CN110283900A - 一种检测ALDH2基因rs671多态性的引物及其应用 - Google Patents
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Abstract
本发明提供了一种检测ALDH2基因rs671多态性的引物，包括一对上下游引物，还提供了应用，用于ALDH2基因rs671多态性的检测，该检测方法为：提取人体外周血的总DNA，提取的总DNA为模板，以上下游引物进行降落式PCR扩增，得到PCR扩增产物，进行酶切后，电泳法检测ALDH2基因rs671多态性。本发明的ALDH2基因rs671多态性的引物在应用于ALDH2基因rs671多态性的检测中，用本发明的检测ALDH2基因rs671多态性的引物，扩增采用降落式PCR扩增，一步到位，PCR扩增产物不需要纯化可以直接进行酶切，采用限制性核酸内切酶，20分钟之内就可以完成酶切，缩短了检测周期，且成本低，能够实现ALDH2基因rs671多态性的快速检测。
Description
技术领域
本发明属于分子检测技术领域，具体涉及一种检测ALDH2基因rs671多态性的引物及其应用。
背景技术
基因突变是指，由于DNA分子中发生碱基对的增添、缺失或替换，而引起的基因结构的改变，基因突变在自然界各物种中普遍存在。不论是真核生物还是原核生物的突变，也不论是什么类型的突变，基因突变都具有随机性、低频性和可逆性等共同的特性。基因突变后会导致体内代谢的改变，比如酒精在体内的代谢，饮酒后乙醇主要在小肠吸收，其中90％～95％在肝内代谢。乙醇在肝脏内被胞质内的ADH(己二酸二酰肼)和微粒体内的CYP2EI为主的微粒体乙醇氧化系统分解为乙醛，乙醛主要经过ALDH(乙醛脱氢酶)代谢为乙酸，后者进入三羧酸循环，代谢为二氧化碳和水。人类ALDH基因家族中有12个成员，其中ALDH2(乙醛脱氢酶2)是和嗜酒密切相关并具有多态性的主要代谢酶，在亚洲人群有高度遗传多态性。ALDH2基因位于染色体12q24.2，有2个等位基因，若ALDH2的基因rs671多态性的碱基为G/G即为野生型，判定为对乙醛的分解能力高，但是，若是杂合型G/A、突变型的碱基的纯合型A/A，则对于乙醛的分解能力低，从而使ALDH2丧失酶活性，使乙醛在体内蓄积，体内过量的乙醛会增加酒精性肝病的患病风险。
现有检测ALDH2基因多态性的方法通常采用PCR荧光探针法，往往需要多次扩增，对产物进行优化才能满足检测需要，成本较高；也有采用酶切法，但是扩增引物往往不理想，扩增程序待优化，导致需要二次扩增或者对扩增产物进行纯化才能进行酶切反应。
发明内容
本发明所要解决的技术问题在于针对上述现有技术的不足，提供一种检测ALDH2基因rs671多态性的引物及其应用，该ALDH2基因rs671多态性的引物在应用于ALDH2基因rs671多态性的检测中，用本发明的检测ALDH2基因rs671多态性的引物，扩增采用降落式PCR扩增，一步到位，PCR扩增产物不需要纯化可以直接进行酶切，采用限制性核酸内切酶，20分钟之内就可以完成酶切，缩短了检测周期，且成本低，能够实现ALDH2基因rs671多态性的快速检测。
为解决上述技术问题，本发明采用的技术方案是：一种检测ALDH2基因rs671多态性的引物，所述引物为一对上下游引物，上游引物的核苷酸序列为：5′-AACCCATAACCCCCAAGAGT-3′，下游引物的核苷酸序列为：5′-CCCAGCAAATGACCGCATAG-3′。
本发明还提供了上述的检测ALDH2基因rs671多态性的引物的应用，所述引物用于ALDH2基因rs671多态性的检测，该检测方法为：
S1、基因提取：提取人体外周血的总DNA；
S2、基因扩增：以S1中得到的提取人体外周血的总DNA为模板，以上下游引物进行降落式PCR扩增，得到PCR扩增产物；所述扩增的程序为：94℃预变性5min；94℃变性30s，60℃退火30s，72℃延伸45s，循环2次；94℃变性30s，59℃退火30s，72℃延伸45s，循环2次；94℃变性30s，58℃退火30s，72℃延伸45s，循环2次；94℃变性30s，57℃退火30s，72℃延伸45s,循环2次；94℃变性30s，56℃退火30s，72℃延伸45s,循环2次；94℃变性30s，58℃退火30s，72℃延伸45s,循环20次；72℃延伸5min；所述扩增的体系为2×PCR反应预混液12μL、上下游引物各1μL、人体外周血的总DNA 1μL、重蒸去离子水补充体积至25μL；
S3、酶切程序：将S2中得到的PCR扩增产物在温度为65℃的条件下酶切20min，得到酶切产物；所述酶切的体系为：限制性核酸内切酶2μL，PCR缓冲液4μL，PCR扩增产物5μL，重蒸去离子水补充体积至30μL；
S4、将S3中得到的酶切产物用1％琼脂糖凝胶电泳法检测ALDH2基因rs671多态性。
优选地，S1中所述人体外周血的用量为100μL。
本发明与现有技术相比具有以下优点：
