Excessive addition split peak formed by the non-templated nucleotide addition property of Taq DNA polymerase after PCR amplification

被引：2

作者：

Zhou, Yongsong ^{[1
,2
]}

Bo, Fan ^{[2
]}

Tian, Tian ^{[1
]}

Wu, Buling ^{[1
]}

Zhu, Bofeng ^{[1
,2
,3
]}

机构：

[1] Southern Med Univ, Shenzhen Stomatol Hosp Pingshan, Shenzhen, Guangdong, Peoples R China

[2] Southern Med Univ, Sch Forens Med, Guangzhou Key Lab Forens Multi Precis Identificat, Guangzhou, Guangdong, Peoples R China

[3] Xi An Jiao Tong Univ, Coll Stomatol, Key Lab Shaanxi Prov Craniofacial Precis Med Res, Xian, Shanxi, Peoples R China

来源：

FRONTIERS IN BIOENGINEERING AND BIOTECHNOLOGY | 2023年 / 11卷

基金：

中国国家自然科学基金;

关键词：

non-template addition; Taq DNA polymerase; artifact; split peak; capillary electrophoresis; FORMAMIDE; RNA; END;

D O I：

10.3389/fbioe.2023.1180542

中图分类号：

Q81 [生物工程学（生物技术）]; Q93 [微生物学];

学科分类号：

071005 ; 0836 ; 090102 ; 100705 ;

摘要：

Because of its non-template addition feature, Taq DNA polymerase can catalyze one or more extra nucleotides onto the 3' terminus of PCR products. An extra peak is observed at DYS391 locus after the PCR products stored for 4 days at 4 degrees C. To explore the formation mechanism of this artifact, PCR primers and amplicon sequences of Y-STR loci are analyzed, furthermore, PCR products storage conditions and termination of PCR are discussed. The extra peak is a + 2 addition product, which we call excessive addition split peak (EASP). The most significant difference between EASP and the incomplete addition of adenine product is that the size of EASP is about one base larger than the true allele, and the EASP locates on the right side of the real allelic peak. The EASP cannot be eliminated by increasing loading mixture volume and conducting heat denaturation prior to electrophoresis injection. However, the EASP is not observed when the PCR is terminated with ethylenediaminetetraacetic acid or formamide. These findings suggest that formation of EASP is a result of 3 ' end non-template extension by Taq DNA polymerase, rather than being the result of DNA fragment secondary structure produced under a suboptimal electrophoresis condition. In addition, the EASP formation is affected by the primer sequences and the storage conditions of PCR products.

引用

页数：13