【摘要】 聚合酶链反应(polymerase chain reaction,PCR)是分子生物学领域的一项具有划时代意义的技术,但定量PCR产物或测定能生成焦磷酸的酶活性仍需要新技术的发展。本文提供了一种PCR产物定性及定量检测方法(Color PCR kit)及其用途;该方法通过焦磷酸(pyrophosphate,PPi)显色测定PCR的副产物PPi。利用试剂盒中的试剂与PPi反应,最终生成物为甲暨(formazan),呈红色。根据显色现象(红色)判断PCR的阳性结果,由目测实现PCR的定性检测;或通过测定490 nm处的吸光度值定量检测PCR过程中生成的副产物PPi的含量,定量检测PCR产物的生成量;目测情况下Color PCR kit可检测到2.5 ng水平的PCR产物,用紫外分光光度计可检测到低限为1 pg;Color PCR kit法比琼脂糖凝胶电泳检测法(低限为4 ng)灵敏。Color PCR kit还可用于连接酶和转移酶活性的测定。更多还原

【Abstract】 Polymerase chain reaction( PCR) is a critical technology that has revolutionized the field of molecular biology. Quantification of PCR products or other pyrophosphate-producing enzyme activity is a major challenge. This study introduced a novel colorimetric PCR kit to detect and quantify PCR products or activities of pyrophosphate-producing enzymes. The principle of this kit is to measure inorganic pyrophosphate( PPi) generated by PCR amplification or other pyrophosphate-producing enzyme activity assay. Total PPi reacts with inosine 5’-monophosphate under catalysis of hypoxanthine-guanine phosphoribosyl transferase to yield hypoxanthine and phosphoribosyl pyrophosphate. The hypoxanthine is then oxidized to uric acid by xanthine oxidase / xanthine dehydrogenase and can be measured by formation of formazan in red color when a tetrazolium salt is used as the oxidant. This kit is suitable for quantifying the activity of DNA polymerase I,T4 DNA ligase,and terminal deoxynucleotidyl transferase( Td T)where PPi is a product. Our data indicated that this color PCR kit is sensitive,accurate,precise,and linear in quantifying PCR products or activity of DNA polymerase I,T4 DNA ligase,and Td T compared to standard techniques,such as agarose-gel electrophoresis,32 P,and3H incorporation methods. This study suggests that this color PCR kit is an alternative multi-purpose tool that allows rapid,simple,and cost-effective detection of large amounts of amplified nucleic acid samples,enzyme activities,and PPi amounts.更多还原