Modulation of non-templated nucleotide addition by taq DNA polymerase: Primer modifications that facilitate genotyping
Modulation of non-templated nucleotide addition by taq DNA polymerase: Primer modifications that facilitate genotyping
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DOI：
10.2144/96206st01
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发表时间：
1996-06-01
期刊：
影响因子：
2.7
通讯作者：
Smith, JR
中科院分区：
文献类型：
--
作者：
Brownstein, MJ;Carpten, JD;Smith, JR
标签DNA聚合酶可以催化核苷酸（主要是腺苷）非模板化地添加到PCR扩增产物的3'末端。最近，我们发现，这种活动，这是引物特异性的，提出了一个潜在的错误来源的基因分型研究的基础上使用短串联重复序列（STR）标记。此外，在回顾我们的数据时，我们发现邻近3'末端C的非模板化核苷酸添加是有利的，而邻近3'末端A的添加不是有利的。然而，很明显，除了3'末端碱基之外，模板的特征也影响产物腺苷酸化的分数。为了确定促进或抑制产物腺苷酸化的共有序列，我们将序列移植到腺苷酸化的标记物的反向引物的5'端和未腺苷酸化的标记物的反向引物的5'端之间。证明难以鉴定能够保护所有标记物的产物免于核苷酸的非模板化添加的单一序列。另一方面，将序列GTTTCTT置于反向引物的5'端导致正向链的3'端的几乎100%腺苷酸化。这种修饰或相关修饰（称为“PIG-加尾”）应有助于准确的基因分型和有效的T/A克隆。
Tag DNA polymerase can catalyze non-templated addition of a nucleotide (principally adenosine) to the 3' end of PCR-amplified products. Recently, we showed that this activity, which is primer-specific, presents a potential source of error in genotyping studies based on the use of short tandem repeat (STR) markers. Furthermore, in reviewing our data, we found that non-templated nucleotide addition adjacent to a 3' terminal C is favored and that addition adjacent to a 3' terminal A is not. It was clear, however, that features of the template in addition to the 3' terminal base also affect the fraction of product adenylated. To define consensus sequences that promote or inhibit product adenylation, we transplanted sequences between the 5' ends of the reverse primers of markers that are adenylated and those of markers that are not adenylated. It proved difficult to identify a single sequence capable of protecting the products of all markers from non-templated addition of nucleotide. On the other hand, placing the sequence GTTTCTT on the 5' end of reverse primers resulted in nearly 100% adenylation of the 3' end of the forward strand. This modification or related ones (called ''PIG-tailing'') should facilitate accurate genotyping and efficient T/A cloning.