摘要
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的影响不明显,由PCR产物形成的12μl异质双链DNA可用蛋白质总量约900ng的CEL I粗提物进行有效切割。常规PCR缓冲液与已报道的CEL I缓冲液的效果基本相近,实际工作中可选用PCR缓冲液作CEL I酶切反应液;缓冲液pH值对错配切割效果具有明显的影响,适宜pH约近中性(6.5～7.5)。Zn2+对错配切割具有促进作用,但不是必需的,最适浓度为0.25mmol/L。CEL I粗提物切割错配的反应时间以42℃下酶切20min为宜。比较试验显示,本研究建立技术的点突变检测效果与Transgenomic公司试剂盒SurveyorTM基本相同。
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.Zn^2 ＋ 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.
出处
《核农学报》
CAS
CSCD
北大核心
2011年第1期37-42,共6页
Journal of Nuclear Agricultural Sciences
基金
农业部农业公益性行业科研专项经费项目(200803034)
国际原子能机构研究合同项目(R13631)
关键词
CELI粗提物
切割活性
条件优化
突变检测
CEL I crude extract
mismatch cleavage activity
condition optimization
mutation detection relative
