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PRINS and Immunocytochemistry 463 11. Kadandale, J. S., Wachtel, S. S., Tunca, Y., Sid Wilroy Jr., R., Martens, P. R., and Tharapel, A. T. (2000) Localization of SRY by primed in situ labeling in XX and XY sex reversal. Am. J. Med. Genet. 95, 71–74. 12. Speel, E. J. M., Ramaekers, F. C. S., and Hopman, A. H. N. (1995) Cytochemical detection systems for in situ hybridization, and the combination with immunocytochemistry. Who is still afraid of red, green and blue? Histochem. J. 27, 833–858. 13. Speel, E. J. M., Herbergs, J., Ramaekers, F. C. S., and Hopman, A. H. N. (1994) Combined immunocytochemistry and fluorescence in situ hybridization for simultaneous tricolor detection of cell cycle, genomic, and phenotypic parameters of tumor cells. J. Histochem. Cytochem. 42, 961–966. 14. Herbergs, J., Speel, E. J. M., Ramaekers, F. C. S., De Bruïne, A. P., Arends, J. W., and Hopman, A. H. N. (1996) Combination of lamin immunocytochemistry and in situ hybridization for the analysis of chromosome copy numbers in tumor cell areas with high nuclear density. Cytometry 23, 1–7. 15. Speel, E. J. M., Lawson, D., Ramaekers, F. C. S., Gosden, J. R., and Hopman, A. H. N. (1997) Combined immunocytochemistry and PRINS DNA synthesis for simultaneous detection of phenotypic and genomic parameters in cells, in PRINS and in situ PCR protocols, Methods in Molecular Biology, Vol. 71 (Gosden, J. R., ed.), Humana Press, Totowa, NJ, pp. 53–60. 16. Roy, L., Coullin, P., Vitrat, N., Hellio, R., Debili, N., Weinstein, J., et al. (2001) Asymmetrical segregation of chromosomes with a normal metaphase/anaphase checkpoint in polyploid megakaryocytes. Blood 97, 2238–2247. 17. Coullin, P., Roy, L., Pellestor, F., Candelier, J.-J., Bed’’hom, B., Guillier-Gencik, Z., et al. (2002) PRINS, the other in situ DNA labeling method that is useful in cellular biology. Am. J. Med. Genet. 107, 127–135. 18. Jeppesen, P. (1994) Immunofluorescence techniques applied to mitotic chromosome preparations, in Chromosome Analysis Protocols, Methods in Molecular Biology (Gosden, J. R., ed.), Humana Press, Totowa, NJ, pp. 253–286. 19. Mitchell, A. R. (1997) Chromosomal PRINS DNA labeling combined with indirect immunocytochemistry, in PRINS and in situ PCR protocols, Methods in Molecular Biology (Gosden, J. R., ed.), Humana Press, Totowa, NJ, pp. 61–70. 20. Glaser, T., Housman, D., Lewis, W. H., Gerhard, D., and Jones, C. (1989) A fine-structure deletion map of chromosome 11p: analysis of J1 series hybrids. Somat. Cell Mol. Genet. 15, 477–501. 21. Boerman, O. C., Mijnheere, E. P., Broers, J. L. V., Vooijs, G. P., and Ramaekers, F. C. S. (1991) Biodistribution of a monoclonal antibody (RNL-1) against the neural cell adhesion molecule (NCAM) in athymic mice bearing human small-cell lung-cancer xenografts. Int. J. Cancer 48, 457– 462. 22. Dorin, J. R., Inglis, J. D., and Porteous, D. J. (1989) Selection for precise chromosomal targeting of a dominant marker by homologous recombination. Science 243, 1357–1360. 23. Carney, D. N., Gazdar, A. F., Bepler, G., Guccion, J. G., Marangos, P. J., Moody, T. W., et al. (1985) Establishment and identification of small cell lung cancer cell lines having classic and variant features. Cancer Res. 45, 2913–2923. 24. Koch, J., Hindkjder, J., Kölvraa, S., and Bolund, L. (1995) Construction of a panel of chromosome-specific oligonucleotide probes (PRINS-primers) useful for the identification of individual human chromosomes in situ. Cytogenet. Cell. Genet. 71, 142–147. 25. Pellestor, F., Girardet, A., Lefort, G., Andréo, B., and Charlieu, J.-P. (1995) Selection of chromosome specific primers and their use in simple and double PRINS technique for rapid in situ identification of human chromosomes. Cytogenet. Cell. Genet. 70, 138–142.
