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Ligase Chain Reaction 145 2. The LCx procedure requires that the negative control and the calibrator be run in duplicate with each carousel of specimen. 3. The negative control and calibrator are activated by the addition of 100 µL of LCx activation reagent. It is important to make sure correct volume is added or the run may be invalid. After addition, the contents of the bottles are then recapped and vortexed for 20 s. Each bottle of activated negative control or calibrator is designed to be used up to 48 h if stored at 2–8°C. 4. A positive control that monitors the entire assay procedure including the specimen processing step should be tested. A known positive urine specimen can be processed in parallel and tested with unknown specimens. The positive control should give a positive assay value (S/CO ratio >1.00). Each laboratory should establish a target value and limits from each control batch using statistical control rules. These target values and limits should be maintained throughout storage. Alternative choices for a positive control are type strains of the microorganism targeted in the assay, e.g., N. gonorrhoeae. 3.3.6. LCx Amplification Procedure 1. Turn the LCx Thermal Cycler on for at least 15 min prior to use. 2. Collect all LCx amplification vials containing samples, negative control and calibrator from Area 1 and transfer to Area 2 for thermal cycling. 3. LCx thermal cycling conditions should be edited to the following amplification parameters: 93°C for 1 s, 59°C for 1 s, 62°C for 1 min, 10 s for 40 cycles. 4. Place the amplification vials into the thermal cycler, and initiate run. After completion of the thermal cycler run, amplification product may remain at 15–30°C for up to 72 h prior to LCx detection. 3.3.7. Detection of Amplification Product 1. Refer to the LCx Analyzer Operations Manual for detailed instrument operation procedures. Before running the LCx Analyzer, check to see that LCx Inactivation Diluent (1) contains a minimum of 100 mL and the LCx System Diluent (2) contains a minimum of 250 mL. Remove the LCx Amplification Vials from the LCx Thermal Cycler. 2. Place LCx Reaction Cells into a Carousel; lock the carousel. 3. Pulse-centrifuge the LCx amplification vials in a microcentrifuge for 10–15 s before placing into the LCx reaction cells. 4. Place the amplification vials into the LCx reaction cells in the following order: negative controls in positions 1 and 2, calibrators in positions 3 and 4, and specimens in the remaining positions. 5. Place the carousel into the LCx Analyzer. 6. Lock the amplification vial Retainer. 7. Remove the LCx detection reagent Pack from 2–8°C storage, gently invert it five times, and open the reagent pack bottles in the numeric order: 1, 2, 3, 4. 8. Look for any film that may have formed over the opening of the reagent bottles. If present burst the bubble. 9. Place the LCx detection reagent pack into the LCx Analyzer. 10. Press Assay, then sample management to log in samples for the run. Press RUN on the LCx Analyzer control panel. Final assay results will be printed in approx 60 min. 11. Store the detection reagent pack at 2–8°C in original packaging, decontaminated with 1% v/v hypochlorite or separate from unopened LCx kits. 12. Remove the Carousel, individually remove the LCx reaction cells, and dispose appropriately. Rinse the carousel with water after each use.
146 Benjamin, Smith, and Waites 3.3.8. Calculation of Results 1. N. gonorrhoeae. The LCx Assay uses MEIA detection on the LCx Analyzer to detect DNA. All calculations are performed automatically. 2. The presence or absence is determined by relating the LCx Assay results for the specimen to the Cutoff value. The Cutoff value is the mean RATE (c/s/s) of the LCx calibrator duplicates multiplied by 0.25. 3. Calculation of the Cutoff value: Cutoff value = 0.25 × (Mean of LCx Gonorrhea Calibrator RATES) The S/CO value is determined by calculating a ratio of the sample RATE to the Cutoff value S CO Sample RATE Cutoff Value 3.3.9. Interpretation of Results N. gonorrhoeae S/CO Ratio Result Report >1.20 LCx positive N. gonorrhoeae DNA detected, and positive for N. gonorrhoeae by LCR amplification and MEIA detection.
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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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Page 105 and 106: 106 Wang and Young full-length cDNA
Page 107 and 108: 108 Wang and Young Fig. 1. (A) Nort
Page 109 and 110: 110 Wang and Young Fig. 3. Expresse
Page 111 and 112: 112 Wang and Young 2. First-strand
Page 113 and 114: 114 Wang and Young 3′-RACE outlin
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Page 117 and 118: 118 Dassanayake and Samaranayake co
Page 119 and 120: 120 Dassanayake and Samaranayake 5.
Page 121 and 122: 122 Dassanayake and Samaranayake Re
Page 123 and 124: 124 Iannone et al. Fig. 1. Diagram
Page 125 and 126: Table 1 Oligonucleotides Descriptio
Page 127 and 128: 128 Iannone et al. 5. For microsphe
Page 129 and 130: 130 Iannone et al. 4. To adjust for
Page 131 and 132: 132 Iannone et al. 6. Target concen
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Page 135 and 136: 136 Benjamin, Smith, and Waites amp
Page 137 and 138: 138 Benjamin, Smith, and Waites the
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Page 141 and 142: 142 Benjamin, Smith, and Waites 2.
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Page 151 and 152: 152 Olmos et al. Fig. 1. RT-hemines
Page 153 and 154: 154 Olmos et al. This nested RT-PCR
Page 155 and 156: 156 Olmos et al. Table 2 Volume and
Page 157 and 158: 158 Olmos et al. 8. The sensitivity
Page 159 and 160: 160 Olmos et al.
Page 161 and 162: 162 Abe 5. Moloney murine leukemia
Page 163 and 164: 164 Abe Table 2 Detection Rate of H
Page 165 and 166: 166 Abe 4. For HBV, nested PCR usin
Page 167 and 168: 168 Tellier et al. of Pfu (and ther
Page 169 and 170: 170 Tellier et al. 2. Cline, J., Br
Page 171 and 172: 172 Tellier et al. 38. Tamiya, S.,
Page 173 and 174: 174 Tellier et al. 13. Thin-wall PC
Page 175 and 176: 176 Tellier et al. 13 min for the l
Page 177 and 178: 178 Tellier et al.
Page 179 and 180: 182 Stirling efficiency of the reac
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Page 183 and 184: 186 Cremer and Moos Fig. 1. Rearran
Page 185 and 186: 188 Cremer and Moos 4. Count cells
Page 187 and 188: 190 Cremer and Moos Fig. 3. Alignme
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Page 191 and 192: 194 Cremer and Moos 5. Compare the
Page 193 and 194: 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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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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