NEW RAPID METHODS FOR DNA SEQUENCING BASED ON EXONUCLEASE-III DIGESTION FOLLOWED BY REPAIR SYNTHESIS
NEW RAPID METHODS FOR DNA SEQUENCING BASED ON EXONUCLEASE-III DIGESTION FOLLOWED BY REPAIR SYNTHESIS
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DOI：
10.1093/nar/10.6.2065
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发表时间：
1982-01-01
影响因子：
14.9
通讯作者：
WU, R
中科院分区：
文献类型：
--
作者：
GUO, LH;WU, R
我们描述了改进的酶法测序DNA。它们的基础是用核酸外切酶III对双链DNA进行部分酶切，产生3 ‘端缩短到不同长度的DNA分子，然后进行修复合成以延长和标记3 ’端。用限制性内切酶对DNA进行不对称切割后，用凝胶电泳分离标记产物，并从自射线图上读取序列。整个过程，从无限制的DNA开始，然后通过凝胶电泳，只需要一天的时间就可以对DNA分子的两条链进行测序。这些方法特别适合于对质粒载体上克隆的DNA进行测序，极大地扩展了Sangeretal的双脱氧核苷酸链终止法的实用性。(Proc国家的。学会科学。美国74,5463,1977)。利用这些方法，我们确定了包含酵母SUP3酪氨酸tRNA基因的410碱基对片段的序列。
We describe improve enzymatic methods for sequencing DNA. They are based on partial digestion of duplex DNA with exonuclease III to produce DNA molecules with 3′ ends shortened to varying lengths, followed by repair synthesis to extend and label the 3′ ends. After asymmetrical cleavage of the DNA with a restriction enzyme, the labeled products are separated by gel electrophoresis and the sequence read from the autoradiogram. The entire procedure, beginning with unrestricted DNA and followed through gel electrophoresis, takes only one day for sequencing both strands of the DNA molecule. These methods are especially suitable for sequencing DNA cloned in plasmid vectors, and they greatly extend the usefulness of the dideoxynucleotide chain termination method of Sangeretal. (Proc. Natl. Acad. Sci. USA 74, 5463, 1977). Using these methods we have determined the sequence of a 410 base pair fragment which includes the yeast SUP3 tyrosine tRNA gene.