本发明的检测ALDH2基因rs671多态性的引物在应用于ALDH2基因rs671多态性的检测中，用本发明的检测ALDH2基因rs671多态性的引物，扩增采用降落式PCR扩增，一步到位，PCR扩增产物不需要纯化可以直接进行酶切，采用限制性核酸内切酶，20分钟之内就可以完成酶切，缩短了检测周期，且成本低，能够实现ALDH2基因rs671多态性的快速检测。
下面结合附图和实施例对本发明作进一步详细说明。
附图说明
图1是本发明的ALDH2基因rs671多态性的电泳图。
具体实施方式
实施例1
本实施例的检测ALDH2基因rs671多态性的引物，所述引物为一对上下游引物，上游引物的核苷酸序列为：5′-AACCCATAACCCCCAAGAGT-3′，下游引物的核苷酸序列为：5′-CCCAGCAAATGACCGCATAG-3′。
本实施例还提供了上述的检测ALDH2基因rs671多态性的引物的应用，所述引物用于ALDH2基因rs671多态性的检测，该检测方法为：
S1、基因提取：取人体外周血100μL，提取人体外周血的总DNA；
S2、基因扩增：以S1中得到的提取人体外周血的总DNA为模板，以上下游引物进行降落式PCR扩增，得到PCR扩增产物；所述扩增的程序为：94℃预变性5min；94℃变性30s，60℃退火30s，72℃延伸45s，循环2次；94℃变性30s，59℃退火30s，72℃延伸45s，循环2次；94℃变性30s，58℃退火30s，72℃延伸45s，循环2次；94℃变性30s，57℃退火30s，72℃延伸45s,循环2次；94℃变性30s，56℃退火30s，72℃延伸45s,循环2次；94℃变性30s，58℃退火30s，72℃延伸45s,循环20次；72℃延伸5min；所述扩增的体系为2×PCR反应预混液12μL、上下游引物各1μL、人体外周血的总DNA 1μL、重蒸去离子水补充体积至25μL；所述2×PCR反应预混液市购。
S3、酶切程序：将S2中得到的PCR扩增产物在温度为65℃的条件下酶切20min，得到酶切产物；所述酶切的体系为：限制性核酸内切酶2μL，PCR缓冲液4μL，PCR扩增产物5μL，重蒸去离子水补充体积至30μL；
S4、将S3中得到的酶切产物用4％琼脂糖凝胶电泳法检测ALDH2基因rs671多态性。
如图1所示，由ALDH2基因rs671多态性的检测结果可知，图中孔道M为DNA分子量标记，孔道1为S2中得到的PCR扩增产物，作为对照，孔道2表示人体的ALDH2基因rs671多态性为杂合型G/A，孔道4表示人体的ALDH2基因rs671多态性为突变型的碱基的纯合型A/A，杂合型G/A和突变型的碱基的纯合型A/A的人体对于乙醛的分解能力低，从而使ALDH2丧失酶活性，使乙醛在体内蓄积，体内过量的乙醛会增加酒精性肝病的患病风险，孔道3表示人体的ALDH2基因rs671多态性为野生型G/G，该人体对乙醛的分解能力高。
以上所述，仅是本发明的较佳实施例，并非对本发明作任何限制。凡是根据发明技术实质对以上实施例所作的任何简单修改、变更以及等效变化，均仍属于本发明技术方案的保护范围内。
Claims (3)
1.一种检测ALDH2基因rs671多态性的引物，其特征在于，所述引物为一对上下游引物，上游引物的核苷酸序列为：5′-AACCCATAACCCCCAAGAGT-3′，下游引物的核苷酸序列为：5′-CCCAGCAAATGACCGCATAG-3′。
2.一种如权利要求1所述的检测ALDH2基因rs671多态性的引物的应用，其特征在于，所述引物用于ALDH2基因rs671多态性的检测，该检测方法为：
S1、基因提取：提取人体外周血的总DNA；
S2、基因扩增：以S1中得到的提取人体外周血的总DNA为模板，以上下游引物进行降落式PCR扩增，得到PCR扩增产物；所述扩增的程序为：94℃预变性5min；94℃变性30s，60℃退火30s，72℃延伸45s，循环2次；94℃变性30s，59℃退火30s，72℃延伸45s，循环2次；94℃变性30s，58℃退火30s，72℃延伸45s，循环2次；94℃变性30s，57℃退火30s，72℃延伸45s,循环2次；94℃变性30s，56℃退火30s，72℃延伸45s,循环2次；94℃变性30s，58℃退火30s，72℃延伸45s,循环20次；72℃延伸5min；所述扩增的体系为2×PCR反应预混液12μL、上下游引物各1μL、人体外周血的总DNA1μL、重蒸去离子水补充体积至25μL；
S3、酶切程序：将S2中得到的PCR扩增产物在温度为65℃的条件下酶切20min，得到酶切产物；所述酶切的体系为：限制性核酸内切酶2μL，PCR缓冲液4μL，PCR扩增产物5μL，重蒸去离子水补充体积至30μL；
S4、将S3中得到的酶切产物用4％琼脂糖凝胶电泳法检测ALDH2基因rs671多态性。
3.根据权利要求2所述的应用，其特征在于，S1中所述人体外周血的用量为100μL。
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