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163 Table 1 Primer Sequences Used for Nested PCR Primer Sequence Primer pair Product size For HBV DNA (X region) MD24 5′-TGCCAACTGGATCCTTCGCGGGACGTCCTT-3′ (nt 1392-1421) MD24/MD26 (1st) 233bp MD26 5′-GTTCACGGTGGTATAAATG-3′ (nt 1625-1607) HBx1 5′-GTCCCCTTCTTCATCTGCCGT-3′ (nt 1487-1507) HBx1/HBx2 (2nd) 117 bp HBx2 5′-ACGTGCAGAGGTGAAGCGAAG-3′ (nt 1604-1584) For HCV RNA (5′-untranslated region) 19 5′-GCGACACTCCACCATAGAT-3′ (nt 2-20) 19/20 (1st) 329 bp 20 5′-GCTCATGGTGCACGGTCTA-3′ (nt 330-312) 21 5′-CTGTGAGGAACTACTGTCT-3′ (nt 28-46) 21/22 (2nd) 268 bp 22 5′-ACTCGCAAGCACCCTATCA-3′ (nt 295-277) Nucleotide positions deduced from HBVadr4 (see ref. 4) for HBV and HC-J1 for HCV (see ref. 5) Direct PCR from Serum 163
164 Abe Table 2 Detection Rate of Hepatitis Virus Nucleic Acids in Serum. Comparison of Conventional PCR and Direct PCR HBV DNA HCV RNA HGV RNA Conventional PCR Positive 121 56 32 Direct PCR-Positive 121 51 30 Agreement Rate 100% 91% 94% Fig. 1. Efficiency of serum volume and diluent as a factor in HCV-PCR reaction. A different amount of serum sample ranging in volume from 1 to 5 µL of serum was used. Each aliquot of serum, except for the 5 µL of volume, was diluted with PBS or H 2 O to the final volume of 5 µL, respectively. M = 50-bp DNA ladder. 2. Perform the first PCR reaction by adding 45 µL of mastermix 1 to each 5-µL sample of heat-inactivated serum from Subheading 3.1. Then, program the thermocycler to incubate the samples for 50 min at 37°C for the initial RT step and then preheat at 95°C for 10 min to activate AmpliTaq Gold followed by 50 cycles consisting of 94°C for 20 s, 55°C for 20 s, and 72°C for 30 s using a Thermal Cycler. RT-PCR is performed with a one-step method combined with cDNA synthesis reaction, followed by the PCR reaction in a single tube (see Note 2). That is, for RNA of HCV, the first PCR is combined with the RT step in the same tube containing 50 µL of a reaction buffer as shown above. To obtain an automatic hot-start reaction, we use the AmpliTaq Gold DNA polymerase instead of regular thermostable DNA polymerase. 3. For the second reaction, 2.5 µL (1/20 volume) of the first PCR product is added to a tube containing second PCR buffer. The thermocycling for 50 cycles is performed as above, but omitting the initial 50 minute incubation at 37°C and the annealing temperature is raised to 60°C instead of 55°C for the second round of PCR. 4. The PCR products are electrophoresed on a 2% agarose gels staining with ethidium bromide and evaluated under ultraviolet light. The sizes of PCR products are estimated according to the migration pattern of 50-bp DNA ladder. 4. Notes 1. Examination of the optimal serum quantity reveals that 1 to 2 µL of serum give a readily amplifiable band of HCV RNA but 3 µL of serum give a faint band and 4 to 5 µL of serum do not yield any band (Fig. 1). This result is much better when PBS is used to dilute serum than that obtained using H 2 O. It is similar even if this result is in the case of HBV. 2. The whole process of the direct RT-PCR can be completed within 6 h by combination with the one-step amplification method and the second round PCR. 3. Detection rate of HCV RNA and HBV DNA by direct PCR are consistent with the results obtained by conventional PCR (Fig. 2 and Table 2).
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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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Page 121 and 122: 122 Dassanayake and Samaranayake Re
Page 123 and 124: 124 Iannone et al. Fig. 1. Diagram
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Page 157 and 158: 158 Olmos et al. 8. The sensitivity
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Page 169 and 170: 170 Tellier et al. 2. Cline, J., Br
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Page 173 and 174: 174 Tellier et al. 13. Thin-wall PC
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Page 195 and 196: 198 McDonagh the dilution method is
Page 197 and 198: 200 McDonagh 2.2. Basic PCR 1. dNTP
Page 199 and 200: 202 McDonagh Fig. 2. Quantitative P
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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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