Maat J, Smith A J
Nucleic Acids Res. 1978 Dec;5(12):4537-45. doi: 10.1093/nar/5.12.4537.
A rapid enzymatic approach is described for the sequence analysis of a 5' terminally labelled restriction fragment. It involves limited nicking of the strands of the molecule throughout the sequence by pancreatic DNAase I. The 3' hydroxyl groups exposed by each nick are then used to prime chain extension by DNA polymerase I in four separate reactions. Each reaction uses one of the four chain terminating dideoxynucleoside triphosphates (ddNT-PSs), together with the four deoxynucleoside triphosphates (dNTPs). In a single reaction all the 3' ends are terminated in positions of the same base, which is different for each of the four reactions. When the products of these reactions are resolved by gel electrophoresis according to size, a sequence can be deduced from the pattern of radioactive bands. Sequences can be determined onwards from 10-20 residues from the 5' labelled end. The length of sequence which can be determined is only limited by the resolution of the gel.
本文描述了一种用于对5'末端标记的限制片段进行序列分析的快速酶促方法。该方法包括通过胰DNA酶I在整个序列中对分子链进行有限的切口。每个切口暴露的3'羟基随后用于在四个单独的反应中由DNA聚合酶I引发链延伸。每个反应使用四种链终止双脱氧核苷三磷酸（ddNTPs）中的一种，以及四种脱氧核苷三磷酸（dNTPs）。在单个反应中，所有3'末端都在相同碱基的位置终止，这在四个反应中的每一个中都是不同的。当这些反应的产物根据大小通过凝胶电泳分离时，可以从放射性条带的模式推断出序列。可以从5'标记末端的10 - 20个残基开始确定序列。能够确定的序列长度仅受凝胶分辨率的限制。
