhǎng)度多態(tài)性(PCR-Restriction Fragment Length Polymorphism, PCR-RFLP),單鏈構(gòu)象多態(tài)性分析(Single-Strand Conformation Polymorphisms, SSCP),兩對(duì)交叉引物PCR(PCR with Confronting Two-Pair Primers, PCR-CTPP),擴(kuò)增產(chǎn)物長(zhǎng)度多態(tài)性(Amplified Product Length Polymorphism, APLP),變性高效液相色譜(Denaturing High Performance Liquid Chromatography, DHPLC)等具有一定的局限性,如耗時(shí)長(zhǎng),操作繁瑣,準(zhǔn)確度低,成本相對(duì)較高等。與這些方法相比,HRM分析方法具有一定的優(yōu)勢(shì)：成本低,只需飽和染料不需要昂貴的探針；在PCR反應(yīng)結(jié)束后,不需要后續(xù)工作而直接進(jìn)行熔解曲線反應(yīng)；快速、高通量,一次可以同時(shí)檢測(cè)96或384個(gè)樣本；閉管操,防止樣本DNA遭受污染；經(jīng)過HRM分析處理后的PCR擴(kuò)增產(chǎn)物還可以直接進(jìn)行DNA測(cè)序,十分方便快捷。HRM具有較高的敏感性和特異性,目前已廣泛應(yīng)用于生命科學(xué)、醫(yī)學(xué)、農(nóng)學(xué)、畜牧業(yè)等各個(gè)領(lǐng)域的研究工作中。基于HRM上述特點(diǎn),本研究擬應(yīng)用HRM技術(shù),建立ADH1B和ALDH2的檢測(cè)方法,對(duì)預(yù)測(cè)個(gè)體患酒精相關(guān)疾病的風(fēng)險(xiǎn)和研究酶變異與酒精相關(guān)疾病的關(guān)系提供方便,同時(shí)為中國(guó)南方酒精相關(guān)疾病患者的遺傳咨詢、臨床診斷提供有用的信息。 材料與方法 1.標(biāo)本：共收集200例全血標(biāo)本。采用標(biāo)準(zhǔn)的飽和酚／氯仿法提取外周血中的基因組DNA。 2.分子分析方法： (1)針對(duì)ADH1B和ALDH2基因以及143GA和1510GA變異位點(diǎn)設(shè)計(jì)PCR擴(kuò)增體系及優(yōu)化PCR條件。通過DNA測(cè)序獲得ADH1B和ALDH2各種基因型樣品。 (2)建立檢測(cè)ADH1B和ALDH2基因的高分辨率熔解曲線分析技術(shù)。針對(duì)ADH1B和ALDH2基因以及143GA和1510GA變異位點(diǎn)設(shè)計(jì)HRM分析的引物,利用經(jīng)測(cè)序已知的基因型樣品優(yōu)化HRM分析條件,從而建立ADH1B和ALDH2基因的高分辨率熔解曲線分型的分析方法。 (3)根據(jù)HRM的分析結(jié)果,從各種基因型中選取一定數(shù)量的樣品進(jìn)行測(cè)序分析,以驗(yàn)證評(píng)價(jià)該方法的準(zhǔn)確性。并且選取每種ADH1B和ALDH2基因野生純合子和突變純合子各10個(gè)樣本進(jìn)行重復(fù)實(shí)驗(yàn)。同時(shí)對(duì)ADH1B基因的野生純合子和突變純合子樣本進(jìn)行了2倍稀釋法,進(jìn)行5個(gè)濃度檢測(cè),探討該方法檢測(cè)敏感性。 3.統(tǒng)計(jì)學(xué)分析：對(duì)構(gòu)建的ADH1B和ALDH2基因已知突變分型檢測(cè)體系進(jìn)行準(zhǔn)確性和穩(wěn)定性分析。利用部分樣本直接測(cè)序?qū)Ρ葋眚?yàn)證其準(zhǔn)確性；采用重復(fù)性實(shí)驗(yàn)以確定該體系的穩(wěn)定性與準(zhǔn)確性。同時(shí)分析東莞地區(qū)隨機(jī)漢族人群的ADH1B和ALDH2的基因頻率和基因型頻率,并應(yīng)用χ2檢驗(yàn)該群體樣品是否符合Hardy-Weinberg平衡。使用的統(tǒng)計(jì)學(xué)分析軟件為SPSS13.0。 4.實(shí)驗(yàn)結(jié)果的綜合分析、總結(jié)。 結(jié)果 建立了穩(wěn)定可靠的雙重HRM檢測(cè)體系。此體系中,所選取的擴(kuò)增目的DNA序列以及所設(shè)計(jì)的引物的特異性高。ADH1B和ALDH2基因的野生純合子G/G,雜合子G/A,突變純合子A/A三種基因型能夠在HRM上進(jìn)行明顯的區(qū)分。 用盲法分析對(duì)該方法進(jìn)行評(píng)價(jià),用此方法分析東莞地區(qū)200份漢族人基因組DNA樣品,從每組基因型中隨機(jī)選出部分樣品進(jìn)行測(cè)序?qū)φ?