Open AccessModulation of Non-Templated Nucleotide Addition by Taq DNA Polymerase: Primer Modifications that Facilitate Genotyping
Author(s) -
Michael J. Brownstein,
John D. Carpten,
Jeffrey R. Smith
Publication year - 1996
Publication title -
biotechniques/biotechniques
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.617
H-Index - 131
eISSN - 1940-9818
pISSN - 0736-6205
DOI - 10.2144/96206st01
Subject(s) - taq polymerase , genotyping , primer (cosmetics) , polymerase chain reaction , adenylylation , biology , hot start pcr , polymerase , nucleotide , dna polymerase , cloning (programming) , reverse transcriptase , computational biology , microbiology and biotechnology , dna , genetics , chemistry , thermus aquaticus , genotype , nested polymerase chain reaction , computer science , gene , organic chemistry , biosynthesis , programming language
Taq DNA polymerase can catalyze nontemplated 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 “PIGtailing”) should facilitate accurate genotyping and efficient T/A cloning.