PHOSPHOROTHIOATE PRIMERS IMPROVE THE AMPLIFICATION OF DNA-SEQUENCES BY DNA-POLYMERASES WITH PROOFREADING ACTIVITY
PHOSPHOROTHIOATE PRIMERS IMPROVE THE AMPLIFICATION OF DNA-SEQUENCES BY DNA-POLYMERASES WITH PROOFREADING ACTIVITY
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DOI：
10.1093/nar/20.14.3551
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发表时间：
1992-07-25
影响因子：
14.9
通讯作者：
SKERRA, A
中科院分区：
文献类型：
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作者：
SKERRA, A
两种具有校对活性的耐热DNA聚合酶-Vent DNA聚合酶和Pfu DNA聚合酶最近引起了人们的关注，主要是因为它们在聚合酶链式反应扩增DNA序列时具有更高的保真度。然而，由于这些酶具有3‘到5’的核酸外切酶活性，这对它们的实际应用造成了严重的不利影响，因为这些酶降解了作为DNA合成引物的寡核苷酸。结果表明，通过在其3‘端引入单硫键，可以有效地防止这种对引物分子的核外攻击。这一策略可以很容易地用常规的DNA合成方法来实现，可能会为这些新型酶在聚合酶链式反应中的广泛使用开辟道路。
Two thermostable DNA polymerases with proofreading activity-Vent DNA polymerase and Pfu DNA polymerase-have attracted recent attention, mainly because of their enhanced fidelities during amplification of DNA sequences by the polymerase chain reaction. A severe disadvantage for their practical application, however, results from the observation that due to their 3' to 5' exonuclease activities these enzymes degrade the oligodeoxynucleotides serving as primers for the DNA synthesis. It is demonstrated that this exonucleolytic attack on the primer molecules can be efficiently prevented by the introduction of single phosphorothioate bonds at their 3' termini. This strategy, which can be easily accomplished using routine DNA synthesis methodology, may open the way to a widespread use of these novel enzymes in the polymerase chain reaction.