基于PCR和位点特异性引物延伸反应的SNP检测方法的建立

doi:10.3969/j.issn.1673-8640.2018.06.015

摘要/Abstract

摘要：

目的建立一种基于聚合酶链反应（PCR）、位点特异性引物延伸反应（ASE）和核酸试纸条检测技术的单核苷酸多态性（SNP）检测方法。方法先通过PCR对包含SNP位点的特异基因序列进行扩增;然后通过带有A标记的ASE引物针对SNP位点的不同基因类型进行特异性延伸;延伸后的产物可以与B标记的探针进行杂交结合,形成同时携带A和B标记的杂交产物;而该杂交产物可以通过核酸试纸条进行目视化检测,从而完成对SNP基因型的检测。结果通过对10倍浓度梯度稀释的人类基因组DNA进行检测,PCR-ASE的检测敏感性为88 ng/反应;通过在同一条核酸试纸条上针对同一SNP位点2种不同基因型的检测,达到了多重检测的目的;PCR-ASE对19名志愿者的5个不同SNP位点的检测结果与基因测序法完全一致。结论 PCR-ASE 是一种简单、准确的SNP基因型检测方法。

关键词: 引物特异性延伸反应, 单核苷酸多态性, 核酸试纸条检测技术

Abstract:

Objective To establish polymerase chain reaction（PCR）,allele-specific extension（ASE） and nucleic acid detection strip-based detection for single nucleotide polymorphisms（SNP）. Methods The specific gene sequence containing SNP sites was amplified by PCR. For each SNP site,ASE primers labeled with A were extended. The ASE reaction products can bind to probe labeled with B,and hybridization products containing A and B simultaneously were formed. The products can be detected by disposable amplicon cross-contamination proof device containing a nucleic acid detection strip,and the detection of SNP genotypes can be accomplished. Results Ten-fold serial dilutions of quantified human genomic DNA were used to determine the sensitivity of PCR-ASE（88 ng/reaction）. The ability of duplex PCR-ASE with the products can be detected by a single nucleic acid detection strip. A total of 19 samples representing 5 common SNP were detected by PCR-ASE,and the results had the consistency of 100% with DNA sequencing. Conclusions PCR-ASE is simple and accurate detection for SNP.

Key words: Allele-specific extension, Single nucleotide polymorphism, Nucleic acid detection strip-based detection

中图分类号:

R446.1

单洪波, 金亚南. 基于PCR和位点特异性引物延伸反应的SNP检测方法的建立[J]. 检验医学, 2018, 33(6): 530-535.

SHAN Hongbo, JIN Yanan. Establishment of PCR and ASE-based detection for SNP[J]. Laboratory Medicine, 2018, 33(6): 530-535.

图/表 4

参考文献 14

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引物名称	基因型	序列信息（5'~3'）	长度（bp）
P53 282PF		TCCTGCTTGCTTACCTCGCTTAG	23
P53 282PR		CTGATTTCCTTACTGCCTCTTGC	23
P53 282-C	G/A	FITC-GCTTTGAGGTGcG	13
P53 282-T		FITC-GCTTTGAGGTGtG	13
P53 282DP		TCCCAGGACAGGCA-BIOTIN	14
rs 6323PF		GCGATCCCTCCGACCTTGACT	21
rs 6323PR		CTTCTTCCAGAAGGCCTCCTTG	22
rs 6323-G	G/T	FITC-CCCATTGGAAGgC	13
rs 6323-T		FITC-CCCATTGGAAGtC	13
rs 6323DP		AGAGAGAAACCAGTTA-BIOTIN	16
rs 4532PF		GGTGGGGAGGACTCCTGGA	19
rs 4532PR		TGTTCGTATCCTCACTGCCTGT	22
rs 4532-A	A/G	FITC-ATTCCCTGCTTaG	13
rs 4532-G		FITC-ATTCCCTGCTTgG	13
rs 4532DP		AGTCCTCATCTTCCT-BIOTIN	15
rs 6318PF		GCATGAGCAACGTATTGTGTATAAG	25
rs 6318PR		GACGATTGAAAGTGCTGGCCAG	22
rs 6318-G	G/C	FITC-GGTTTGGCAATgT	13
rs 6318-C		FITC-GGTTTGGCAATcT	13
rs 6318DP		CGTCTGGGAATTTGA-BIOTIN	15

引物名称	基因型	序列信息（5'~3'）	长度（bp）
P53 282PF		TCCTGCTTGCTTACCTCGCTTAG	23
P53 282PR		CTGATTTCCTTACTGCCTCTTGC	23
P53 282-C	G/A	FITC-GCTTTGAGGTGcG	13
P53 282-T		FITC-GCTTTGAGGTGtG	13
P53 282DP		TCCCAGGACAGGCA-BIOTIN	14
rs 6323PF		GCGATCCCTCCGACCTTGACT	21
rs 6323PR		CTTCTTCCAGAAGGCCTCCTTG	22
rs 6323-G	G/T	FITC-CCCATTGGAAGgC	13
rs 6323-T		FITC-CCCATTGGAAGtC	13
rs 6323DP		AGAGAGAAACCAGTTA-BIOTIN	16
rs 4532PF		GGTGGGGAGGACTCCTGGA	19
rs 4532PR		TGTTCGTATCCTCACTGCCTGT	22
rs 4532-A	A/G	FITC-ATTCCCTGCTTaG	13
rs 4532-G		FITC-ATTCCCTGCTTgG	13
rs 4532DP		AGTCCTCATCTTCCT-BIOTIN	15
rs 6318PF		GCATGAGCAACGTATTGTGTATAAG	25
rs 6318PR		GACGATTGAAAGTGCTGGCCAG	22
rs 6318-G	G/C	FITC-GGTTTGGCAATgT	13
rs 6318-C		FITC-GGTTTGGCAATcT	13
rs 6318DP		CGTCTGGGAATTTGA-BIOTIN	15

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