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Direct labeling during 1 PCR cycle Relative high sensitivity (target 1–5 kb) Multiple-target detection (up to 3) High specificity and efficiency Use of primers with different Accurate DNA localization annealing temperatures Well-preserved morphology Relative accurate DNA localization Multi-target detection (up to 32) Short turnaround time (few hours) Disadvantages Limited primer elongation due to nicks Only reasonable accessibility of PCR Relative long turnaround time (1–2 days) and target DNA bound to the glass slide reagents and poor primer elongation due Relative low sensitivity (single-copy DNA) to suboptimal protein removal at the start Relative poor morphology on tissue Increasing number of PCR cycles improves Subsequent PRINS reactions needed primer elongation but also diffusion of PCR for multiple-target PRINS products. This results in: Nonspecific incorporation of labeled Low amplification efficiency, variable nucleotides (nicks, mispriming) leading reproducibility and poor DNA localization to limited signal-to-noise ratio Relative poor morphology due to heating steps in the PCR procedure Further disadvantages: Nonspecific incorporation of labeled nucleotides at nicks (direct in situ PCR) and amplification due to mispriming Only single-target detection Relative long turnaround time (1–2 days) Comments Muliple oligonuclotides and signal Signal amplification may improve Signal amplification have improved amplification have improved signal-to- signal-to-noise ratio signal-to-noise ratio and evaluation noise ratio and enabled single copy of results gene detection PRINS and In Situ PCR 413
414 Speel, Ramaekers, and Hopman technique has also been applied to identify endogenous DNA and RNA sequences in human cells (for reviews, see refs. 8–10,13). In situ PCR techniques are theoretically straightforward and comprise (1) sample fixation and pretreatment to improve the accessibility of the target nucleic acid sequences by the PCR primers, nucleotides, and Taq polymerase enzymes and to avoid diffusion of PCR-generated amplificants; (2) PCR amplification in the cell by Taq polymerase using the target DNA as a template; and (3) direct (by incorporation of labeled nucleotides during PCR) or indirect (by ISH with labeled nucleic acid probes) detection of the amplified nucleic acid molecules. Visualization of labeled target or probe DNA can be performed as described in Subheading 2.1. Thus, in principle direct in situ PCR is identical to the cycling PRINS procedure described above when cellular DNA is directly labeled during PCR cycling (Fig. 1). Consequently, the conditions for optimal chromosome labeling by (cycling) PRINS will also apply for the direct in situ PCR approach, although they need to be adapted for the specimen of interest and the fixative used to process cells and tissues. 3.2. Sample Fixation and Processing It has been reported that 4 to 10% buffered formaldehyde is the fixative of choice for successful in situ PCR on cells and (frozen) tissues and that these specimens should be adhered to coated (e.g., organosilane) glass slides to prevent loss of tissue adherence during the in situ PCR procedure (5,9). In addition, cell and tissue preparations need to be subjected to a protease (pepsin, trypsin, or protease K) treatment to create holes in cellular membranes and remove cross-linked DNA-binding proteins from nuclear DNA, thereby facilitating the accessibility of the nucleic acids in situ by the PCR and detection reagents, as well as the ISH probes (in the indirect in situ PCR procedure). Importantly, for every protease and tissue, the optimal balance between time and concentration should be determined (usually 5 to 30 min of 2 mg/mL protease at room temperature or 37°C) to avoid overdigestion, leading to poor tissue morphology and possible leakage (diffusion) of amplified products from the cell in which they were generated, or insufficient protein removal, resulting in a decreased or completely absent in situ signal caused by very inefficient or failure of amplification (5,9). A striking point to notice here is that the generally used protease pretreatment for in situ PCR is less powerful than the one applied usually for optimal ISH on formaldehyde-fixed, paraffin-embedded preparations, in which prior to the protease digestion treatments with 1 M sodium thiocyanate and, optionally, 85% formic acid/0.3% H 2 O 2 are performed to achieve optimal conditions for ISH while preserving nuclear morphology (44). Although this may still be sufficient to allow the PCR reagents to reach the target DNA, we know from our own results that efficient in situ primer extension rather requires a more powerful tissue pretreatment step than used for ISH (see Subheading 2.2. and 3.3.2.). Indeed, we have not been able to detect human centromere repeats by PRINS labeling using tissue pretreatment conditions optimal for ISH with centromere-specific plasmid probes (unpublished results), indicating that in situ primer extension during the first couple of cycles of PCR on the slide is most likely impossible or very inefficient. This might in part explain the low amplification efficiency (restricted sensitivity) often obtained by in situ PCR, which furthermore can be caused by diffusion of the synthesized DNA amplificants outside the cell (see Fig. 1 and below).
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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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358 Mazars and Theillet of genomic
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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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