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Solid-Phase Minisequencing 365 8. Release the detection step primer from the template by adding 60 µL of 50 mM NaOH and incubating for 3 min at room temperature. 9. Transfer the eluted primer to the scintillation vials, add scintillation reagent, and measure the radioactivity, that is, the amount of incorporated label, in a liquid scintillation counter (see Note 9). 10. The result is obtained as counts per minute (cpm) values. The cpm value of each reaction expresses the amount of the incorporated [ 3 H] dNTP. Calculate the ratio (R) between the mutant and normal nucleotide cpms. In a sample of a subject homozygous for the mutant nucleotide the R will be >10, in a homozygote for the normal nucleotide R
366 Suomalainen and Syvänen temperatures for the primer annealing may be required. 9. Streptavidin-coated microtiter plates made of scintillating polystyrene are available (Wallac, Finland). In this case the final washing, denaturation and transfer of the eluted detection primer can be omitted, but a scintillation counter for microtiter plates is needed (11). 10. The ratio between the cpm values for the two nucleotides reflects the ratio between the two sequences in the original sample. Therefore, the solid-phase minisequencing method can be used for quantitative PCR analyses (2–6). The R value is affected by the specific activities of the [ 3 H] dNTPs used, and if either the mutant or the normal sequence allows the detection step primer to be extended by more than one [ 3 H] dNTP, this will obviously also affect the R value. Both of these factors can easily be corrected for when calculating the ratio between the two sequences. Alternatively, a standard curve is constructed by mixing the two sequences in known ratios and plotting the obtained R-values as a function of the ratios to obtain a linear standard curve (3,5,6). The test results can then be interpreted from the standard curve without the need of taking the specific activities of the number of [ 3 H] dNTPs incorporated into account. References 1. Syvänen, A.-C., Aalto-Setälä, K., Harju, L., Kontula, K., and Söderlund, H. (1990) A primer-guided nucleotide incorporation assay in the genotyping of apolipoprotein E. Genomics 8, 684–692. 2. Suomalainen, A. and Syvänen, A.-C. (2000) Quantitative analysis of human DNA sequences by PCR and solid-phase minisequencing. Mol. <strong>Bio</strong>technol. 15, 123–131. 3. Suomalainen, A., Kollmann, P., Octave, J.-N., Söderlund, H., and Syvänen, A.-C. (1993) Quantification of mitochondrial DNA carrying the tRNA Lys 8344 point mutation in myoclonus epilepsy and ragged-red-fiber disease. Eur. J. Hum. Genet. 1, 88–95. 4. Syvänen, A.-C. (1999) From gels to chips: “Minisequencing” primer extension for analysis of point mutations and single nucleotide polymorphisms. Hum. Mutat. 13, 1–10. 5. Syvänen, A-C., Söderlund, H., Laaksonen, E., Bengtström, M., Turunen, M., and Palotie, A. (1992) N-ras gene mutations in acute myeloid leukemia: accurate detection by solid-phase minisequencing. Int. J. Cancer 50, 713–718. 6. Suomalainen, A., Majander, A., Pihko, H., Peltonen, L., and Syvänen, A.-C. (1993) Quantification of tRNA Leu 3243 point mutation of mitochondrial DNA in MELAS patients and its effects on mitochondrial transcription. Hum. Mol. Genet. 2, 525–534. 7. Chen, X. and Kwok, P. Y. (1997) Template-directed dye-terminator incorporation (TDI) assay: a homogeneous DNA diagnostic method based on fluorescence resonance energy transfer. Nucleic Acids Res. 15, 347–353. 8. Pastinen, T., Perola, M., Niini, P., Terwilliger, J., Salomaa, V., Vartiainen, E., et al. (1998) Array-based multiplex analysis of candidate genes reveals two independent and additive risk factors for myocardial infarction in the Finnish population. Hum. Mol. Genet. 7, 1453–1462. 9. Pastinen, T., Partanen, J., and Syvänen, A.-C. (1996) Multiplex, fluorescent solid-phase minisequencing for efficient screening of DNA sequence variation. Clin. Chem. 42, 1391–1397. 10. Sitbon, G., Hurtig, M., Palotie, A., Lönngren, J., and Syvänen, A.-C. (1997) A colorimetric mini-sequencing assay for the mutation in codon 506 of the coagulation factor V gene. Thrombosis Haemostasis 77, 701–703. 11. Ihalainen, J., Siitari, H., Laine, S., Syvänen, A.-C., and Palotie, A. (1994) Towards automatic detection of point mutations: use of scintillating microplates in solid-phase minisequencing. <strong>Bio</strong>Techniques 16, 938–943.
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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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34 Pearson and Stirling 11. Microfu
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36 Going 3. Methods 3.1. Section Cu
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38 Going pipet filler. Spread the p
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40 Going digitally to record the di
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42 Going
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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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52 Stirling and Bartlett
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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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62 Schmerer
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64 Stirling
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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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84 Hyndman and Mitsuhashi Fig. 1. H
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86 Hyndman and Mitsuhashi Fig. 3. H
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88 Hyndman and Mitsuhashi can be ge
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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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96 Grunenwald than absolutely neces
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98 Grunenwald 2. Foord, O. S. and R
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100 Grunenwald
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102 Stirling Table 1 Thermal Cycler
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104 Stirling
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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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110 Wang and Young Fig. 3. Expresse
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112 Wang and Young 2. First-strand
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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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122 Dassanayake and Samaranayake Re
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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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132 Iannone et al. 6. Target concen
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134 Iannone et al.
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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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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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Page 309 and 310: 316 Aubele and Smida 2.2. Chemicals
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Page 313 and 314: 320 Nakashima, Akahoshi, and Tanaka
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Page 317 and 318: 324 Stirling 9. TAE (20×): 484 g o
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Page 321 and 322: 328 Han and Robinson Fig. 1. Basic
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Page 337 and 338: 346 Daniels References 1. Orita, M.
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Page 353 and 354: 362 Suomalainen and Syvänen Fig. 1
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Page 359 and 360: 368 Shen et al. ladder. Also, direc
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Page 363 and 364: 372 Shen et al. extended primers, w
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Page 371 and 372: 380 Quivy and Becker 7. Precipitate
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Page 377 and 378: 386 Walsh by most, but not all M13
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Page 381 and 382: 390 Walsh 5. For palindromic restri
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Page 385 and 386: 394 McAleer, Coffey, and Dunham Fig
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Page 391 and 392: 400 McAleer, Coffey, and Dunham
Page 393 and 394: 402 Stirling 5. Homopolymer Regions
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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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