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PCR Fluorescence Differential Display 237 37 PCR Fluorescence Differential Display Kostya Khalturin, Sergej Kuznetsov, and Thomas C. G. Bosch 1. Introduction Differential display of mRNA via polymerase chain reaction (DD-PCR) has become a powerful procedure for the quantitative detection of differentially expressed genes in distinct cell populations (1–4). The standard procedure includes selective reverse transcription of polyadenylated RNA using specific anchored oligo(dT) primers, PCR amplification of cDNA using the oligo(dT) primer and an arbitrary upstream primer, resolution of PCR products on denaturing sequencing gels, and radioactive detection methods. To avoid hazardous radioisotopes, several nonradioactive methods for identification of differential display cDNAs have been reported, including ethidium bromide visualization in agarose gels (5), silver staining (6,7) and chemiluminescent detection (8,9) of cDNA bands. Several protocols have been published reporting the use of automated DNA sequencers for differential display of cDNAs labeled with infrared dyes (10) or fluorescent tags (11–13). A major drawback for using automated sequencers for this purpose is the inability to recover the amplified cDNAs from the gel. As an alternative, the programmable GenomyxLR DNA sequencer (Genomyx, Foster City, CA), which allows high resolution of cDNAs and easy localization and excision of radiolabeled bands from the gel, is becoming widely used in differential display studies (3). For nonradioactive detection of differential cDNA bands on the GenomyxLR DNA sequencer, cDNAs can be fluorescently labeled by using tetramethylrhodamine (TAMRA)-anchored primers. Alternatively, cDNAs can be fluorescently labeled by using TAMRA-dUTP (Perkin–Elmer), which is incorporated into extending cDNA during the PCR (14). Fluorescently labeled PCR products are separated on a GenomyxLR DNA sequencer (Genomyx), detected by the GenomyxSC Fluorescent Imaging Scanner, and directly excised from the gel for further characterization using an actual and virtual grid system. The flowchart shown in Fig. 1 outlines the experimental procedure. 2. Materials 1. peqGold RNAPure kit (PEQLAB Biotechnologie GmbH, Germany) or other reagents for total RNA/mRNA extraction. 2. DEPC water. From: Methods in Molecular Biology, Vol. 226: PCR Protocols, Second Edition Edited by: J. M. S. Bartlett and D. Stirling © Humana Press Inc., Totowa, NJ 237
238 Khalturin, Kuznetsov, and Bosch Fig. 1. Flowchart of the fluorescent differential display procedure. 3. First-Strand cDNA synthesis Kit (Amersham Pharmacia Biotech Inc.). 4. Tailing primers T 12 2NN (e.g., T 12 CA) at concentration 25 µM (see Note 2). 5. Arbitrary 10-mer primers (e.g., OPA Kit by Operon Technologies Ltd., Amelada, CA) at concentrations of 5 µM. 6. dNTPs stock solution (1 mM of each). 7. Taq DNA Polymerase and 10, PCR buffer (Amersham Pharmacia Biotech Inc.). 8. TAMRA-dUTP (400 µM stock solution, Perkin–Elmer). 9. MgCl 2 (25 mM stock solution). 10. GeneQuantII photometer (Pharmacia) or equivalent device for measuring RNA concentration.
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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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16 McDonagh Fig. 1. Unidirectional
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18 McDonagh stored. This means that
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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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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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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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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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182 Stirling efficiency of the reac
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290 Edwards and Bartlett ing less p
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292 Edwards and Bartlett Stepwise m
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296 Rithidech and Dunn 11. Ethidium
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298 Rithidech and Dunn Fig. 1. PCR
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308 Edwards and Bartlett 3. Sartor,
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312 Schmerer References 1. Kunkel,
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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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328 Han and Robinson Fig. 1. Basic
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342 Daniels to sequence a 500-bp PC
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358 Mazars and Theillet of genomic
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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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402 Stirling 5. Homopolymer Regions
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406 Speel, Ramaekers, and Hopman Ta
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410 Speel, Ramaekers, and Hopman po
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414 Speel, Ramaekers, and Hopman te
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428 Bull and Paskins 2.3. Detection
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452 Gilchrist and Befus References
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468 Pearson and Stirling into PCR p
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470 Wang 2. Materials 2.1. PCR 1. D
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472 Wang Fig. 1. A brief outline of
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474 Wang
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476 Horton, Raju, and Conti-Fine Fi
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480 Horton, Raju, and Conti-Fine 1.
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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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524 Jones and Winistorfer 7. Goulde
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526 Burke and Barik Fig. 1. The bas
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530 Burke and Barik purified mutant
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532 Burke and Barik
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