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Direct Automated Cycle Sequencing 345 Fig. 1. Analyzed sequence data for a 350-bp PCR fragment amplified from genomic human DNA. The data were obtained using one of the primers used for PCR amplification. 345
346 Daniels References 1. Orita, M., Suzuki, Y., Sekeiya, T., and Hayashi, K. (1989) Rapid and sensitive detection of point mutations and DNA polymorphisms using the polymerase chain reaction. Genomics 5, 874–879. 2. Kwok, P. Y., Carlson, C., Yager, T. D., Ankener, W., and Nickerson, D. A. (1994) Comparative analysis of human DNA variations by fluorescence based sequencing of PCR products. Genomics 23, 138–144. 3. Martin-Gallardo, A., McCombie, W. R., Gocayne, J. D., Fitzgerald, M. G., Wallace, S., Lee, B. M. B., et al. (1992) Automated DNA sequencing and analysis of 106 kilobases from human chromosome 19q 13.3. Nat. Genet. 1, 34–39. 4. NIH/CEPH Collaborative Mapping Group. (1992) A comprehensive genetic linkage map of the human genome. Science 258, 67–86. 5. Mullis, K. B. and Faloona, F. A. (1987) Specific synthesis of DNA in vitro via a polymerase catalysed chain reaction. Methods Enzymol. 155, 335–350. 6. Sanger, F., Nicklen, S., and Coulson, A. R. (1977) DNA sequencing with chain terminating inhibitors. Proc. Natl. Acad. Sci. USA 74, 5463–5467. 7. Maxam, A. M. and Gilbert, W. (1977) A new method for sequencing DNA. Proc. Natl. Acad. Sci. USA 74, 560–564. 8. Carothers, A. M., Urlaub, G., Mucha, J., Grunberger, D., and Chasin, L. A. (1989) Point mutation analysis in a mammalian gene: Rapid preparation of total RNA, PCR amplification of cDNA, and Taq sequencing by a novel method. <strong>Bio</strong>Techniques 7, 494– 499. 9. McBride, L. J., Koepf, S. M., Gibbs, R. A., Nyugen, P., Salser, W., Mayrand, P. E., et al. (1989) Automated DNA sequencing methods involving polymerase chain reaction. Clin. Chem. 35, 2196–2201. 10. Tracy, T. E. and Mulcahy, L. S. (1991) A simple method for direct automated sequencing of PCR fragments. <strong>Bio</strong>Techniques 11, 68–75. 11. Rosenthal, A. and Charnock Jones, D. S. (1992) New protocols for DNA sequencing with dye terminators. DNA Sequence 3, 61–64. 12. Kelley, J. M. (1994) Automated dye terminator DNA sequencing, in Automated DNA Sequencing and Analysis (Adams, M. A., Fields, C., and Venter, J. C., eds.), Academic, London, pp. 175–181. 13. Hawkins, T. L. and Sulston, J. E. (1991) The resolution of compressions in automated fluorescent sequencing. Nucleic Acids Res. 19, 2784.
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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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Page 287 and 288: 294 Edwards and Bartlett
Page 289 and 290: 296 Rithidech and Dunn 11. Ethidium
Page 291 and 292: 298 Rithidech and Dunn Fig. 1. PCR
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Page 295 and 296: 302 Edwards and Bartlett Studies th
Page 297 and 298: 304 Edwards and Bartlett 11. Hot st
Page 299 and 300: 306 Edwards and Bartlett Fig. 1. Ex
Page 301 and 302: 308 Edwards and Bartlett 3. Sartor,
Page 303 and 304: 310 Schmerer the investigation conc
Page 305 and 306: 312 Schmerer References 1. Kunkel,
Page 307 and 308: 314 Schmerer
Page 309 and 310: 316 Aubele and Smida 2.2. Chemicals
Page 311 and 312: 318 Aubele and Smida References 1.
Page 313 and 314: 320 Nakashima, Akahoshi, and Tanaka
Page 315 and 316: 322 Nakashima, Akahoshi, and Tanaka
Page 317 and 318: 324 Stirling 9. TAE (20×): 484 g o
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Page 321 and 322: 328 Han and Robinson Fig. 1. Basic
Page 323 and 324: 330 Han and Robinson sequence diffe
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Page 331 and 332: 340 Stirling concentrations interfe
Page 333 and 334: 342 Daniels to sequence a 500-bp PC
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Page 339 and 340: 348 Kösel et al. Fig. 1. Schematic
Page 341 and 342: 350 Kösel et al. 3. Methods 3.1. P
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Page 353 and 354: 362 Suomalainen and Syvänen Fig. 1
Page 355 and 356: 364 Suomalainen and Syvänen detect
Page 357 and 358: 366 Suomalainen and Syvänen temper
Page 359 and 360: 368 Shen et al. ladder. Also, direc
Page 361 and 362: 370 Shen et al. 3. Mineral oil. 4.
Page 363 and 364: 372 Shen et al. extended primers, w
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Page 369 and 370: 378 Quivy and Becker 2. Solution of
Page 371 and 372: 380 Quivy and Becker 7. Precipitate
Page 373 and 374: 382 Quivy and Becker 7. Prepare a p
Page 375 and 376: 384 Quivy and Becker
Page 377 and 378: 386 Walsh by most, but not all M13
Page 379 and 380: 388 Walsh PCR products are now flus
Page 381 and 382: 390 Walsh 5. For palindromic restri
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Page 385 and 386: 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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Table 2 Comparison of PRINS, in Sit
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414 Speel, Ramaekers, and Hopman te
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Table 3 Summary of Control Experime
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418 Speel, Ramaekers, and Hopman 3.
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420 Speel, Ramaekers, and Hopman ad
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422 Speel, Ramaekers, and Hopman 25
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426 Bull and Paskins Fig. 1. Result
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428 Bull and Paskins 2.3. Detection
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430 Bull and Paskins 6. Shake the s
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432 Bull and Paskins
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434 Wiedorn and Goldmann Fig. 1. IS
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436 Wiedorn and Goldmann 41. Protei
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438 Wiedorn and Goldmann 2. Incubat
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440 Wiedorn and Goldmann 3.15. Prim
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442 Wiedorn and Goldmann ficient se
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444 Wiedorn and Goldmann 25. Kommin
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446 Gilchrist and Befus Fig. 1. Sch
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448 Gilchrist and Befus 2. Cells ar
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450 Gilchrist and Befus Fig. 3. RT
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452 Gilchrist and Befus References
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454 Speel, Ramaekers, and Hopman of
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456 Speel, Ramaekers, and Hopman Fi
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462 Speel, Ramaekers, and Hopman bu
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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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