论文部分内容阅读
目的 探讨硫化修饰的次 3′末端不配对引物能否引发高保真 DNA聚合酶介导的聚合反应的非成熟性终止 ,即所谓聚合反应的“关”效应。方法 采用配对及非末端不配对的 3′硫化修饰引物 ,研究其对不同保真度 DNA聚合酶引物延伸反应的影响。结果 非末端不配对的 3′硫化修饰引物也能引发高保真 DNA聚合酶介导的聚合反应非成熟性终止 ,而对低保真 DNA聚合酶所介导的聚合反应则无明显影响。同时 ,3′硫化修饰的配对引物对不同保真度 DNA聚合酶引物延伸反应均无影响。结论 硫化修饰的次 3′末端不配对引物与 3′末端不配对引物对引物延伸的影响相似 ,同样能引起高保真 DNA聚合酶介导的聚合反应的“关”的效应。显然 ,在单基因遗传病的诊断及单核苷酸多态性 (single nucleotide polymorphism,SNP)的高通量分析等方面 ,硫化修饰的碱基特异性引物与高保真 DNA聚合酶所构成的对 SNP敏感的“开 /关”系统 ,具有广阔的应用前景。
OBJECTIVE: To investigate if immature termination of high-fidelity DNA polymerase-mediated polymerization by sulphided modified minor 3 ’unpaired primers, the so called “off” effect of polymerization. Methods Pairwise and non-paired 3 ’vulcanization primers were used to study the effect of different fidelity DNA polymerase primer extension reactions. Results Non-terminal, unpaired 3 ’vulcanization primers also induced immature termination of high-fidelity DNA polymerase-mediated polymerization without significant effect on the polymerization mediated by low-fidelity DNA polymerase. At the same time, the 3’-sulfated paired primers had no effect on the extension of primer with different fidelity DNA polymerase. Conclusion Sulfidated minor 3 ’uncoordinated primers have similar effects on primer extension as 3’ uncoordinated primers, and can also cause the “off” effect of high-fidelity DNA polymerase-mediated polymerization. Clearly, in the diagnosis of single-gene disease and single-nucleotide polymorphism (single nucleotide polymorphism, SNP) high-throughput analysis, etc., sulfuration modified base-specific primers and high-fidelity DNA polymerase consisting of SNP sensitive “on / off” system, has broad application prospects.