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Cloning and Mutagenesis 467 64 Cloning and Mutagenesis A Technical Overview Helen Pearson and David Stirling 1. Introduction Strategies for cloning polymerase chain reaction (PCR) products and performing in vitro site-directed mutagenesis are legion, and the following chapters outline five robust and reliable protocols. Before embarking on such a strategy, however, it is worth considering if it is entirely necessary. Even high-fidelity, proofreading Taq polymerases carry the risk of misincorporated bases being included, especially late in the PCR, when dNTP concentrations may become limiting. Thus, if the product is cloned, there is a chance that the clone selected contains a misincorporated base. If the purpose of the exercise is simply to determine the sequence of the original template, it may be more appropriate to sequence the PCR product directly and so avoid such cloning bias. If the object is to produce a clone that can be further used in expression studies for instance, it is imperative that all cloned material is sequenced to verify its integrity, prior to expression. 2. Cloning 2.1. T Overhangs Taq polymerases lacking 3′ to 5′ exonuclease activity tends to add nontemplatedirected nucleotides to the ends of double-stranded DNA fragments. If blunt end cloning is to be used, these overhangs need to be “polished,” and Chapter 65 by Kai Wang provides an optimized method using T4 DNA polymerase. Because the predominant nucleotide added in this nontemplate-directed manner is adenosine, many successful protocols have used vectors with a thymidine overhang to direct the cloning. This allows rapid cloning into a shuttle vector, from which the fragment can then be restricted and cloned further. Alternatively, Horton and colleagues present an elegant protocol (Chapter 66) using T-linkers to enable cloning into specific sites. 2.2. Restriction Site Primers Neither blunt-end cloning nor the use of T-overhang vectors allows directional cloning. If the region of template DNA to be amplified contains suitable restriction sites, the product can simple be digested and cloned in the same way as any other DNA fragment. If this is not feasible, it is possible to introduce restriction site sequences From: Methods in Molecular <strong>Bio</strong>logy, Vol. 226: PCR Protocols, Second Edition Edited by: J. M. S. <strong>Bartlett</strong> and D. Stirling © Humana Press Inc., Totowa, NJ 467
468 Pearson and Stirling into PCR products by having these sequences incorporated into the 5′ end of the PCR primer(s). The short restriction site sequence on the 5′ end of the PCR primer will not hybridize, but as long as the 3′ hybridizing region is long enough (i.e., its T m is high enough; ~20 mer), the primer will specifically bind to the appropriate site. The PCR product will thus have an additional DNA sequence at the 5′ end that will contain the endonuclease restriction site. A similar or different restriction site sequence can be added via the other PCR primer. If the other primer has a different restriction sequence, then the PCR fragment can be inserted in a directional-dependent manner in a host plasmid. There are a number of potential problems with this method that should be considered. There is no easy way to prevent internal sites containing similar restriction sequences from being cut when the end of the PCR product are cut. Care should therefore be taken to use restriction sites that are not present in the fragment to be amplified. Restriction sequences are inverse repeat sequences, thus the potential exists for primer dimer association and resultant non-productive annealing. Finally, when restriction sites are located very close to the end of an amplified fragment, the efficiency of cleavage of those sites can be markedly impaired. It may therefore be necessary to include not just the restriction site but an additional 5 to 10 bases to avoid this problem. 3. Mutagenesis In vitro site-directed mutagenesis is an invaluable technique for studying protein structure–function relationships, gene expression, and vector modification. Several methods have appeared in the literature, but many of these methods require singlestranded DNA as the template. The reason for this, historically, has been the need for separating the complementary strands to prevent reannealing. Use of PCR in site-directed mutagenesis accomplishes strand separation by using a denaturing step to separate the complementing strands and allowing efficient polymerization of the PCR primers. PCR site-directed methods thus allow site-specific mutations to be incorporated in virtually any double-stranded plasmid, eliminating the need for M13- based vectors or single-stranded rescue. Three divergent strategies for mutagenesis are outlined in the following chapters; however, several points applicable to all three should be here. First, it is often desirable to reduce the number of cycles during PCR when performing PCR-based site-directed mutagenesis to prevent clonal expansion of any (undesired) second-site mutations. Limited cycling, which would result in reduced product yield, can be offset by increasing the starting template concentration. Second, a selection must be used to reduce the number of parental molecules coming through the reaction. This is of particular importance when the parental molecules are used in high concentrations. Third, because of the tendency of some thermostable polymerases to add nontemplatedirected nucleotides to the ends of double-stranded DNA fragments, it is often necessary to incorporate an end-polishing step into the procedure prior to end-to-end ligation of the PCR-generated product containing the incorporated mutations in one or both PCR primers (see Subheading 2.1.). Finally, even if the presence of the desired mutation is confirmed by restriction digest or sequencing, it is essential to sequence the entire region of manipulated DNA to ensure that there has been no undesirable mutation introduced by the PCR processes.
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63. Primed In Situ Nucleic Acid Lab
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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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70 Bartlett 2. 5× TBE: 54 g of Tri
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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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94 Grunenwald of template DNA, a su
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106 Wang and Young full-length cDNA
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118 Dassanayake and Samaranayake co
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124 Iannone et al. Fig. 1. Diagram
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Table 1 Oligonucleotides Descriptio
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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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136 Benjamin, Smith, and Waites amp
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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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160 Olmos et al.
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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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198 McDonagh the dilution method is
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200 McDonagh 2.2. Basic PCR 1. dNTP
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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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246 Ringquist et al. In this chapte
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256 Case-Green, Pritchard, and Sout
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272 Oien Fig. 1. A schematic diagra
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274 Oien 4. 100% and 70% ethanol. 5
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278 Oien 50-mL conical tubes. Resus
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280 Oien Forward Primer, and then r
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282 Oien 8. PCR. With SAGE, achievi
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288 Edwards and Bartlett technology
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296 Rithidech and Dunn 11. Ethidium
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342 Daniels to sequence a 500-bp PC
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348 Kösel et al. Fig. 1. Schematic
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350 Kösel et al. 3. Methods 3.1. P
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372 Shen et al. extended primers, w
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374 Quivy and Becker Fig. 1. The us
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380 Quivy and Becker 7. Precipitate
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382 Quivy and Becker 7. Prepare a p
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384 Quivy and Becker
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386 Walsh by most, but not all M13
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388 Walsh PCR products are now flus
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390 Walsh 5. For palindromic restri
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392 Walsh
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394 McAleer, Coffey, and Dunham Fig
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402 Stirling 5. Homopolymer Regions
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518 Jones and Winistorfer Fig. 1. D
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520 Jones and Winistorfer Fig. 2. D
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524 Jones and Winistorfer 7. Goulde
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526 Burke and Barik Fig. 1. The bas
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528 Burke and Barik concentration o
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530 Burke and Barik purified mutant
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532 Burke and Barik
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