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PCR Primer Design 81 19 PCR Primer Design David L. Hyndman and Masato Mitsuhashi 1. Introduction The selection of primers for a given polymerase chain reaction (PCR) can determine the efficiency and specificity of the PCR. Although in many cases successful PCR primers have been selected with little understanding of the principles involved, PCR can often only be achieved by using primers that are designed appropriately. Here, we give general recommendations for PCR primer selection and various aspects to be considered when designing primers. 2. General Primer Considerations 2.1. Location The location of PCR primers is sometimes dictated by the purpose of the experiment. If the experiment is simply intended to identify the presence or absence of the sequence, then the location is of no consequence, so long as the amplification works well. If, however, the experiment is part of an assay for a particular allele of a gene, then the amplicon would be required to contain that region of interest. 2.2. Amplicon Size In general, amplicons are from 100 to 1000 bp in length. The lower limit is caused by the typical need to be able to visualize the amplicon on an agarose gel. The upper limit of 1000 bp is the result of difficulties in amplifying large sequences. If an assay that does not require a minimum amplicon size is used, there is no theoretical minimum amplicon size. 2.3. Guanine/Cytosine (G/C) Content Defined as the proportion of bases in the primer that are either G (guanine) or C (cytosine), good PCR primers are generally selected to have a G/C content between 40 and 60%. However, there is no well-defined reason for this, only that it has been considered preferable. 3. Considerations for Optimal PCR The issues concerning PCR primer design can be divided into two categories: efficiency and specificity. Both of these are important to consider in most applications, but often the factors that promote one of these will adversely affect the other. 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 81
82 Hyndman and Mitsuhashi 3.1. Efficiency Efficiency can be viewed as the proportion of templates that are used to synthesize new strands with each round of PCR, assuming the PCR primers are in a high abundance. A situation in which efficiency is the primary concern is the amplification of a purified template in which there is no chance of nonspecific PCR amplifications, so the main issue is that of primer binding to its target. 3.1.1. Melting Temperature Melting temperature, or Tm, is defined for a given DNA duplex as the temperature at which half of the strands are hybridized and half of the strands are not hybridized. The original definition of T m implied that the two complementary strands were in equal proportions. In cases where the two strands are not in equal proportions, such as a primer hybridizing to its target in PCR, the definition of Tm must be altered. Because in a PCR primer concentrations will be orders of magnitude higher than template concentrations, the interpretation is that the Tm is the temperature at which a primer is hybridized to half of the template strands. The T m of a given primer/template combination depends on primer concentration, template concentration, and salt concentration. Generally, the template concentration is considered to be negligible compared with the primer concentration, so the formula for calculating Tm is simplified such that it does not contain a parameter for template concentration. T m can be expressed as follows: ∆H T m = ————— –273.15 + 16.6 log [Na + ] (1,2) ∆S + Rln(c) The primer concentration is c; the ∆H and ∆S refer to the total enthalpy and entropy of hybridization, respectively; and [Na + ] is the sodium ion concentration but can refer to the total concentration of most monovalent cations (such as K + ). If there is Mg 2+ or other divalent cations such as Mn 2+ , the conversion is generally accepted as follows: Na + = 4 × [Mg 2+ ] 2 (3) 3.1.2. Calculating T m with the Nearest Neighbor Model The accurate calculation of T m from a given sequence requires determining the ∆H and ∆S of the hybridization. The most successful method for this is through the use of the nearest neighbor model (4–7). With the nearest neighbor model, every pair of adjacent base pairs makes a specific contribution to the overall ∆H and ∆S of the duplex. The total ∆H and ∆S are calculated by adding all of the component values plus a value for initiation of duplex formation. A table of ∆H and ∆S nearest neighbor values is shown in Table 1 (5). 3.1.3. Efficient PCR Primers and T m The most important issue for designing efficient PCR primers is that they must bind to the target site efficiently under the conditions of the PCR. This generally means not only that they bind at the annealing temperature but that if the annealing temperature
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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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132 Iannone et al. 6. Target concen
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136 Benjamin, Smith, and Waites amp
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138 Benjamin, Smith, and Waites the
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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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150 Benjamin, Smith, and Waites
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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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174 Tellier et al. 13. Thin-wall PC
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176 Tellier et al. 13 min for the l
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178 Tellier et al.
