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PCR Primer Design 87 (10,11). The entire statistical frequency of the entire oligonucleotide is calculated based on the constituent 6-mers by starting with the 5′ terminal 6-mer frequency and multiplying the relative frequency of the 5-mer on the 3′-end of that 6 mer having the next nucleotide. This is repeated until the end of the oligonucleotide is reached. For example, to calculate the frequency (f) of the 8-mer: CATAGCCT f(CATAGCCT) = 4 f(ATAGCC) f(CATAGC) × ——————————————————————— f(ATAGCT) + f(ATAGCG) + f(ATAGCC) + f(ATAGCT) f(CATAGC) 4 f(TAGCCT) f(CATAGC) × ——————————————————————— f(TAGCCA) + f(TAGCCG) + f(TAGCCC) + f(TAGCCT) where f(CATAGC) denotes the frequency of the 6-mer, CATAGC. 3.2.4. 3′-Terminal Effects Partial hybridization of the primer at the 3′-terminus can permit extension by DNA polymerase. This could result in depletion of primers as well as possible nonspecific amplification; therefore, this type of partial hybridization should be minimized as much as possible. There are two considerations for decreasing the chance of partial hybridization of the 3′-terminal: frequency and stability. 3.2.5. 3′-Terminal Frequency If the 3′-terminal region has a sequence that has many occurrences in the DNA that will be in the reaction, then the likelihood of partial hybridization is greater. To minimize this, the primers can be selected such that the 3′-terminal region does not have a high frequency of occurrence within the genome of interest. 3.2.6. 3′-Terminal Stability If the 3′-terminal region has a strong hybridization energy, then 3′-terminal partial hybridizations will be relatively more stable. More stable 3′-terminal hybridization will allow more false priming. Therefore if the primers are chosen such that the 3′-terminal has a low hybridization strength (also referred to as terminal stability), the primer is less likely to be priming as a result of such partial hybridization. Note that in the efficiency section above, a stronger 3′-terminal stability is said to improve efficiency. Whether one should select primers with high or low terminal stability would depend on factors, such as the nature of the experiment (i.e., whether there will be a large amount of other DNA) and the nature of the gene (whether highly specific primers can be found). 3.2.7. Specificity Within the Target Sequence If the primer is able to partially hybridize to a nonspecific region of the template, particularly undesirable effects can occur. If the nonspecific hybridization allows extension along the template such that a PCR product can be formed in conjunction with one of the primers binding at one of the actual binding sites, nonspecific amplicons
88 Hyndman and Mitsuhashi can be generated. This problem is compounded if the nonspecific binding site is within the amplicon itself. For this reason, primers should be checked for nonspecific alternate hybridization sites within the target sequence. 4. Selecting Primers for Multiplex PCR Multiplex PCR, in which several primer sets amplify several amplicons in the same reaction, add a degree of complexity to designing optimal primers. The additional issues to consider are those of possible heterodimer formation between all of the candidate primers and possible alternate hybridization sites within any of the target sequences. Some of the available primer design software provides functions for these types of designs. 5. Primer Design Software Several programs are available for PCR primer design. As mentioned above, we think HYBsimulator is the most powerful such program and does provide PCR primer selection based on all criteria mentioned in this chapter. Other popular programs are Oligo and Primer Premier , which provide a subset of these functions but are slightly easier to use. References 1. Breslauer, K. J., Frank, R., Blocker, H., and Marky, L. (1986) Predicting DNA duplex stability from the base sequence. Proc. Natl. Acad. Sci. USA 83, 3746–3750. 2. Freir, S. M., Kierzed, R., Jaeger, J. A., Sugimoto, N., Caruthers, M. H., Neilson, T., and Turner, D. H. (1986) Improved free-energy parameters for predictions of RNA duplex. Biochemistry 83, 9373–9377. 3. Wetmer, J. (1991) DNA probes: Applications of the principles of nucleic acid hybridization. Crit. Rev. Biochem. Mol. Biol. 26, 227–259. 4. Sugimoto, N., Nakano, S., Katoh, M., Matsumura, A., Nakamuta, H., Ohmichi, T., et al. (1995) Thermodynamic parameters to predict stability of RNA/DNA hybrid duplexes. Biochemistry 34, 11,211–11,216. 5. SantaLucia, J., Allawi, H. T., and Seneviratne, P. A. (1996) Improved nearest-neighbor parameters for predicting DNA duplex stability. Biochemistry 35, 3555–3562. 6. SantaLucia, J., Kierzed, R., and Turner, D. H. (1990) Effects of GA mismatches on the structure and thermodynamics of RNA internal loops. Biochemistry 29, 8813–8819. 7. Sugimoto, N., Kierzed, R., Freier, S. M., and Turner, D. H. (1986) Energetics of internal GC mismatches in ribooligonulceotide helix. Biochemistry 25, 5755–5759. 8. Hyndman, D., Cooper, A., Pruzinsky, S., Coad, D., and Mitsuhashi, M. (1996) Software to determine optimal oligonucleotide sequences based on hybridization simulation data. BioTechniques 20, 1090–1096. 9. Altschul, S. F., Gish, W., Miller, W., Myers, E. W., and Lipman, D. J. (1990) Basic local alignment search tool. J. Mol. Biol. 215, 403– 410. 10. Han, J., Hsu, C., Zhu, Z., Longshore, J., and Finley, H. (1994) Over-representation of the disease associated (CAG) and (CGG) repeats in the human genome. Nucleic Acids Res. 22, 1735–1740. 11. Han, J., Zhu, Z., Hsu, C., and Finley, W. (1994) Selection of antisense oligonucleotides on the basis of genomic frequency of the target sequence. Antisense Res. Devel. 4, 53–65.
