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Arrays for Genotyping 257 Fig. 2. The layout of reaction cells used for the synthesis of arrays. The synthesis support is pressed to the reaction cell by a clamp. The inlet and outlet ports are attached to a DNA synthesizer. DNA synthesis on the support is achieved using the synthesizer to add and remove reagents from the reaction cell; the reagents flood the surface of the support. After the required sequence has been added the clamp is released and the cell moved and reclamped to allow further synthesis. Apparatus described in ref. 6 can be used to aid this process. leaves the oligonucleotides attached to the polypropylene via the initial, ammonia stable, phosphoramidite linkage to the 5′ end of the oligonucleotide and the amine of the polypropylene support. 1.1.1. Arrays for Biallelic (SNP) Typing The simplest fabrication method involves the synthesis of each allele specific oligonucleotide (ASO) one at a time at a separate site on the surface. A patch of the first oligonucleotide is synthesized on the support using a cell to direct the synthesis reagents. The cell is then moved to a position adjacent to the first oligonucleotide and the second oligonucleotide synthesized. A long narrow cell (Fig. 2, left) minimizes
258 Case-Green, Pritchard, and Southern reagent use and allows the array to be cut into strips in a direction perpendicular to the array synthesis. Alternatively, a sequence that flanks the variable base is synthesized; the cell is displaced by half a cell’s width and a base corresponding to one of the alleles is added, the cell is then moved to cover the other half and the other varying base added. The cell can then be returned to its original position and any further bases common to both allele specific probe oligonucleotides added. 1.1.2. STR Array Fabrication The basic requirements for an array suitable for use in STR typing are shown in Fig. 3. All the oligonucleotides of the array include a sequence complementary to the region immediately adjacent to the repeats. This registration sequence forms duplex between the target and the array oligonucleotides and aligns the start of the repeat of the oligonucleotides with that in the target. The array oligonucleotides vary in the number of repeat units that they contain on top of the flanking registration sequence. Synthesis of these arrays can be carried out in a combinatorial manner. Figure 4 shows a scheme for the synthesis of an array for typing the FES locus (10). 1.2. Preparation of Target DNA To achieve good hybridization yields and allow efficient extension of the array oligonucleotides, a single-stranded target is required. Depending on the type of assay the target may be either labeled or unlabeled. Because the DNA for analysis is usually genomically derived material, an amplification step is normally performed. For hybridization assays, the most common target species is body labeled RNA, prepared by carrying out a polymerase chain reaction (PCR) of the required region using a primer that includes an RNA polymerase promoter sequence and transcribing the product with the appropriate enzyme. The reaction mixture also contains labeled nucleotide triphosphate. For assays involving enzymes, a single-stranded unlabeled DNA target is required. A two-step procedure can be used where the sample is first amplified using the PCR and the unwanted strand digested away using an exonuclease (11). The primer for the strand to be retained is synthesized with approximately the last five internucleotide linkages at the 5′ end as phosphorothioate (12), which protect the strand against nuclease degradation. After PCR, T7 gene 6 exonuclease is added to the product to digest away the unwanted, (unphosphorothioated) strand. The resulting single-stranded DNA can often be used without further purification but, if required, standard purification techniques could be used, for example, Sephadex or Qiaquick columns. 1.3. Hybridization Assays Target alleles are distinguished by their ability to hybridize to complementary allele specific oligonucleotides (ASOs) on the array (Fig. 5A) (8). Hybridization yield and discrimination depend on temperature, salt concentration, time, and target concentration. The sequence of the oligonucleotide also affects both yield and discrimination. Many alleles can be simultaneously examined on the same array, but this requires careful choice of both oligonucleotide sequence and hybridization and washing conditions to achieve the maximum discrimination under one set of conditions. Computer-based
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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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Page 203 and 204: 206 Bartlett Table 1 Example Assay
Page 205 and 206: 208 Bartlett 3.4. Calculation of Re
Page 207 and 208: 210 Bartlett 11. Cerenkov counting
Page 209 and 210: 212 Kerr Fig. 1. Fluorescent probes
Page 211 and 212: 214 Kerr after the PCR. This reduce
Page 213 and 214: 218 Bartlett As with any experiment
Page 215 and 216: 220 Bartlett Even once novel regula
Page 217 and 218: 222 Bartlett Notwithstanding these
Page 219 and 220: 224 Bartlett
Page 221 and 222: 226 Dominguez et al. Table 1 Primer
Page 223 and 224: 228 Dominguez et al. 4. Oligonucleo
Page 225 and 226: 230 Dominguez et al. Fig. 2. cDNA n
Page 227 and 228: 232 Dominguez et al. 3.4. Gel Elect
Page 229 and 230: 234 Dominguez et al. 3. If the cont
Page 231 and 232: 236 Dominguez et al. 11. Joshi, C.
Page 233 and 234: 238 Khalturin, Kuznetsov, and Bosch
Page 241 and 242: 246 Ringquist et al. In this chapte
Page 243 and 244: 248 Ringquist et al. 5. Total RNA s
Page 245 and 246: 250 Ringquist et al. 3. Purificatio
Page 247 and 248: 252 Ringquist et al. 2. The present
Page 249 and 250: 254 Ringquist et al.
Page 251: 256 Case-Green, Pritchard, and Sout
Page 255 and 256: 260 Case-Green, Pritchard, and Sout
Page 267 and 268: 272 Oien Fig. 1. A schematic diagra
Page 269 and 270: 274 Oien 4. 100% and 70% ethanol. 5
Page 271 and 272: 276 Oien 2. Purify polyA + mRNA fro
Page 273 and 274: 278 Oien 50-mL conical tubes. Resus
Page 275 and 276: 280 Oien Forward Primer, and then r
Page 277 and 278: 282 Oien 8. PCR. With SAGE, achievi
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Page 281 and 282: 288 Edwards and Bartlett technology
Page 283 and 284: 290 Edwards and Bartlett ing less p
Page 285 and 286: 292 Edwards and Bartlett Stepwise m
Page 287 and 288: 294 Edwards and Bartlett
Page 289 and 290: 296 Rithidech and Dunn 11. Ethidium
Page 291 and 292: 298 Rithidech and Dunn Fig. 1. PCR
Page 293 and 294: 300 Rithidech and Dunn
Page 295 and 296: 302 Edwards and Bartlett Studies th
Page 297 and 298: 304 Edwards and Bartlett 11. Hot st
Page 299 and 300: 306 Edwards and Bartlett Fig. 1. Ex
Page 301 and 302: 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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