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

T-Linker Strategy 481
small amounts of agar, will inhibit the reaction. Be sure to include one reaction of a
nonrecombinant (“blue”) colony as a negative control.
13. Heat to 94°C for 1 min.
14. Amplify for 30 cycles using the following parameters: 94°C for 15 s, 55°C for 15 s,
and 72°C for 45 s.
4. Notes
1. Why not use a T-vector? The method of choice for routine cloning of PCR products is
probably a T-vector. One of the more important considerations is that with a T-vector you
do not need to digest the insert with a restriction enzyme, so you do not need to worry about
whether or not the insert contains that site. Also, reamplification with the T-linker provides
another set of opportunities for the polymerase to introduce errors. However, T-linkers have
several differences that can provide advantage in certain circumstances.
a. The efficiency of ligation can theoretically be increased because much higher concentrations
of linkers can be achieved.
b. The efficiency of ligation does not need to be as high because the ligated product can
be reamplified with one of the linker oligonucleotides to give a product that has added
restriction sites at the ends.
c. Oligonucleotides, such as those used to construct the T-linker, are quite stable, and
remain usable for many years. Stability has been a problem with some of the commercially
available T-vectors.
d. Because the ends of a PCR product can be precisely defined and/or modified, and
because T-linkers can be custom-made, it is possible to “split” a DNA sequence, such
as a restriction site, between the PCR product and the T-linker so that a complete site
is formed only at one end of the final product, without having to include the entire
site in the primer made for amplifying a specific gene. This can save on the cost of
oligonucleotides but still allow directional cloning.
e. T-linkers should be more flexible in terms of using a variety of restriction sites in a
variety of vectors.
2. The directional cloning of the coding sequence for the α3 acetylcholine receptor subunit
illustrated in Fig. 2 was accomplished by selecting the locations of the primers so that a
HindIII site was created at only one end. However, by random chance, in one case of four,
a PCR product made with primers not designed to complete the NdeI site will have a g at
a given end, and will thus end up with an NdeI site. Similarly, one of 16 randomly chosen
products will have NdeI sites at both ends. Thus, 3 of 16 randomly chosen PCR products
will have a g at only one end, and could be cloned directionally using this T linker. A set
of such linkers, with each depending on the presence of a different single nucleotide
at the end of the PCR product, could therefore be used to directionally clone 75% of
randomly chosen PCR products. Potential restriction enzymes for such complementable
sites in T-linkers would be:
Site ends in
Restriction enzymes
. . . t(g) NdeI, PvuII (blunt), Pm1I (blunt)
. . . t(c) EcoRI, EcoRV (blunt), AatII, SacI
. . . t(a) SnaBI (blunt)
. . . t(t) Hind III, SspI (blunt)
Three linkers would make up a complete set, that is, you do not need an a linker because
any product that only has a at one end automatically has one of the other three bases at the