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Direct and Indirect In Situ PCR 443 9a. Furuta, Y., Shinohara, T., Sano, K., Meguro, M., and Nagashima, K. (1990) In situ hybridization with digoxigenin-labelled DNA probes for detection of viral genomes. J. Clin. Pathol. 43, 806–809. 10. Allan, G. M., Todd, D., Smyth, J. A., Mackie, D. P., Burns, J., and McNulty, M. S. (1989) In situ hybridization: an optimised detection protocol for a biotinylated DNA probe renders it more sensitive than a comparable 35 S-labeled probe. J. Virol. Methods 24, 181–190. 11. Burns, J., Graham, A., Frak, C., Fleming, F., Evans, M., and McGee, J. (1987) Detection of low copy human papilloma virus DNA and mRNA in routine paraffin sections of cervix by non-isotopic in situ hybridization. J. Clin. Pathol. 40, 858–864. 12. Furuta, Y., Shinohara, T., Sano, K., Meguro, M., and Nagashima, K. (1990) In situ hybridization with digoxigenin-labelled DNA probes for detection of viral genomes. J. Clin. Pathol. 43, 806–809. 13. Nuovo, G. J. and Richart, R. M. (1989) A comparison of biotin and 35 S-based in situ hybridization methodologies for detection of human papilloma-virus DNA. Lab. Invest. 61, 471–476. 14. Syrjänen, S., Partanen, P., Mäntyjärvi, R., and Syrjanen K. (1988) Sensitivity of in situ hybridization techniques using biotin- and 35S-labeled human papillomavirus (HPV) DNA probes. J. Virol. Methods 19, 225–238. 15. Wiedorn, K. H., Lang, D., Köhl, H., Galle, J., and Vollmer, E. (1999) Sensitive in situ detection of modulated expression levels of interleukin 6 (IL6) and interleukin 8 (IL8) in bronchial epithelium and leucocytes using cDNA specific primers and direct RT in-Situ- PCR. Elec. J. Pathol. Histol. 993–996. 16. Herrington, C. S., Burns, J., Graham, A. K., Evans, M., and McGee, J. O. (1989) Interphase cytogenetics using biotin and digoxigenin labeled probes I: relative sensitivity of both reporter molecules for detection of HPV 16 in CaSki cells. J. Clin. Pathol. 42, 592–600. 17. Holm, R., Karlsen, F., and Nesland, J. M. (1992) In situ hybridization with nonisotopic probes using different detection systems. Mod. Pathol. 5, 315–319. 18. Lassus, J., Niemi, K.-M., Marjamaki, A., Syrjanen, S., Kartamaa, M., Lehmus, A., et al. (1992) Comparison of four in situ hybridization methods, based on digoxigenin- and biotin-labelled probes, in detecting HPV DNA in male condylomata acuminata. Int. J. STD AIDS 3, 196–203. 19. Mifflin, T. E., Bowdwn, J., Lovell, M. A., Burns, D. E., Hayden, F. G., Graschel, D. H. M, et al. (1987) Comparison of radioactive and biotinylated DNA probes for the detection of cytomegalovirus. Clin. Biochem. 20, 231–235. 20. Niedobitek, G., Finn, T., Herbst, H., and Stein, H. (1989) In situ hybridization using biotinylated probes. An evaluation of different detection systems. Pathol. Res. Pract. 184, 343–348. 21. Komminoth, P. and Long, A. A. (1993) In situ polymerase chain reaction. An overview of methods, applications and limitations of a new molecular technique. Virchows Archiv. 64, 67–73. 22. Komminoth, P. and Long, A. A. (1995) In situ polymerase chain reaction—methodology, applications and nonspecific pathways, in PCR Applications Manual, Boehringer Mannheim, Mannheim, pp. 97–106. 23. Long, A. A., Komminoth, P., and Wolfe, H. J. (1993) Comparison of indirect and direct in situ polymerase chain reaction in cell preparations and tissue sections. Detection of viral DNA gene rearrangements and chromosomal translocations. Histochemistry 99, 51–162. 24. Söllström, 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, 1153–1154.
444 Wiedorn and Goldmann 25. Komminoth, P. and Werner, M. (1997) Target and signal amplification: approaches to increase the sensitivity of in situ hybridization. Histochem. Cell Biol. 108, 325–333. 26. Komminoth, P., Adams, V., Long, A. A., Roth, J., Saremaslani, P, Flury, R., et al. (1994) Evaluation of methods for hepatitis C virus (HCV) detection in liver biopsies: Comparison of histology, immunohistochemistry, in situ hybridization, reverse (RT) PCR and in situ RT PCR. Pathol. Res. Pract. 190, 1017–1025. 27. Wiedorn, K. H., Schaaf, B., Kühl, H., Spuck, S., Galle, J., Dalhoff, K., et al. (1999) Granulocyte Colony Stimulating Factor (G-CSF) induced modulation of cytokine expression: A semiquantitative in situ analysis using target amplification by reverse transcriptase in situ PCR (RT IS PCR) as well as signal amplification by in situ hybridization (ISH) with catalyzed reporter deposition (CARD). Verhandlungen der Deutschen Gesellschaft für Zytologie 21, 63–65. 28. Wiedorn, K. H., Goldmann, T., Kühl, H., Rohrer, Ch., and Vollmer, E. (2000) Single-copy virus detection by GenPoint in situ hybridisation on the Eppendorf Mastercycler. BioTech Int. 12, 12–13. 29. Wiedorn, K. H. (1996) In situ Hybridisierungshandbuch zum MISHA in situ Thermocycler, in Misha Multipler In-Situ-Hybridisierungsautomat für die In-Situ-Hybridisierung und Polymerase-Kettenreaktion (PCR), Shandon, Frankfurt. 30. Stapleton, M. J., Levin, M. C., and Jacobson, S. (1994) DNA Amplification within cells on slides: Advances in evaporation and temperature control. Cell Vision 1, 177–181.
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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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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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370 Shen et al. 3. Mineral oil. 4.
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372 Shen et al. extended primers, w
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374 Quivy and Becker Fig. 1. The us
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376 Quivy and Becker that decreases
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378 Quivy and Becker 2. Solution of
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380 Quivy and Becker 7. Precipitate
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382 Quivy and Becker 7. Prepare a p
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384 Quivy and Becker
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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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Page 385 and 386: 394 McAleer, Coffey, and Dunham Fig
Page 387 and 388: 396 McAleer, Coffey, and Dunham Tab
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Page 391 and 392: 400 McAleer, Coffey, and Dunham
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Page 415 and 416: 426 Bull and Paskins Fig. 1. Result
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Page 423 and 424: 434 Wiedorn and Goldmann Fig. 1. IS
Page 425 and 426: 436 Wiedorn and Goldmann 41. Protei
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Page 435 and 436: 446 Gilchrist and Befus Fig. 1. Sch
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Page 441 and 442: 452 Gilchrist and Befus References
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Page 445 and 446: 456 Speel, Ramaekers, and Hopman Fi
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Page 455 and 456: 468 Pearson and Stirling into PCR p
Page 457 and 458: 470 Wang 2. Materials 2.1. PCR 1. D
Page 459 and 460: 472 Wang Fig. 1. A brief outline of
Page 461 and 462: 474 Wang
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Page 467 and 468: 480 Horton, Raju, and Conti-Fine 1.
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Page 471 and 472: 484 Horton, Raju, and Conti-Fine
Page 473 and 474: 486 Preston amplification approach
Page 475 and 476: 488 Preston 1. 10× PCR buffer: 100
Page 477 and 478: 490 Preston Fig. 1. Design of degen
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Page 481 and 482: 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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