Directional cloning of native PCR products with preformed sticky ends (Autosticky PCR)
Directional cloning of native PCR products with preformed sticky ends (Autosticky PCR)
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DOI：
10.1007/s004380050930
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发表时间：
1999-01-01
期刊：
影响因子：
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通讯作者：
Kálmán, M
中科院分区：
文献类型：
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作者：
Gál, J;Schnell, R;Kálmán, M
提出了一种新的定向克隆方法。DNA模板中的基本位点使DNA聚合酶在互补链合成过程中停留在该位点。由于PCR产物链的5′端含有内置扩增引物，引物内的基本位点导致PCR产物末端形成5′单链悬垂，使其能够直接连接到合适的裂解克隆载体上，而无需进一步修饰。这种“自粘PCR”（AS-PCR）克服了限制性内切酶末端敏感性或扩增序列内部限制性内切位点引起的问题，并且能够产生任何所需的5'悬垂。
A novel method for the directional cloning of native PCR products was developed. Abasic sites in DNA templates make DNA polymerases stall at the site during synthesis of the complementary strand. Since the 5' ends of PCR product strands contain built-in amplification primers, abasic sites within the primers result in the formation of 5' single-stranded overhangs at the ends of the PCR product, enabling its direct ligation to a suitably cleaved cloning vector without any further modification. This "autosticky PCR" (AS-PCR) overcomes the problems caused by end sensitivity of restriction enzymes, or internal restriction sites within the amplified sequences, and enables the generation of essentially any desired 5' overhang.