Guo L H, Wu R
Nucleic Acids Res. 1982 Mar 25;10(6):2065-84. doi: 10.1093/nar/10.6.2065.
We describe improve enzymatic methods for sequencing method 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 procedures, 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 Sanger et al. (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.
我们描述了改进的用于DNA测序的酶法。这些方法基于用核酸外切酶III对双链DNA进行部分消化，以产生3'末端缩短至不同长度的DNA分子，随后进行修复合成以延伸并标记3'末端。在用限制性酶对DNA进行不对称切割后，通过凝胶电泳分离标记产物，并从放射自显影片上读取序列。从无限制的DNA开始并经过凝胶电泳的整个过程，对DNA分子的两条链进行测序仅需一天时间。这些方法特别适用于对克隆在质粒载体中的DNA进行测序，并且极大地扩展了桑格等人（《美国国家科学院院刊》74, 5463, 1977）的双脱氧核苷酸链终止法的用途。使用这些方法，我们确定了一个包含酵母SUP3酪氨酸tRNA基因的410个碱基对片段的序列。