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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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8 Carroll and Casimir of PCR. Such
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10 Carroll and Casimir cost more th
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12 Carroll and Casimir are no longe
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14 Carroll and Casimir Should these
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16 McDonagh Fig. 1. Unidirectional
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18 McDonagh stored. This means that
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20 McDonagh
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22 Stirling which will either not a
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24 Stirling
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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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32 Bartlett and White
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Page 41 and 42: 40 Going digitally to record the di
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Page 49 and 50: 48 Pearson 3. Method The method of
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Page 121 and 122: 122 Dassanayake and Samaranayake Re
Page 123 and 124: 124 Iannone et al. Fig. 1. Diagram
Page 125 and 126: Table 1 Oligonucleotides Descriptio
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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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142 Benjamin, Smith, and Waites 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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190 Cremer and Moos Fig. 3. Alignme
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192 Cremer and Moos Fig. 4. Testing
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194 Cremer and Moos 5. Compare the
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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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234 Dominguez et al. 3. If the cont
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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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248 Ringquist et al. 5. Total RNA s
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250 Ringquist et al. 3. Purificatio
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252 Ringquist et al. 2. The present
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254 Ringquist et al.
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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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284 Oien
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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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294 Edwards and Bartlett
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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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300 Rithidech and Dunn
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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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314 Schmerer
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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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322 Nakashima, Akahoshi, and Tanaka
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324 Stirling 9. TAE (20×): 484 g o
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326 Stirling
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328 Han and Robinson Fig. 1. Basic
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330 Han and Robinson sequence diffe
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332 Han and Robinson 4. Notes 1. PC
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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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352 Kösel et al. Fig. 2. Sequencin
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354 Kösel et al. 5. Kwok, S. and H
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356 Mazars and Theillet Fig. 1. Sch
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358 Mazars and Theillet of genomic
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360 Mazars and Theillet
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362 Suomalainen and Syvänen Fig. 1
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364 Suomalainen and Syvänen detect
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366 Suomalainen and Syvänen temper
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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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372 Shen et al. extended primers, w
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374 Quivy and Becker Fig. 1. The us
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376 Quivy and Becker that decreases
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378 Quivy and Becker 2. Solution of
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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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396 McAleer, Coffey, and Dunham Tab
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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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Table 2 Comparison of PRINS, in Sit
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414 Speel, Ramaekers, and Hopman te
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Table 3 Summary of Control Experime
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418 Speel, Ramaekers, and Hopman 3.
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420 Speel, Ramaekers, and Hopman ad
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422 Speel, Ramaekers, and Hopman 25
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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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434 Wiedorn and Goldmann Fig. 1. IS
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436 Wiedorn and Goldmann 41. Protei
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438 Wiedorn and Goldmann 2. Incubat
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440 Wiedorn and Goldmann 3.15. Prim
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442 Wiedorn and Goldmann ficient se
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444 Wiedorn and Goldmann 25. Kommin
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446 Gilchrist and Befus Fig. 1. Sch
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448 Gilchrist and Befus 2. Cells ar
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450 Gilchrist and Befus Fig. 3. RT
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452 Gilchrist and Befus References
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454 Speel, Ramaekers, and Hopman of
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456 Speel, Ramaekers, and Hopman Fi
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462 Speel, Ramaekers, and Hopman bu
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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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476 Horton, Raju, and Conti-Fine Fi
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478 Horton, Raju, and Conti-Fine th
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480 Horton, Raju, and Conti-Fine 1.
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482 Horton, Raju, and Conti-Fine ot
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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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498 Preston 21. Hung, T., Mak, K.,
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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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522 Jones and Winistorfer The yield
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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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