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

476 Horton, Raju, and Conti-Fine
Fig. 1. Cloning of PCR products with a T-linker. (A) Oligonucleotide design. (B) Addition
of the T-linker to a PCR product. The parenthetical g and NdeI indicate that if a g is the first base
in the PCR product, the NdeI site will be completed. (C) Sequence from a pBluescript KS II+
plasmid containing a T-linker-reamplified insert cloned into its EcoRI site. This sequence was
obtained using an fmol cycle sequencing kit (Promega, Madison, WI). The base representing
the overhanging t of the T-linker is indicated by the solid triangle to the right of the sequence.
Since the PCR product begins with a g, a complete NdeI site is formed.
this way, very minor restraints in the PCR primer sequences (having one start with g,
but not the other) can be used to complete the site at only one end of the reamplified
product. One simple application of this idea is directional cloning (see Notes 2 and 3).
The use of this T-linker is illustrated in Fig. 1. A portion of a T-cell receptor V region
was amplified from Jurkat tumor line cDNA using primers Vb8-1cpe5 and hpVbe3.
The ligated products were reamplfied with TL-A and cloned into the EcoRI site of
pBluescript KS II + . The sequence of the resulting product is shown in Fig. 1C. At
the bottom of the gel is sequence from the polylinker of the vector to the EcoRI site,
followed by the T-linker. The overhanging t is marked. The g completing the NdeI site
is the first base of Vb81cp.5.
1.1.2. HisTL
Our second example adds another level of sophistication, as shown in Fig. 2. Here
an additional ability of the T-linker is brought into use. A new and useful sequence
is added in addition to the restriction sites, namely a “histidine tag” sequence, which
will be used for affinity purification of the expressed recombinant α3 protein on a Ni 2+
column (Qiagen, Chatsworth, CA). Note that the HindIII site is created only at the
3′ end of the product in this case, because only the 3′ primer begins with t. The
histidine tag portion is removed from the 3′ end by cutting with HindIII. No special
sequences have been added to the sequence-specific primers hacpe5 and hacpe3, but
their positions have been chosen to take advantage of the design of the HisT-linkers.
The initial PCR-amplified sequence begins with a full codon to put the sequence in