CN1661094A - 等位特异性扩增与纳米金探针结合的基因点突变检测方法 - Google Patents

等位特异性扩增与纳米金探针结合的基因点突变检测方法 Download PDF

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CN1661094A
CN1661094A CN 200410077509 CN200410077509A CN1661094A CN 1661094 A CN1661094 A CN 1661094A CN 200410077509 CN200410077509 CN 200410077509 CN 200410077509 A CN200410077509 A CN 200410077509A CN 1661094 A CN1661094 A CN 1661094A
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gene
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primer
stranded dna
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CN100395349C (zh
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邢达
周政
朱德斌
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South China Normal University
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Abstract

本发明涉及一种等位特异性扩增与纳米金探针结合的基因点突变检测方法，包括：根据突变位点设计等位特异性引物，该引物的3’端碱基与突变型基因的突变碱基互补，而与野生型基因不互补；进行等位特异性扩增，突变型基因顺利扩增出双链DNA(称扩增产物1)；野生型基因不被扩增造成反应产物中含有未被消耗的单链DNA－引物(称扩增产物2)；向纳米金溶胶中依次加入扩增产物和电解质溶液，加入扩增产物1将导致溶液颜色由红色变蓝色；加入扩增产物2导致溶液仍保持红色。该法检测基因点突变直观，快速，简便，实验成本低，为点突变检测及单核苷酸多态性分析提供了一种实用的新方法。

Description

等位特异性扩增与纳米金探针结合的基因点突变检测方法
                        技术领域
本发明涉及一种用于基因点突变检测和单核苷酸多态性分析的等位特异性扩增与纳米金探针结合的基因点突变检测方法。
                        背景技术
目前，检测基因点突变的常用方法有：限制性片段长度多态性、变性梯度凝胶电泳、化学错配裂解、酶错配裂解、单链构象多态性、等位特异性探针杂交、连接酶链式反应和竞争性寡核苷酸引物等。这些基于电泳分析的传统检测方法大多存在操作复杂、费时、需要使用放射性元素或溴化乙锭等有害物质的缺点。
                        发明内容
本发明的目的在于提供一种用于基因点突变检测和单核苷酸多态性分析的等位特异性扩增与纳米金探针结合的基因点突变检测方法。该法操作简便、安全、快速，结果直观，无需特殊仪器，检测费用低。
本发明的等位特异性扩增与纳米金探针结合的基因点突变检测方法包括如下步骤：
(1)根据突变位点设计等位特异性引物，该引物的3’端碱基与突变型基因的突变碱基互补，而与野生型基因不互补。
(2)进行等位特异性扩增，突变型基因顺利扩增出双链DNA(称扩增产物1)；而野生型基因不被扩增，反应产物中含有大量未被消耗的单链DNA—引物(称扩增产物2)。
(3)向纳米金溶胶中依次加入扩增产物和电解质溶液。当加入扩增产物1时，因为纳米颗粒表面没有吸附DNA，加入电解质溶液将导致纳米金聚集，溶液颜色由红色变蓝色；而当加入扩增产物2时，单链DNA被吸附到纳米金颗粒表面，使其保持稳定，加入电解质溶液后溶液仍保持红色。
本发明是将等位特异性扩增和纳米金探针结合进行基因点突变检测，其检测基本原理如下：在纳米金颗粒形成聚集的过程中，由于表面共振吸收光谱峰值的改变，其溶液颜色随聚集程度逐渐由红色变成蓝色。在纳米金溶胶中加入电解质溶液，能够引起纳米颗粒的聚集，溶胶颜色变蓝；而表面吸附了DNA的纳米金颗粒能在电解质溶液中保持稳定状态，溶液颜色不变。当纳米金分别与单、双链DNA混合后，加入电解质溶液，由于单链DNA和双链DNA与纳米金颗粒存在不同的静电作用，导致单链DNA能被吸附到纳米金颗粒表面，使得纳米金颗粒在电解质溶液中不发生聚集；而双链DNA不被吸附，纳米金颗粒在电解质溶液中发生聚集。在进行等位基因特异性扩增时，3’碱基在DNA延伸过程中起着重要作用，当引物3’末端碱基与模板互补时，DNA延伸，产生扩增产物；否则无扩增产物产生。因此，采用突变型引物对待测基因进行等位特异性扩增，突变型基因扩增产物中大部分是双链DNA；而野生型基因由于不被扩增，产物中大部分是未被消耗的单链引物。向纳米金溶胶中依次加入扩增产物和电解质溶液，单链DNA使混合液不变色，双链DNA使混合液变蓝；即以野生型基因为模板扩增的样品仍然保持红色，以突变型基因为模板扩增的样品颜色变蓝。根据野生型和突变型基因分别呈现出不同的颜色，可以直接判断待测样品的基因型。
与现有方法相比，该法具有操作简便、安全、快速，结果直观，无需特殊仪器，检测费用低等优点。
                      具体实施方式
我们将等位特异性扩增与纳米金探针结合的基因点突变检测方法应用于检测K-ras基因密码子12处的突变热点，具体步骤如下：
(1)根据K-ras基因序列，设计突变型引物；
(2)分别以野生型和突变型基因序列为模板，进行等位特异性扩增；
(4)向200μl纳米金溶胶中加入10μl上述扩增产物；
(5)加入60μl PBS溶液(10mM，0.2M NaCl)，观察颜色变化，用数码相机拍摄实验结果。
结果显示：突变型基因颜色由红色变成蓝色；野生型基因仍然保持红色。

Claims (1)

1、一种等位特异性扩增与纳米金探针结合的基因点突变检测方法，其特征在于包括如下步骤：
(1)根据突变位点设计等位特异性引物，该引物的3’端碱基与突变型基因的突变碱基互补，而与野生型基因不互补；
(2)进行等位特异性扩增，突变型基因顺利扩增出双链DNA(称扩增产物1)；野生型基因不被扩增造成反应产物中含有未被消耗的单链DNA-引物(称扩增产物2)；
(3)向纳米金溶胶中依次加入扩增产物和电解质溶液，加入扩增产物1将导致溶液颜色由红色变蓝色；加入扩增产物2导致溶液仍保持红色。
CNB2004100775092A 2004-12-22 2004-12-22 等位特异性扩增与纳米金探针结合的基因点突变检测方法 Expired - Fee Related CN100395349C (zh)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008029374A2 (en) 2006-09-08 2008-03-13 Universidade Nova De Lisboa Detection and quantification system of biological matter constituted by one or more optical sensors and one or more light sources, associated process and related applications
CN104928349A (zh) * 2015-05-29 2015-09-23 西安交通大学 一种基于纳米金颗粒的单核苷酸多态性检测方法
CN108220328A (zh) * 2017-12-15 2018-06-29 中国烟草总公司郑州烟草研究院 一种鉴定基因组编辑纯合突变体的方法

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008029374A2 (en) 2006-09-08 2008-03-13 Universidade Nova De Lisboa Detection and quantification system of biological matter constituted by one or more optical sensors and one or more light sources, associated process and related applications
CN104928349A (zh) * 2015-05-29 2015-09-23 西安交通大学 一种基于纳米金颗粒的单核苷酸多态性检测方法
CN104928349B (zh) * 2015-05-29 2018-01-05 西安交通大学 一种基于纳米金颗粒的单核苷酸多态性检测方法
CN108220328A (zh) * 2017-12-15 2018-06-29 中国烟草总公司郑州烟草研究院 一种鉴定基因组编辑纯合突变体的方法

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