DNA-SEQUENCE ANALYSIS WITH A MODIFIED BACTERIOPHAGE-T7 DNA-POLYMERASE
DNA-SEQUENCE ANALYSIS WITH A MODIFIED BACTERIOPHAGE-T7 DNA-POLYMERASE
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DOI：
10.1073/pnas.84.14.4767
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发表时间：
1987-07-01
影响因子：
11.1
通讯作者：
RICHARDSON, CC
中科院分区：
文献类型：
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作者：
TABOR, S;RICHARDSON, CC
化学修饰的噬菌体T7 DNA聚合酶具有三种特性，使其成为通过链终止法进行DNA测序的理想选择。这种酶是高度加工性的，催化数千个核苷酸的聚合而不解离。由于修饰，3“至5”核酸外切酶活性被消除。经修饰的聚合酶有效地使用核苷酸类似物，其增加凝胶中条带的电泳分辨率。因此，双脱氧核苷酸封端的片段在几个到数千个核苷酸长度的范围内具有高度均匀的放射性强度。实际上，由于终止于暂停点或二级结构障碍，没有背景。用dITP代替dGTP的过程合成消除了条带压缩，使得从单个方向明确确定序列成为可能。
A chemically modified phage T7 DNA polymerase has three properties that make it ideal for DNA sequencing by the chain-termination method. The enzyme is highly processive, catalyzing the polymerization of thousands of nucleotides without dissociating. By virtue of the modification the 3'' to 5'' exonuclease activity is eliminated. The modified polymerase efficiently uses nucleotide analogs that increase the electrophoretic resolution of bands in gels. Consequently, dideoxynucleotide-terminated fragments have highly uniform radioactive intensity throughout the range of a few to thousands of nucleotides in length. There is virtually no background due to terminations at pause sites or secondary-structure impediments. Processive synthesis with dITP in place of dGTP eliminates band compressions, making possible the unambiguous determination of sequences from a single orientation.