DETECTION OF SPECIFIC POLYMERASE CHAIN-REACTION PRODUCT BY UTILIZING THE 5'-]3' EXONUCLEASE ACTIVITY OF THERMUS-AQUATICUS DNA-POLYMERASE
DETECTION OF SPECIFIC POLYMERASE CHAIN-REACTION PRODUCT BY UTILIZING THE 5'-]3' EXONUCLEASE ACTIVITY OF THERMUS-AQUATICUS DNA-POLYMERASE
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DOI：
10.1073/pnas.88.16.7276
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发表时间：
1991-08-01
影响因子：
11.1
通讯作者：
GELFAND, DH
中科院分区：
文献类型：
--
作者：
HOLLAND, PM;ABRAMSON, RD;GELFAND, DH
耐热酶Thermus Aquaticus DNA聚合酶的5‘->；3’外切酶活性可用于聚合酶链式反应产物检测系统，以在扩增的同时产生特定的可检测信号。在聚合酶链式反应分析中引入寡核苷酸探针，该寡核苷酸探针在3‘端不可延伸，在5’端标记，并被设计成在目标序列内杂交。在扩增过程中，对其中一条聚合酶链式反应产物链上的探针进行退火，可以产生适合核酸外切酶活性的底物。在扩增过程中，水曲霉菌DNA聚合酶的5‘-->；3’外切酶活性将探针降解成可与未降解的探针区分开来的较小片段。该检测方法灵敏、特异，是对更为繁琐的检测方法的重大改进。
The 5' --> 3' exonuclease activity of the thermostable enzyme Thermus aquaticus DNA polymerase may be employed in a polymerase chain reaction product detection system to generate a specific detectable signal concomitantly with amplification. An oligonucleotide probe, nonextendable at the 3' end, labeled at the 5' end, and designed to hybridize within the target sequence, is introduced into the polymerase chain reaction assay. Annealing of probe to one of the polymerase chain reaction product strands during the course of amplification generates a substrate suitable for exonuclease activity. During amplification, the 5' --> 3' exonuclease activity of T. aquaticus DNA polymerase degrades the probe into smaller fragments that can be differentiated from undegraded probe. The assay is sensitive and specific and is a significant improvement over more cumbersome detection methods.