John M. S. Bartlett.pdf - Bio-Nica.info

Cloning PCR Products 385
56
Cloning PCR Products for Sequencing in M13 Vectors
David Walsh
1. Introduction
Although numerous methods are now available for direct sequencing of polymerase
chain reaction (PCR) products, cloning of amplified DNA for sequencing in M13 vectors
remains an attractive approach because of the high quality of sequence information
generated from single-stranded bacteriophage DNA templates.
Cloning of PCR products is in theory straightforward but in practice is often
problematical, as widely reported (1–4). Difficulties are generally ascribed to modifications
of the DNA termini by Taq DNA polymerase. After completion of thermal cycling,
the enzyme may remain associated with DNA ends and thus interfere with subsequent
ligations, unless specific steps are included for its removal or inactivation. Carryover of
Taq DNA polymerase and residual dNTPs into restriction digests can also result in
end filling of 5′-overhangs (5), severely reducing the efficiency of cohesive-ended
cloning strategies using restriction sites within PCR primers. Removal of Taq DNA
polymerase by proteinase K digestion (6) or repeated phenol/chloroform extractions
(5) circumvents these problems.
Furthermore, the terminal transferase activity of Taq DNA polymerase catalyzes the
nontemplate-directed addition of a single nucleotide, almost invariably deoxyadenosine
(dA), to the 3′ ends of amplified DNA molecules (7). The resulting “ragged ends” must
be removed if blunt-end ligation to SmaI-cut vector is required. This is best achieved
by utilizing the strong 3′ to 5′ exonuclease activity of T4 DNA polymerase, which
in the presence of low concentrations of dNTPs removes 3′ overhangs from doublestranded
DNA. Cloning of amplified DNA into linearized 5′-dephosphorylated vector
also necessitates the presence of 5′ phosphate groups on the PCR products, achieved by
kinasing either the primers before amplification or the PCR product itself. Conveniently,
3′-dA removal by T4 DNA polymerase and 5′-phosphorylation by T4 polynucleotide
kinase can be performed simultaneously (8).
Difficulties in cloning PCR products as blunt-ended molecules may be avoided
by incorporating restriction sites into the PCR primers and cloning products more
efficiently as cohesive-ended molecules. The major problem encountered here is the
failure of some restriction endonucleases to cleave toward the ends of DNA fragments.
The presence of 4 bp 5′ to the recognition sequence is sufficient for efficient cutting
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
385