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Digestion-based SNP Genotyping Method   

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实验原理:SNP差异是在实验过程中经常遇到的基因型多态性类型,传统方法可以通过dCAPS或者测序的方法进行鉴定,但不是每一个SNP位点都能设计出dCAPS引物,而测序成本太高,难以进行规模化鉴定。来自芹菜的核酸酶CEL I可以特异识别DNA中的错配碱基而切割DNA,基于此,可以通过设计跨SNP位置的引物经PCR扩增后通过CEL I酶切跑胶检测来确定基因型。杂合基因型AB的PCR产物,会形成有碱基错配的异源双链DNA,可被CEL I识别并切割,跑胶会出现两条带。野生基因型AA和突变基因型BB在进行PCR扩增时加入一个已知亲本(A)的DNA到所有DNA中,然后PCR扩增,创造人工杂合基因型,如果此时可以被CEL I切割,则表明这份检测样品的基因型为BB,如果依然不能被CEL I切割,则表明此份DNA为AA基因型,因此通过两轮PCR(不加亲本A和加亲本A的DNA)结合CEL I酶切,可以准确的鉴定3种基因型。
实验目的:SNP基因分型

关键词: SNP, 基因分型, 异源双链

材料与试剂

  1. HEPES
  2. KCl
  3. MgSO4
  4. Triton X-100
  5. BSA
  6. 核酸酶CEL I (来自芹菜) 或者SURVEYOR® Nuclease Kits (Transgenomic, catalog number: 706020)
    注:如果使用CEL I则需要自己制备酶的粗提物 (Till等, 2006)。
  7. CEL I酶粗提物缓冲液 (见溶液配方)

仪器设备

  1. PCR仪
  2. 电泳仪

实验步骤

  1. 设计PCR引物,使得扩增产物跨越待检测的SNP位点,按常规体系和方法进行PCR反应。在普通扩增PCR程序后面加上一步变性-退火步骤以便SNP位点异源双链DNA的形成:99°C,10 min;70 °C,20 s (70次循环,每次降温0.3 °C);降到15 °C保存。
  2. 为了鉴定基因型,每个样品需要进行两次PCR,一次正常操作 (不加已知亲本),另一次混入等量的一个已知亲本的DNA再进行PCR。
  3. CEL I酶切检测PCR产物
    PCR产物
    9 μl
    CEL I
    0.3 μl
    Buffer
    1.5 μl
    ddH2O
    4.2 μl
    45 °C反应15 min,加入3 μl,0.5 M EDTA (pH 8.0)终止反应
  4. 常规琼脂糖电泳检测酶切产物,按照实验原理所述进行基因型判断。

溶液配方

  1. CEL I酶粗提物10x缓冲液
    0.1 M HEPES pH 7.5
    0.1 M KCl
    0.1 M MgSO4
    0.02% Triton X-100
    0.002 mg/ml BSA

致谢

感谢中国科学院植物研究所刘春明研究员对本方法体系建立所提供的帮助。

参考文献

  1. Till, B. J., Zerr, T., Comai, L. and Henikoff, S. (2006). A protocol for TILLING and Ecotilling in plants and animals. Nat Protoc 1(5): 2465-2477.
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
引用格式:王磊, 符德保, 欧阳亦聃. (2018). 基于酶切方法的SNP基因分型. Bio-101: e1010109. DOI: 10.21769/BioProtoc.1010109.
How to cite: Wang, L., Fu, D. B. and Ouyang, Y. D. (2018). Digestion-based SNP Genotyping Method. Bio-101: e1010109. DOI: 10.21769/BioProtoc.1010109.
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