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T-Linker Strategy 475
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A T-Linker Strategy for Modification
and Directional Cloning of PCR Products
Robert M. Horton, Raghavanpillai Raju, and Bianca M. Conti-Fine
1. Introduction
The propensity of Taq polymerase to add 3′-A overhangs (1,2) to polymerase chain
reaction (PCR)-amplified DNA has made possible a simple method for cloning PCR
products into a T-vector (Invitrogen, San Diego, CA) (3–5). Here, we present a related
strategy that uses T-linkers to add sequences, such as restriction sites, to the ends
of PCR products (see Note 1). A single-base T overhang at the end of a synthetic
double-stranded oligonucleotide linker allows ligation of the linker to the unpolished
ends of a PCR product. This avoids the expense of adding the “extra” sequences to
sequence-specific primers.
1.1. Examples
Two T-linker designs are presented here. In each case, the T-linker is a doublestranded
synthetic oligonucleotide composed of complementary oligos (either TL-A
and TL-B for the NdeT linker or HisTL-A and HisTL-B for the HisT-linker) with a
single 3′ overhanging t at one end.
1.1.1. NdeTL
The basic principles involved in using a T-linker are shown using the Nde-T-linker
in Fig. 1. This T-linker contains complete EcoRI and NotI sites, and a third site (NdeI)
is partially present, except for its final g; the overhanging t is part of this site. The 5′
end of TL-B is phosphorylated (indicated by an asterisk) so that it can be ligated. The
other end of the linker contains a sticky HindIII-compatible end, which was not used
in the approach described here. However, because this 5′ overhang is filled in by the
polymerase during PCR, these extra bases serve as a “clamp” or spacer, which permits
the EcoRI site to be cut.
The overhanging t of the linker matches the a added by the polymerase and directs
the ligation of TL-B, allowing reamplification of the sequence using TL-A as a primer.
The resulting molecule contains the original PCR-amplified sequence flanked by
inverted repeats of the T-linker.
If one of the sequence-specific primers has a g at its 5′ end, an NdeI site will be
formed. This site is “split” between the T-linker and the PCR product to be cloned. In
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
475