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本文叙述一种修改过的DNA序列测定——M13双脱氧终止法的原理和操作。首先克隆,将实验DNA片段插入载体M13mp,再用重组体转染E.coli JM101,经筛选、纯化和增殖,离心去菌体,上清经沉淀得单链模板,与M13通用引物一起退火,在dNTP、ddNTP和AMV逆转录酶存在时合成不同长度的DNA片段,用变性凝胶电泳展开,一次可读300以上硷基序列。
This article describes a modified DNA sequencing assay - the principle and operation of the M13 dideoxy termination method. First cloned, the experimental DNA fragment inserted into the vector M13mp, and then recombinant E. coli JM101 transfection, after screening, purification and proliferation, the bacteria were centrifuged, the supernatant was precipitated single-stranded template, and M13 universal primer annealing together, DNA fragments of different lengths were synthesized in the presence of dNTP, ddNTP and AMV reverse transcriptase, and denatured gel electrophoresis was performed to read more than 300 base sequences at a time.