焦磷酸显色法检测PCR产物及酶活性

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。

Development of a New Colorimetric PCR Kit for the Detection and Quantification of PCR Products and Activities of Pyrophosphate-Producing Enzymes

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 (TdT) 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 TdT compared to standard techniques, such as agarose-gel electrophoresis, 32P, and 3H 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.