Substrate nucleotide-determined non-templated addition of adenine by Taq DNA polymerase: Implications for PCR-based genotyping and cloning
Substrate nucleotide-determined non-templated addition of adenine by Taq DNA polymerase: Implications for PCR-based genotyping and cloning
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DOI：
10.2144/96214rr03
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发表时间：
1996-10-01
期刊：
影响因子：
2.7
通讯作者：
Collins, FS
中科院分区：
文献类型：
--
作者：
Magnuson, VL;Ally, DS;Collins, FS
Applied Biosystems PRISM（TM）基于荧光的基因分型系统以及Invitrogen TA Cloning（R）载体系统受到Taq DNA聚合酶在延伸后向PCR产物的3'末端添加腺嘌呤核苷酸的趋势的影响。大部分PCR产物链中腺嘌呤的不完全添加会在基因分型过程中产生等位基因识别问题，并可能降低此类产物的克隆效率。本文报道的实验表明，某些末端核苷酸可以抑制或增强腺嘌呤添加的Taq和PCR引物设计可用于调节这种活性。当使用GENOTYPER（TM）软件时，我们提出的方法可以大大改善有问题的微卫星标记的等位基因调用。
The Applied Biosystems PRISM(TM) florescence-based genotyping system as well as the Invitrogen TA Cloning(R) vector system are influenced by the tendency of Taq DNA polymerase to add an adenine nucleotide to the 3' end of PCR products after extension. Incomplete addition of adenine to a majority of PCR product strands creates problems in allele-calling during genotyping and potentially diminishes the cloning efficiency of such products. Experiments reported here show that certain terminal nucleotides can either inhibit or enhance adenine addition by Taq and that PCR primer design can be used to modulate this activity. The methods we propose can substantially improve allele-calling for problematic microsatellite markers when using GENOTYPER(TM) software.