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PRINS and In Situ PCR 411 PRINS reaction (Fig. 1, refs. 20,29,37,38,40). The repeated PRINS reactions appear to label not only the target sequence (as observed after the last PRINS cycle) but also the DNA that has been synthesized during the preceding cycles. Several protocols have been described with suggestions for optimal gene localization, including (1) the use of single or multiple primers generating DNA products of 250 to 550 bp in length; (2) alternative fixation and pretreatment protocols (e.g., ethanol and microwave treatment instead of methanol:acetic acid [31] fixation); 3) the use of a hot start (applying the reaction mixture and/or Taq polymerase at the annealing or a higher temperature to the slide to avoid mispriming or primer oligomerization during PRINS/PCR) before 1 to 20 PRINS cycles, eventually preceding by PCR with the same primers but without labeled nucleotides; and (4) adaptation of the PRINS reaction volume and conditions on the glass slide during cycling. However, despite these recommendations, a main concern comprises the reproducibility of cycling PRINS, because in most studies variable and relatively low frequencies (10–70%) of chromosomes or cells harboring PRINS signals were reported. Besides the discussed possibilities that may result in background (nicks, mispriming), the major disadvantage of applying multiple cycles of PRINS (or PCR, see later) on cell and tissue preparations is the inevitable diffusion of newly synthesized DNA products, inextricably bound up with the denaturation steps, from the site of synthesis inside and/or outside the cells, followed by possible extracellular generation of amplificants (Fig. 1. refs. 9,20,29,41–43). Several studies have provided evidence for this phenomenon by demonstrating the expected DNA products using gel electrophoresis of the reaction mixture after cycling PRINS. The observation of stronger but often more diffuse and less discretely localized PRINS signals in only a low percentage of chromosomes or cell nuclei fits in with the view that labeled DNA in these cases is retained at or near the site of synthesis by possible entrapment in the chromatin or nonspecific binding to the surrounding cellular structures, whereas diffusion of DNA away from the site of synthesis has been taken place in the remaining negative chromosomes or nuclei. That diffusion occurs may be explained by the fact that methanolacetic acid (31) fixation is an effective procedure to extract proteins, thus limiting the possibillities to entrap synthesized DNA molecules efficiently in the chromosome structures. Although postfixation in paraformaldehyde after cycling PRINS has been suggested to reduce diffusion of produced DNA (17), this will of course only be of help in the chromosomes and nuclei harboring signals. Thus, because of several drawbacks concerning the efficiency and reproducibility of cycling PRINS in localizing either low or single-copy DNA sequences in situ, a single PRINS reaction with multiple primers combined with tyramide signal amplification is recommended for this purpose (as a rapid alternative to ISH with locus-specific probes). 3. In Situ PCR 3.1. In Situ PCR vs Cycling PRINS In line with the development of PRINS, several groups working in the fields of pathology and microbiology have introduced the successful combination of PCR and ISH to visualize specific amplified nucleic acid sequences in cell and particularly formaldehyde-fixed, paraffin-embedded tissue preparations. Most studies have focused on the detection of (pro)viral (foreign) nucleic acid sequences, but in addition the
Table 2 Comparison of PRINS, in Situ PCR, and ISH for Localization of DNA Target Sequences in Situ PRINS In situ PCR ISH Main application Chromosome and gene identification and Detection of altered genes or foreign As for PRINS and in situ PCR, and quantification in cells and chromosomes (e.g., viral) DNA in paraffin-embedded localization of DNA sequences to study for use in clinical and cancer genetics and tissue section for use in molecular 3D organization of the interphase nucleus preimplantation genetic diagnosis pathology Crucial steps Primer diffusion to DNA target Primer diffusion to DNA target Probe diffusion to DNA target (see Fig. 1) Primer hybridization Primer hybridization Probe hybridization Primer elongation and DNA labeling Primer elongation (and DNA labeling) by Taq polymerase by Taq polymerase PCR while limiting diffusion of newly synthesized DNA products Procedure Specimen, fixative, and Ch: Methanolacetic acid (31) Ce, Ti: 4–10% (para)formaldehyde Ch, Ce: Methanolacetic acid (31), preferred protein removal Ce, Ti: Methanolacetic acid (31) and tuned proteinase K digestion to allow Ce: 70% ethanol and mild pepsin procedure and mild pepsin digestion reagents to access the target DNA Ti: 4–10% (para)formaldehyde combined (Ch = chromosomes, but to limit diffusion of PCR products with strong pepsin and 1M sodium Ce = cells, thiocyanate or microwave Ti = tissues) Main probe type Single or multiple Oligonucleotide primer pair Cloned genomic or cDNA (1–100 kb) in Oligonucleotide(s) (20–30 mer) (20–30 mer) plasmid, cosmid, PAC or BAC vector DNA labeling in situ Specimen denaturation Incorporation of labeled Hybridization of a denatured, labeled Primer annealing nucleotides during PCR (10–30 cycles) DNA probe on the pretreated, denatured Incorporation of labeled on the pretreated specimen specimen nucleotides by DNA polymerase (= direct in situ PCR), or PCR followed by FISH detection of amplified DNA (= indirect in situ PCR) Specificity Primer annealing Primer annealing and/or ISH after PCR Probe hybridization and stringent washes Label detection Direct (fluorochromes) or Idem Idem indirect (antibody detection and signal amplification) Advantages Good accessibility of reagents by High sensitivity (target few hundred bp) Good accessibility of reagents by optimal protein removal optimal protein removal 412 Speel, Ramaekers, and Hopman
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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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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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Page 393 and 394: 402 Stirling 5. Homopolymer Regions
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462 Speel, Ramaekers, and Hopman bu
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464 Speel, Ramaekers, and Hopman 26
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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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