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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。
Objective: To develop an allele-specific polymerase chain reaction (AS-PCR) buffer system using whole blood and filter paper as template. Methods: A new buffer HPEC [p H 9.5, Mg2 + 3.5 mmol·L -1, trehalose was prepared by using the blood sample directly as template to improve the specificity of AS-PCR by introducing mismatched bases. 2.5μmol·L -1, (NH 4) 2SO49.6 mmol·L -1, Tween-20 0.01%] were used to detect the single nucleotide polymorphisms (SNPs) The purpose of the fragment for amplification. RESULTS: This method successfully achieved AS-PCR genotyping using blood and filter paper as a template. Conclusion: This buffer eliminates the need for pre-amplification genomic extraction and purification, simplifies procedures and reduces the complexity of DNA contamination opportunities and detection techniques.