464 Speel, Ramaekers, and Hopman 26. Krejci, K. and Koch, J. (1999) An in situ study of variant telomeric repeats in human chromosomes. Genomics 58, 202–206. 27. Ramael, M., Van Steelandt, H., Stuyven, G., Van Steenkiste, M., and Degroote, J. (1999) Detection of human papilloma virus (HPV) genomes by the primed in situ labelling technique. Pathol. Res. Pract. 195, 801–807. 28. Bobrow, M. N., Harris, T. D., Shaughnessy, K. J., and Litt, G. J. (1989) Catalyzed reporter deposition, a novel method of signal amplification. Application to immunoassays. J. Immunol. Methods 125, 279–285. 29. Speel, E. J. M., Hopman, A. H. N., and Komminoth, P. (1999) Amplification methods to increase the sensitivity of in situ hybridization. Play card(s). J. Histochem. Cytochem. 47, 281–288. 30. 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 Biology (Darby, I. A., ed.), Humana Press, Totowa, NJ, pp. 195–216.
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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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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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396 McAleer, Coffey, and Dunham Tab
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398 McAleer, Coffey, and Dunham Tab
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400 McAleer, Coffey, and Dunham
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402 Stirling 5. Homopolymer Regions
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406 Speel, Ramaekers, and Hopman Ta
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408 Speel, Ramaekers, and Hopman Fi
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410 Speel, Ramaekers, and Hopman po
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Page 409 and 410: 420 Speel, Ramaekers, and Hopman ad
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Page 415 and 416: 426 Bull and Paskins Fig. 1. Result
Page 417 and 418: 428 Bull and Paskins 2.3. Detection
Page 419 and 420: 430 Bull and Paskins 6. Shake the s
Page 421 and 422: 432 Bull and Paskins
Page 423 and 424: 434 Wiedorn and Goldmann Fig. 1. IS
Page 425 and 426: 436 Wiedorn and Goldmann 41. Protei
Page 427 and 428: 438 Wiedorn and Goldmann 2. Incubat
Page 429 and 430: 440 Wiedorn and Goldmann 3.15. Prim
Page 431 and 432: 442 Wiedorn and Goldmann ficient se
Page 433 and 434: 444 Wiedorn and Goldmann 25. Kommin
Page 435 and 436: 446 Gilchrist and Befus Fig. 1. Sch
Page 437 and 438: 448 Gilchrist and Befus 2. Cells ar
Page 439 and 440: 450 Gilchrist and Befus Fig. 3. RT
Page 441 and 442: 452 Gilchrist and Befus References
Page 443 and 444: 454 Speel, Ramaekers, and Hopman of
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
Page 463 and 464: 476 Horton, Raju, and Conti-Fine Fi
Page 465 and 466: 478 Horton, Raju, and Conti-Fine th
Page 467 and 468: 480 Horton, Raju, and Conti-Fine 1.
Page 469 and 470: 482 Horton, Raju, and Conti-Fine ot
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
Page 479 and 480: 492 Preston 2. Remove the AmpliTaq
Page 481 and 482: 494 Preston 3.3. Cloning and DNA Se
Page 483 and 484: 496 Preston MgCl 2 concentrations b
Page 485 and 486: 498 Preston 21. Hung, T., Mak, K.,
Page 487 and 488: 500 Ravassard et al. Fig. 1. Constr
Page 489 and 490: 502 Ravassard et al. size dispersio
Page 491 and 492: 504 Ravassard et al. 9. ddH 2 O. 10
Page 493 and 494: 506 Ravassard et al. 3.2.2. RNA Mix
Page 495 and 496: 508 Ravassard et al. 4. Wash twice
Page 497 and 498: 510 Ravassard et al.
Page 499 and 500: 512 Pont-Kingdon Fig. 1. Constructi
Page 501 and 502: 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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