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PRINS and In Situ PCR 415 3.3. In Situ Amplification and Detection of Intracellular PCR Products PCR protocols with optimal stringency of primer annealing and using generally longer extension times, increasing concentrations of Taq polymerase and MgCl 2 , and /or the addition of bovine serum albumin in the reaction mixture on the cell and tissue preparations as compared with solution-phase PCR have been established by using the newly designed programmable thermal cyclers as described for (cycling) PRINS (see Subheading 2.3. and refs. 5,9,30). After PCR amplification, visualization of intracellular PCR products is achieved either directly through immunohistochemical detection of labeled nucleotides (see Subheading 2.1.) that have been incorporated into PCR products during thermal cycling (direct in situ PCR) or indirectly by ISH with a labeled probe and subsequent immunohistochemical detection (see Chapter 63 and refs. 5,9). 3.3.1 Direct In Situ PCR Although direct in situ PCR is more rapid than indirect in situ PCR by eliminating the need for subsequent ISH, this procedure has proved unreliable with respect to the specificity of the results obtained (9,43,45). Even when the hot start procedure is performed (see Subheading 2.4.), the direct detection approach yields significant false-positive results, especially when working with tissue sections that have been dried at 56 to 65°C for several hours (introduction of nicks in the DNA) (5,9). This is the result of a number of artifacts, including incorporation of labeled nucleotides into (1) nonspecific PCR products resulting from mispriming (“endogenous priming” artifacts) and primer oligomerization, which seems to occur less likely inside nuclei (5) but may play a role during extracellular amplification of diffused DNA products); (2) singleand double-stranded nicks introduced by tissue fixation, cutting, and drying; and (3) fragmented DNA undergoing “repair” by DNA polymerase (“repair” artifacts). Repair artifacts may particularly be evident in apoptotic cells or samples that have been pretreated with DNase before in situ reverse transcriptase PCR for mRNA detection (9,45). These artifacts may only slightly be reduced by using an exonuclease-free DNA polymerase or by carrying out a DNA ligase reaction to close the nicks or a ddNTP reaction to prevent a subsequent extension reaction (9,17,33). Thus, caution and adequate use of appropriate controls are recommended in the interpretation of data produced by direct in situ PCR (Table 3 and Subheading 3.4.). 3.3.2. Indirect In Situ PCR The indirect in situ PCR technique, therefore, is the preferred approach to use, because the intracellular target-specific PCR products are identified by ISH, whereas the nonspecific amplificants will not be detected. Probes targeted to regions in between the primers used for PCR, usually oligonucleotide probes of 20 to 40 bases, represent the ideal ISH probes for reasons of specificity because they assure that the detected signal is the PCR product and not the result of for example primer oligomerization. ISH with these small probes, however, is usually less sensitive than with cDNA or genomic probes. Because primer oligomerization seems not to occur inside nuclei during PCR (10), the latter probes are recommended because of the increased number of reporter molecules they carry, leading to a higher detection sensitivity. Immunohistochemical
Table 3 Summary of Control Experiments Required for DNA Detection by PRINS, in situ PCR, and ISH (Modified from ref. 9). Method Control experiment Purpose General Use of known positive and negative control samples Control for specificity and sensitivity as well as mixtures of these (cells) in different ratios Immunophenotpying the cell types of interest Control for specificity and sensitivity Solution-phase PCR on extracted DNA of sample under Control for sensitivity, false negative results and DNA quality study or directly on mildly fixed cell suspension Omission of primary antibody in immunohistochemical Control for background induced by endogenous enzyme activity detection (colorimetric detection) or nonspecific sticking of secondary and/or tertiary detection reagents to sample and/or glass slide Use of no, random, or irrelevant (labeled) primers/probes, Control for background induced by endogenous enzyme activity or vector sequences without a target-specific insert (colorimetric detection) or non-specific sticking of probe and/or detection reagents to sample and/or glass slide (Cycling) PRINS Omission of DNA polymerase Control for background induced by endogenous enzyme activity and (colorimetric detection) or nonspecific sticking of detection Direct in situ PCR reagents to sample and/or glass slide Omission of primers Control for artifacts related to endogenous priming, DNA repair and extension of nicks present in the DNA Indirect in situ PCR Omission of DNA polymerase Control for effect of amplification and sensitivity of ISH 416 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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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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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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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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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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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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206 Bartlett Table 1 Example Assay
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208 Bartlett 3.4. Calculation of Re
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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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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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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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296 Rithidech and Dunn 11. Ethidium
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298 Rithidech and Dunn Fig. 1. PCR
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310 Schmerer the investigation conc
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312 Schmerer References 1. Kunkel,
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320 Nakashima, Akahoshi, and Tanaka
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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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350 Kösel et al. 3. Methods 3.1. P
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360 Mazars and Theillet
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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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