INCOMPLETE PRIMER EXTENSION DURING INVITRO DNA AMPLIFICATION CATALYZED BY TAQ POLYMERASE - EXPLOITATION FOR DNA SEQUENCING
INCOMPLETE PRIMER EXTENSION DURING INVITRO DNA AMPLIFICATION CATALYZED BY TAQ POLYMERASE - EXPLOITATION FOR DNA SEQUENCING
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DOI：
10.1093/nar/17.23.9613
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发表时间：
1989-12-11
影响因子：
14.9
通讯作者：
ECKSTEIN, F
中科院分区：
文献类型：
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作者：
OLSEN, DB;ECKSTEIN, F
通过使用Taq聚合酶的聚合酶链反应获得的DNA片段的聚丙烯酰胺凝胶电泳显示存在比预期产物短的多个片段。即使当琼脂糖凝胶电泳分析仅显示单一条带时，也观察到这些失败的延伸产物。如果在两个寡核苷酸引物（其中一个是5“-32 P标记的）存在下，在反应期间碱基特异性地掺入硫代磷酸酯基团，则可以利用过早终止片段的产生来对PCR产物进行测序。在扩增循环完成后，向反应混合物中加入蛇毒磷酸二酯酶，使每个片段从3“-末端转移到硫代磷酸酯基团，从而可以通过聚丙烯酰胺凝胶电泳读取序列。
Polyacrylamide gel electrophoresis of DNA fragments obtained by the polymerase chain reaction using Taq polymerase revealed the presence of multiple fragments shorter than the expected product. These abortive extension products were observed even when analysis by agarose gel electrophoresis showed only a single band. The production of prematurely terminated fragments can be exploited for the sequencing of PCR products if phosphorothioate groups are incorporated base specifically during the reaction in the presence of two oligonucleotide primers, one of which is 5''-32P-labeled. The addition of snake venom phosphodiesterase to the reaction mixture after completion of the amplification cycles digests each fragment from the 3''-end to a phosphorothioate group so that the sequence can be read by polyacrylamide gel electrophoresis.