《中国畜牧兽医》 ›› 2016, Vol. 43 ›› Issue (10): 2566-2571.doi: 10.16431/j.cnki.1671-7236.2016.10.009

• 生物技术 • 上一篇    下一篇

应用巢式PCR法检测猪单个胚胎中的基因突变

任红艳, 肖红卫, 李晓敏, 徐振宇, 毕延震, 华再东, 郑新民   

  1. 湖北省农业科学院畜牧兽医研究所, 动物胚胎工程及分子育种湖北省重点实验室, 武汉 430064
  • 收稿日期:2016-03-04 出版日期:2016-10-20 发布日期:2016-10-28
  • 通讯作者: 郑新民 E-mail:anbit20@163.com
  • 作者简介:任红艳(1981-),女,陕西富平人,博士,副研究员,研究方向:动物生物技术,E-mail:renhongyan507@163.com
  • 基金资助:

    转基因生物新品种培育重大专项(2016ZX08006001-005、2016ZX08006002-006、2016ZX08010003-006);湖北省农业科技创新中心(2016-620-000-001-027);湖北省科技支撑计划(2014BBB010);湖北省农科院青年基金(2014NKYJJ02、2016NKYJJ19)

Establishment and Application of Nested PCR Method for Detecting Mutation in Single Cell

REN Hong-yan, Xiao Hong-wei, LI Xiao-min, XU Zhen-yu, BI Yan-zhen, HUA Zai-dong, ZHENG Xin-min   

  1. Key Laboratory of Animal Embryo Engineering and Molecular Breeding of Hubei Province, Institute of Animal and Veterinary Sciences, Hubei Academy of Agricultural Sciences, Wuhan 430064, China
  • Received:2016-03-04 Online:2016-10-20 Published:2016-10-28

摘要:

单胚胎基因突变检测技术是以单个胚胎的裂解产物为模板进行DNA或RNA分析的方法,该技术的建立将为在单细胞水平进行基因表达和突变检测提供简便而快捷的方法。本研究设计了2对巢式引物,以显微注射了Cas9 mRNA和猪SS基因2个靶点的gRNA的四细胞期孤雌胚胎为试验材料,通过蛋白酶K对猪单个胚胎进行裂解后直接进行巢式PCR分型,并检测到猪胚胎中SS基因的删除突变。该方法可以最大限度减少假阴性和假阳性结果的出现,在猪基因突变检测中具有重要作用。

关键词: 巢式PCR; 单细胞裂解; CRISPR/Cas9技术; 基因突变

Abstract:

Gene mutation detection in single cell is a new method used for DNA or RNA analysis based on single cell lysis products,the establishment of this technology will provide an easy and quick way for gene expression and mutation detection in single cell level.In this paper,pig 4-cell-stage parthenogenetic embryos microinjected with Cas9 mRNA and 2 target gRNA from pig SS gene were used as experimental material,and two pairs of nested primers were designed for nested PCR.Using this detection method,long fragment deletion in SS gene was detected.This method can minimize the occurrence of false negative and false positive results,which may play an important role in the pig gene mutation detection.

Key words: nested PCR; single cell lysis; CRISPR/Cas9 system; gene mutation

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