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182 Stirling efficiency of the reac
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184 Stirling
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186 Cremer and Moos Fig. 1. Rearran
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188 Cremer and Moos 4. Count cells
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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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204 McDonagh
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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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210 Bartlett 11. Cerenkov counting
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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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218 Bartlett As with any experiment
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220 Bartlett Even once novel regula
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222 Bartlett Notwithstanding these
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224 Bartlett
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226 Dominguez et al. Table 1 Primer
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228 Dominguez et al. 4. Oligonucleo
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230 Dominguez et al. Fig. 2. cDNA n
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232 Dominguez et al. 3.4. Gel Elect
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236 Dominguez et al. 11. Joshi, C.
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238 Khalturin, Kuznetsov, and Bosch
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246 Ringquist et al. In this chapte
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252 Ringquist et al. 2. The present
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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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276 Oien 2. Purify polyA + mRNA fro
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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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290 Edwards and Bartlett ing less p
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292 Edwards and Bartlett Stepwise m
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296 Rithidech and Dunn 11. Ethidium
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298 Rithidech and Dunn Fig. 1. PCR
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302 Edwards and Bartlett Studies th
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304 Edwards and Bartlett 11. Hot st
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306 Edwards and Bartlett Fig. 1. Ex
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308 Edwards and Bartlett 3. Sartor,
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310 Schmerer the investigation conc
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312 Schmerer References 1. Kunkel,
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316 Aubele and Smida 2.2. Chemicals
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318 Aubele and Smida References 1.
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320 Nakashima, Akahoshi, and Tanaka
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324 Stirling 9. TAE (20×): 484 g o
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328 Han and Robinson Fig. 1. Basic
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334 Han and Robinson
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338 Stirling of simple and improved
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340 Stirling concentrations interfe
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342 Daniels to sequence a 500-bp PC
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344 Daniels 4. Load all of the samp
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346 Daniels References 1. Orita, M.
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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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368 Shen et al. ladder. Also, direc
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370 Shen et al. 3. Mineral oil. 4.
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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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398 McAleer, Coffey, and Dunham Tab
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400 McAleer, Coffey, and Dunham
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402 Stirling 5. Homopolymer Regions
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406 Speel, Ramaekers, and Hopman Ta
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408 Speel, Ramaekers, and Hopman Fi
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410 Speel, Ramaekers, and Hopman po
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414 Speel, Ramaekers, and Hopman te
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418 Speel, Ramaekers, and Hopman 3.
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420 Speel, Ramaekers, and Hopman ad
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426 Bull and Paskins Fig. 1. Result
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428 Bull and Paskins 2.3. Detection
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430 Bull and Paskins 6. Shake the s
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432 Bull and Paskins
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452 Gilchrist and Befus References
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468 Pearson and Stirling into PCR p
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470 Wang 2. Materials 2.1. PCR 1. D
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472 Wang Fig. 1. A brief outline of
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474 Wang
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484 Horton, Raju, and Conti-Fine
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486 Preston amplification approach
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488 Preston 1. 10× PCR buffer: 100
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490 Preston Fig. 1. Design of degen
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492 Preston 2. Remove the AmpliTaq
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494 Preston 3.3. Cloning and DNA Se
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496 Preston MgCl 2 concentrations b
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500 Ravassard et al. Fig. 1. Constr
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502 Ravassard et al. size dispersio
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504 Ravassard et al. 9. ddH 2 O. 10
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506 Ravassard et al. 3.2.2. RNA Mix
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508 Ravassard et al. 4. Wash twice
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510 Ravassard et al.
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512 Pont-Kingdon Fig. 1. Constructi
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514 Pont-Kingdon 9. Invert tube and
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516 Pont-Kingdon
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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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