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T4 DNA Polymerase 469
65
Using T4 DNA Polymerase
to Generate Clonable PCR Products
Kai Wang
1. Introduction
Polymerase chain reaction (PCR) mediated through Taq DNA polymerase has
become a simple and routine method for cloning, sequencing, and analyzing genetic
information from very small amounts of materials (1). Taq DNA polymerase, like some
other DNA polymerases, lacks 3′ to 5′ exonuclease activity and will add nontemplatedirected
nucleotides to the ends of double-stranded DNA fragments. Because of the
strong preference of the Taq polymerase for dATP, the nucleotide added is almost
exclusively an adenosine (2). This results in generating “ragged” unclonable amplification
products (2,3). Restriction endonuclease sites are often incorporated into the
amplification primers so that clonable PCR products can be generated by restriction
enzyme cleavage (4). However, the possible secondary sites located within amplified
products often complicate the cloning and interpretation of PCR results. A cloning
system exploiting the template-independent terminal transferase activity of Taq
polymerase has been reported (5–7). However, a special vector with thymidine (T)
overhanging ends has to be used in the process.
T4 DNA polymerase has very strong exonuclease and polymerase activities in a
broad range of reaction conditions (8). By adapting its strong enzymatic activities,
a simple and efficient method to generate clonable PCR fragments with T4 DNA
polymerase has been developed (9). The T4 DNA polymerase not only repairs the ends
of the PCR products, but it also removes the remaining primers in the reaction with its
strong single-stranded exonuclease activity. Therefore, this method usually does not
require multiple sample handling, buffer changes, or gel purification steps. Instead, a
simple alcohol precipitation step is used to purify the PCR products.
The blunt-end cloning protocol can be modified for sticky-end cloning. Even
though this may increase cloning efficiency to a certain extent, a purification step,
to remove excess deoxynucleotides from PCRs, has to be added before adding T4
DNA polymerase.
From: Methods in Molecular Biology, Vol. 226: PCR Protocols, Second Edition
Edited by: J. M. S. Bartlett and D. Stirling © Humana Press Inc., Totowa, NJ
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