CEL I粗提物用于点突变检测的技术

本文以含有单个错配的DNA异质双链为底物鉴定芹菜CEL I粗提物的错配切割酶学活性,并对影响其切割错配的反应条件进行优化,建立以CEL I粗提物为基础的点突变酶学检测技术.结果表明:自制的CEL I粗提物能有效识别和切割DNA异质双链中碱基错配.当异质双链DNA量恒定时,在一定的范围内CEL I粗提物用量对错配切割效果...

A DETECTION METHOD FOR POINT MUTATIONS BASED ON CEL I CRUDE EXTRACT

Enzymatic activity for mismatch cleavage of CEL I crude extract from celery was characterized using DNA hexteroduplexes, which contained a single mismatch as subtracts. An enzymatic method based on the CEL I crude extract for detection of point mutations was developed after optimization of reaction conditions for its mismatch cleavage. Results showed that the mismatch in DNA hexteroduplexes could be effectively recognized and cleaved by the self-made CEL I crude extract. Effect of amounts of CEL I crude extract within a certain range was not significant when the quantity of DNA hexteroduplexes was definite, and usually the amount of CEL I crude extract containing total protein about 900ng could be used for effective cleavage of the mismatch in DNA hexteroduplexes formed by 12μl PCR products. Common PCR buffer was similar to CEL I buffer reported for the mismatch cleavage and the former would be feasible in practice. Effect of buffer pH on the mismatch cleavage was significant and the near neutral pH (6.5~7.5) was suitable for the mismatch cleavage by CEL I crude extract. Zn2+ could promote the mismatch cleavage, with a suitable concentration about 0.25mmol/L, but not necessary. The feasible reaction time was 20min at 42℃ for the mismatch cleavage by CEL I crude extract. Comparison test showed that the result of detecting point mutations by our method was almost the same as that by the kit surveyorTM from Transgenomic Company.