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PRINS and In Situ PCR 423 45. Sällström, J. F., Zehbe, I., Alemi, M., and Wilander, E. (1993) Pitfalls of in situ polymerase chain reaction (PCR) using direct incorporation of labelled nucleotides. Anticancer Res. 13, 1153. 46. Speel, E. J. M., Hopman, A. H. N., and Komminoth, P. (2000) Signal amplification for DNA and mRNA in situ hybridization, in In Situ Hybridization Protocols. Methods in Molecular <strong>Bio</strong>logy (Darby, I. A., ed.), Humana Press, Totowa, NJ, pp. 195–216. 47. Sperry, A., Jin, L., and Lloyd, R. V. (1996) Microwave treatment enhances detection of RNA and DNA by in situ hybridization. Diagn. Mol. Pathol. 5, 291–296. 48. Pilling, A., Endersby-Wood, H., and Jones, S. (1997) Increased sensitivity of in situ hybridization: implications for in situ amplification. Diagn. Mol. Pathol. 6, 174. 49. Nuovo, G. J. (2001) Co-labeling using in situ PCR: A review. J. Histochem. Cytochem. 49, 1329–1339. 50. Komminoth, P., Long, A. A., Ray, R., and Wolfe, H. J. (1992) In situ polymerase chain reaction detection of viral DNA, single copy genes and gene rearrangements in cell suspensions and cytospins. Diagn. Mol. Pathol. 1, 85–97. 51. Wiedorn, K. H., Kühl, H., Galle, J., Caselitz, J., and Vollmer, E. (1999) Comparison of in situ hybridization, direct and indirect in situ PCR as well as tyramide signal amplification for the detection of HPV. Histochem. Cell <strong>Bio</strong>l. 111, 89–95. 52. Patterson, B., Till, M., Otto, P., Goolsby, C., Furtado, M., McBride, L., and Wolinsky, S. (1993) Detection of HIV-1 DNA and messenger RNA in individual cells by PCR-driven in situ hybridization and flow cytometry. Science 260, 976–979. 53. Speel, E. J. M., Richter, J., Moch, H., Egenter, C., Saremaslani, P., Rütimann, K., et al. (1999) Genetic differences in endocrine pancreatic tumor subtypes detected by comparative genomic hybridization. Am. J. Pathol. 155, 1787–1794. 54. Zehbe, I., Hacker, G. W., Su, H., Hauser-Kronberger, C., Hainfeld, J. F., and Tubbs, R. (1997) Sensitive in situ hybridization with catalyzed reporter deposition, streptavidinnanogold, and silver acetate autometallography. Am. J. Pathol. 150, 1553–1561. 55. Lizardi, P. M., Huang, X., Zhu, Z., Bray-Ward, P., Thomas, D. C., and Ward, D. C. (1998) Mutation detection and single-molecule counting using isothermal rolling-circle amplification. Nat. Genet. 19, 225–232. 56. Höfler, H., Pütz, B., Mueller, J., Neubert, W., Sutter, G., and Gais, P. (1995) In situ amplification of measles virus RNA by the self-sustained sequence replication reaction. Lab. Invest. 73, 577–585. 57. Tecott, L. H., Barchas, J. D., and Eberwine, J. H. (1988) In situ transcription: specific synthesis of complementary DNA in fixed tissue sections. Science 240, 1661–1664. 58. Mogensen, J., Kolvraa, S., Hindkjœr, J., Petersen, S., Koch, J., Nygård, M., et al. (1991) Nonradioactive, sequence-specific detection of RNA in situ by primed in situ labeling (PRINS). Exp. Cell Res. 196, 92–98. 59. Player, A. N., Shen, L.-P., Kenny, D., Antao, V. P., and Kolberg, J. A. (2001) Single-copy gene detection using branched DNA (bDNA) in situ hybridization. J. Histochem. Cytochem. 49, 603–611. 60. Mangham, D. C. and Isaacson, P. G. (1999) A novel immunohistochemical detection system using mirror image complementary antibodies (MICA). Histopathology 35, 129–133. 61. Pastore, J. N., Clampett, C., Miller, J., Porter, K., and Miller, D. (1995) A rapid immunoenzyme double labeling technique using enzyme polymer system (EPOS) reagents. J. Histotechnol. 18, 35–40. 62. Kämmerer, U., Kapp, M., Gassel, A. M., Richter, T., Tank, C., Dietl, J., and Ruck, P. (2001) A new rapid immunohistochemical staining technique using the EnVision antibody complex. J. Histochem. Cytochem. 49, 623–630.
424 Speel, Ramaekers, and Hopman 63. Van Gijlswijk, R. P. M., Van Gijlswijk-Janssen, D. J., Raap, A. K., Daha, M. R., and Tanke, H. J. (1996) Enzyme-labeled antibody-avidin conjugates: new flexible and sensitive immunochemical reagents. J. Immunol. Methods 189, 117–127. 64. Chen, B.-X., Szabolcs, M. J., Matsushima, A. Y., and Erlanger, B. F. (1996) A strategy for immunohistochemical signal enhancement by end-product amplification. J. Histochem. Cytochem. 44, 819–824.
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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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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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Page 363 and 364: 372 Shen et al. extended primers, w
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Page 371 and 372: 380 Quivy and Becker 7. Precipitate
Page 373 and 374: 382 Quivy and Becker 7. Prepare a p
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Page 377 and 378: 386 Walsh by most, but not all M13
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Page 381 and 382: 390 Walsh 5. For palindromic restri
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Page 385 and 386: 394 McAleer, Coffey, and Dunham Fig
Page 387 and 388: 396 McAleer, Coffey, and Dunham Tab
Page 389 and 390: 398 McAleer, Coffey, and Dunham Tab
Page 391 and 392: 400 McAleer, Coffey, and Dunham
Page 393 and 394: 402 Stirling 5. Homopolymer Regions
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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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