結(jié)果符合率為100%,證實(shí)了該方法的準(zhǔn)確性很好；進(jìn)行重復(fù)實(shí)驗(yàn),發(fā)現(xiàn)三次實(shí)驗(yàn)的變異系數(shù)(CV)的范圍從0.021%到0.062%,證實(shí)了該方法的穩(wěn)定性很好。在2倍稀釋試驗(yàn)中,5個(gè)濃度均可以準(zhǔn)確分型,可以知道6.25ng濃度仍然可以用于此檢測(cè)體系。 χ2檢驗(yàn)結(jié)果顯示所選群體符合Hardy-Weinberg平衡,ADH1B與ALDH2基因頻率與以前所報(bào)道的數(shù)據(jù)基本符合,從另一個(gè)方面說明了該方法的準(zhǔn)確性。 結(jié)論 飲酒已被認(rèn)為是危害人類健康的嚴(yán)重危險(xiǎn)因素,容易導(dǎo)致多種酒精相關(guān)疾病。酒精在體內(nèi)劑量效應(yīng)和作用時(shí)間,不僅僅與酒量和飲酒頻率相關(guān),還與易感基因ADH1B和ALDH2代謝能力有強(qiáng)關(guān)聯(lián)性。ADH1B和ALDH2兩種基因的變異與酒精依賴性疾病密切相關(guān),所以對(duì)ADH1B和ALDH2兩種基因進(jìn)行快速分型,在中國(guó)人群中進(jìn)行快速篩查,不僅能夠?qū)凭嚓P(guān)疾病風(fēng)險(xiǎn)進(jìn)行評(píng)估,還有助于研究ADH1B和ALDH2基因與其他相關(guān)疾病的致病機(jī)理。 高分辨率熔解曲線分析技術(shù)是近幾年興起一種基于核酸的物理性質(zhì)的基因突變檢測(cè)技術(shù)。這種檢測(cè)技術(shù)沒有局限于突變堿基位點(diǎn)與類型,不需使用序列特異性探針,在PCR結(jié)束后直接進(jìn)行高分辨率熔解曲線分析來完成對(duì)樣品基因型的分析。HRM因其操作簡(jiǎn)便、快速,成本低,結(jié)果準(zhǔn)確,高通量,并且閉管操作而備受關(guān)注。高分辨率熔解曲線分析技術(shù)只需要在普通PCR基礎(chǔ)上增加一個(gè)飽和染料既可以進(jìn)行未知突變掃描,也可以對(duì)已知突變進(jìn)行基因分型,還可以分析短片段重復(fù)序列。HRM分析的這些優(yōu)勢(shì)使它具有極強(qiáng)的可操作性,近年來成為國(guó)內(nèi)外新興的各研究領(lǐng)域?qū)W、方法學(xué)研究和應(yīng)用熱點(diǎn)。 本課題研究中建立的針對(duì)ADH1B和ALDH2基因的高分辨率熔解曲線分析檢測(cè)方法能夠快速、準(zhǔn)確地同時(shí)對(duì)ADH1B和ALDH2基因進(jìn)行檢測(cè),并且實(shí)驗(yàn)的重復(fù)性和穩(wěn)定性好、敏感性高、體系可靠。 本研究對(duì)中國(guó)南方人群進(jìn)行檢測(cè),發(fā)現(xiàn)所檢測(cè)的200人中不存在ADH1B*1/*1和ALDH2*2/*2組合的個(gè)體,所以推測(cè)這種基因型組合在中國(guó)南方是很少見的。有關(guān)文獻(xiàn)報(bào)道,ADH1B*1等位基因會(huì)增加食道癌,肝癌等癌癥發(fā)生的風(fēng)險(xiǎn)性。同時(shí)很多的研究表明ALDH2*1/*2或ALDH2*2/*2個(gè)體由于乙醛的積累導(dǎo)致其患有食道癌等癌癥的風(fēng)險(xiǎn)更大,然而同時(shí)對(duì)這兩個(gè)基因進(jìn)行聯(lián)合研究的很少,因此同時(shí)納入ADH1B和ALDH2兩基因的病例-對(duì)照研究是下一步進(jìn)行疾病風(fēng)險(xiǎn)研究的重點(diǎn)工作。 本研究為ADH1B和ALDH2基因的檢測(cè)建立了一種簡(jiǎn)單、高通量、快速、經(jīng)濟(jì)和靈敏的檢測(cè)方法,有助于酒精相關(guān)疾病的遺傳學(xué)咨詢,流行病學(xué)調(diào)查,并為研究ADH1B和ALDH2基因與其他相關(guān)疾病的關(guān)系提供了一項(xiàng)常規(guī)檢測(cè)手段。 本研究方法的一些處理技巧如改變擴(kuò)增子長(zhǎng)度以及加尾處理具有普遍的代表性與通用性,可為其它基因同時(shí)分型的檢測(cè)方法研究提供借鑒與參考。
[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.
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R346

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