全血和滤纸干血AS-PCR体系的缓冲液研究

发布时间：2018-03-26 11:55

  本文选题：缓冲液　切入点：全血扩增　出处：《中国新药杂志》2016年24期

【摘要】：目的:研制一种以全血和滤纸干血为模板的等位基因特异性PCR(allele-specific polymerase chain reaction,AS-PCR)体系的缓冲液。方法:采用血样直接作为模板,通过引入错配碱基对AS-PCR的特异性进行改进,研制一种新型缓冲液HPEC[p H 9.5,Mg2+3.5 mmol·L~(-1),海藻糖2.5μmol·L~(-1),(NH4)2SO49.6mmol·L~(-1),Tween-20 0.01%],对包含单核苷酸多态性位点(single nucleotide polymorphisms,SNPs)的目的片段进行扩增。结果:该方法成功地实现了以血样和滤纸干血为模板的AS-PCR基因分型。结论:此缓冲液省去了扩增前基因组的提取和纯化,简化了操作过程,降低DNA污染机会和检测技术的复杂性。
[Abstract]:Objective: to develop a buffer solution of allele-specific PCR(allele-specific polymerase chain reactionsis-AS-PCR system with whole blood and filter paper dry blood as template. Methods: the specificity of AS-PCR was improved by introducing mismatch base. A novel buffer solution, HPEC [p H 9.5 mg 23. 5 mmol / L HPEC, trehalose 2. 5 渭 mol L ~ (-1)), NH _ 4N _ 2SO _ 4 9. 6 mmol / L ~ (-1) ~ (-1)] was developed to amplify the target fragment containing single nucleotide polymorphisms (SNPs). Results: this method successfully realized the AS-PCR base using blood samples and filter paper dry blood as templates. Conclusion: this buffer eliminates the extraction and purification of the genome before amplification. It simplifies the operation process, reduces the chance of DNA pollution and the complexity of detection technology.
【作者单位】： 解放军211医院;华侨大学生物医学学院;
【基金】：福建省自然科学基金资助项目(2015J01342)
【分类号】：R440
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本文编号：1667795