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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 <strong>Bio</strong>Labs, 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 (<strong>Bio</strong> 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).
520 Jones and Winistorfer Fig. 2. Diagram illustrating site-directed mutagenesis of two distal sites using recombination PCR. The primers are numbered hemiarrows. Asterisks designate the mutagenesis sites. There is no purification of the PCR products. Notches designate point mismatches in the primers and resulting mutations in the PCR products. Reprinted by permission from Technique 2, 273–278. 4. Visualize the PCR product on an agarose minigel. If 5 µL of the PCR product can be clearly seen following ethidium bromide staining (>15 ng/5 µL), there is enough product. 5. Withdraw 2.5 µL from each PCR tube (typically 10–60 ng/2.5 µL) and then combine the two samples. If the PCR template is linearized by restriction endonuclease digestion outside the region to be amplified, no purification of PCR products is necessary (see Note 5). 6. Transform MAX efficiency competent E. coli (BRL) with the 5-µL sample containing the two PCR products. Maintaining an even molar ratio of one product to another is not necessary. Transformation is carried out following the manufacturer’s instructions with the following modifications:
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TM Methods in Molecular Biology Vol
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Xin Wang and W. Scott Young III 105
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63. Primed In Situ Nucleic Acid Lab
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4 Bartlett and Stirling The thing t
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6 Bartlett and Stirling Fig. 2. Res
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18 McDonagh stored. This means that
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28 Bartlett References 1. US Depart
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30 Bartlett and White 11. 5 M sodiu
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34 Pearson and Stirling 11. Microfu
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36 Going 3. Methods 3.1. Section Cu
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44 Pearson 7. Add 60 µL of chlorof
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46 Bartlett 3. Methods 3.1. RNA Ext
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48 Pearson 3. Method The method of
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50 Stirling and Bartlett 4. Protein
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54 Stirling 3. Methods 3.1. Fungal
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56 McDonagh 2. Add 0.4 mL of TNE bu
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58 Schmerer 2. Materials To apply t
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60 Schmerer 3. The additional purif
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66 Bartlett Table 1 Recommended Aga
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68 Bartlett Table 2 Separation of D
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70 Bartlett 2. 5× TBE: 54 g of Tri
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72 Bartlett 7. Remove upper aqueous
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74 Bartlett 4. Many modern electrop
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76 Bartlett
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78 Stirling 11. Store at -20°C for
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82 Hyndman and Mitsuhashi 3.1. Effi
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90 Grunenwald may be affected by ea
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92 Grunenwald 50°C will generally
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94 Grunenwald of template DNA, a su
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106 Wang and Young full-length cDNA
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108 Wang and Young Fig. 1. (A) Nort
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114 Wang and Young 3′-RACE outlin
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116 Wang and Young
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118 Dassanayake and Samaranayake co
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120 Dassanayake and Samaranayake 5.
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124 Iannone et al. Fig. 1. Diagram
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128 Iannone et al. 5. For microsphe
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130 Iannone et al. 4. To adjust for
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132 Iannone et al. 6. Target concen
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136 Benjamin, Smith, and Waites amp
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140 Benjamin, Smith, and Waites 2.2
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148 Benjamin, Smith, and Waites 8.
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152 Olmos et al. Fig. 1. RT-hemines
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154 Olmos et al. This nested RT-PCR
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156 Olmos et al. Table 2 Volume and
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158 Olmos et al. 8. The sensitivity
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162 Abe 5. Moloney murine leukemia
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164 Abe Table 2 Detection Rate of H
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166 Abe 4. For HBV, nested PCR usin
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168 Tellier et al. of Pfu (and ther
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170 Tellier et al. 2. Cline, J., Br
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172 Tellier et al. 38. Tamiya, S.,
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196 Cremer and Moos 11. Klein, E.,
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198 McDonagh the dilution method is
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200 McDonagh 2.2. Basic PCR 1. dNTP
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202 McDonagh Fig. 2. Quantitative P
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206 Bartlett Table 1 Example Assay
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208 Bartlett 3.4. Calculation of Re
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212 Kerr Fig. 1. Fluorescent probes
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214 Kerr after the PCR. This reduce
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226 Dominguez et al. Table 1 Primer
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288 Edwards and Bartlett technology
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296 Rithidech and Dunn 11. Ethidium
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386 Walsh by most, but not all M13
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390 Walsh 5. For palindromic restri
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392 Walsh
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428 Bull and Paskins 2.3. Detection
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