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Detection of MIS 305 1. Archival DNA template (1 µL). 2. 1× reaction buffer (1 µL of 10× reaction buffer); because the reaction buffer contained 15 mM MgCl 2 , this gave a reaction concentration of 1.5 mM MgCl 2 . 3. 2 µM reverse and forward primer (1 µL) to give a reaction concentration of 0.2 µM for each primer. 4. 0.6 1 µL of 25 mM MgCl 2 to give a final reaction concentration of 3 mM MgCl 2 . 5. 0.2 1 µL of 10 mM dNTP to give an reaction concentration of 200 µL each of dATP, dCTP, dGTP, and dTTP. 6. 0.1 1 µL of 5 units/1 µL hot star Taq polymerase to give 0.5 units per reaction. 7. Volume is made up to 10 1 µL using PCR-grade water. The reaction was started after a 15-min denaturation of DNA at 95°C. DNA amplification was performed in a thermal cycler as follows: 45 cycles of denaturation at 94°C for 30 s; annealing at 47, 50, or 53°C (Table 1) for 30 s and extension at 72°C for 1 min; followed by a final extension for 15 min at 72°C. The PCR is repeated using the same DNA template for each of the primer sets. On removal from the thermal cycler 2 µL of each of the 9 PCR products from each primer set are combined for each template and mixed thoroughly by vortexing. 3.4. PCR Analysis 1. PCR products are then prepared for gel electrophoresis. 2. The following are combined in a 200-µL DNA/RNA free microfuge tube and vortexed: .5 µL of combined PCR products; 1 µL of formamide; 0.5 µL of loading buffer (50 mg/mL blue dextran, 25 mM EDTA); and 0.5 µL of commercial standard (Genescan 400 HD (rox), ABI Perkin–Elmer, Norwalk, CT). 3. The mixture is then incubated at 96°C for 4 min and cooled on ice. 4. The mixture (1.5 µL) is applied to a 4% arcrylamide/bisacrylamide gel and electrophoresed for 2 h on an automated laser-activated fluorescent DNA sequencer (Perkin–Elmer ABI 377 sequencer). 5. Fluorescent gel data were collected automatically during electrophoresis and analyzed using Genescan software. An example of the gel image is shown in Fig. 1. The data gained from Genescan is then exported into Genotyper and further analyzed. 3.5. Assessment of Allele Loss and Microsatellite Instability Allele loss should be assessed as described by Dietmaier et al. (10). In heterozygous individuals, two alleles, that is, two PCR products of different size can be detected in normal DNA. Because PCR fragments of different sizes are amplified with different efficiencies, the ratio of allele peak heights is calculated in matched normal and tumor DNA. Peak heights of the longer length allele peaks are divided by the peak heights of the shorter length allele peaks (see Note 2). The ratio obtained in tumor DNA divided by the allele peak ratio of paired normal DNA gives a result range of 0.00 to 1.00 (see Note 3), that is, (tumor allele 1 peak height/tumor allele 2 peak height)/(normal allele 1 peak height/normal allele 2 peak height). Theoretically a complete allele loss results is a value of 0 and both alleles retained results in a ratio of 1. In cases where the shorter length allele is lost the ratio obtained is greater than 1, this is therefore inverted (1/×) to obtain values within the 0.00 to 1.00 range. A ratio below 0.65 represents an allele signal reduction of 35%, this is considered to be indicative of allele loss (see Note 4). This limit was chosen because
306 Edwards and <strong>Bartlett</strong> Fig. 1. Example of a gel image. the tumor cell content after microdissection is assessed to be greater than 70% and inter assay variations of the detection system is below 5%. An example of LOH detection using the Geneotype ® Software can be seen in Fig. 2. A locus is described as unstable if a novel peak is present or if a peak has undergone a size shift after PCR amplification of tumor DNA compared with PCR amplification of paired normal DNA. A tumor is described as exhibiting MIS if at least 40% of the loci investigated are found to be unstable. 4. Notes 1. Inactivation of proteinase K is a very important step because failure to inactivate proteinase K will cause PCR to fail, with the proteinase K destroying the Taq polymerase. 2. When analyzing Genotyper data, more than one peak may be present for each allele because of polymerase artifact stutter bands. It is therefore difficult to decide the size and the height of the peak to include in the calculation. In general, however, the sizes
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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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Page 309 and 310: 316 Aubele and Smida 2.2. Chemicals
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358 Mazars and Theillet of genomic
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360 Mazars and Theillet
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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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376 Quivy and Becker that decreases
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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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Table 2 Comparison of PRINS, in Sit
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414 Speel, Ramaekers, and Hopman te
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418 Speel, Ramaekers, and Hopman 3.
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420 Speel, Ramaekers, and Hopman ad
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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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444 Wiedorn and Goldmann 25. Kommin
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446 Gilchrist and Befus Fig. 1. Sch
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452 Gilchrist and Befus References
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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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