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

Recombinant PCR 519
In recombination PCR, the sum goal of the two amplifications is to yield two PCR
products where each end of one product is homologous to a distinct end of the other
PCR product. Because the amplifying primer sequences are incorporated into the ends of
a PCR product, so long as primers 1 and 2 contain regions that are complementary to
regions of primers 3 and 4 (or 4 and 3), the PCR products will contain ends that are
homologous to each other, and these primer-determined DNA ends do not need to be
determined by the original donor or recipient templates. The only requirement of this
recombination PCR strategy is that primers 1 and 2 must have regions of complementarity
to primers 3 and 4. Therefore, recombination PCR can be used not only to generate
recombinant constructs, such as gene chimeras, but also for the site-directed mutagenesis
of two distal sites concurrently (Fig. 2) or for the rapid site-directed mutagenesis of single
sites (Fig. 3) (11). In the point mutagenesis protocol illustrated in Fig. 3, the plasmid is
linearized by restriction endonuclease digestion before each PCR amplification. In each
of the two amplifications, the mutating primers (primers 1 and 3) mutate the identical
base pair so that the mutated ends of each product are homologous to each other and
the nonmutating primers (primers 2 and 4) are also designed to produce ends that are
homologous to each other. Both unpurified PCR products are combined to transform
E. coli, generating clones with the mutation of interest.
2. Materials
1. Taq DNA polymerase (AmpliTaq 5 U/mL; Perkin–Elmer, Norwalk CT) (see Note1).
2. 10× PCR buffer II: 500 mM KCl, 100 mM Tris-HCl, pH 8.3.
3. MgCl 2 solution (25 mM).
4. Stocks (10 mM ) of each dATP, dCTP, dTTP, and dGTP, neutralized to pH 7.0 with NaOH.
5. Restriction endonucleases (New England BioLabs, Beverly, MA).
6. PCR primers. In Fig. 1, PCR amplification with primers 1 and 2 results in a product with
24 to 30 bp of homology with the products of primers 3 and 4. For PCR primers that
introduce mutations, see Note 2.
7. Agarose.
8. Ethidium bromide.
9. TAE buffer: 40 mM Tris-acetate, 2 mM EDTA, pH 8.5 (12).
10. Geneclean (Bio 101, La Jolla, CA).
11. TE buffer: 10 mM Tris-HCl, pH 8.0, 1 mM EDTA.
12. MAX Efficiency DH5α Competent E. coli (BRL, Life Technologies). Once a tube is
thawed it should not be reused (see Note 3).
13. SOC Media (13).
14. LB plates with 100 µg/mL ampicillin (14).
15. Luria-Bertani medium (LB broth) (15).
3. Methods
1. Linearize the plasmid template by restriction endonuclease digestion outside the region to
be amplified, if possible. The plasmid digest does not need to be purified prior to its use
as a PCR template (see Notes 4 and 5).
2. Assemble a PCR in a total volume of 50 µL containing the following: 2 ng of plasmid
template, 25 pmol of each primer, 200 µM each dNTP, 1X PCR buffer, 2.5 mM MgCl 2 ,
and 1.25 U DNA Taq DNA polymerase. (see Note 6).
3. Perform PCR amplification using the following parameters (see Note 6): 94°C for 1 min
(initial denaturation), 94°C for 30 s (denaturation), 50°C for 30 s (anneal), 72°C for
1 min/kb of PCR product (extension), 14–20 amplification cycles, and 72°C for 7 min
(final extension step).