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PCR Detection of Tumor Cells 193 Fig. 5. Example for a qPCR of a PB sample from a patient with MM. The specific PCR product is 85 bp. The highest dilution level at which all five replicates are PCR positive is 11000, the lowest dilution level at which all five replicates are PCR negative is 110,000. Analysis of this pattern of PCR results by likelihood maximization and χ 2 -minimization yielded a result of 1.2% of malignant cells. 3.6.1. Testing the Sensitivity of the Assay Using a Cell Line 1. Design primers for a B-cell or myeloma cell line and test for specificity. 2. Isolate DNA from cells of the cell line and from buffy coat of healthy donors and quantitate by OD measurement. 3. Mix DNA from the cell line with buffy coat DNA to simulate samples with different proportions of malignant cells (100%, 10%, 1%, 0.1%, 0.01%, and 0.001%). 4. Analyze all samples by qPCR (see Chapter 6.3.) using the appropriate ASO primers. 5. Compare the results of the qPCR with the simulated tumor loads. 3.6.2. Testing Primer Sensitivity by Comparison to Results of Flow Cytometry 1. Collect a BM sample with a proportion of malignant cells of greater than 2% from a patient in whom ASO primers have been devised. 2. Assess sample for CD38++, κ/γ-restricted cells by flow cytometry. This gives an approximate value for the proportion of malignant cells. 3. Isolate DNA from this sample (see Subheading 3.1.). 4. Quantitate the tumor load by qPCR as described in Subheading 3.1.
194 Cremer and Moos 5. Compare the value determined by qPCR with the result of the analysis by flow cytometry. If the result obtained by qPCR is definitely lower than the one obtained by flow cytometry, then the ASO primer is most probably not able to detect a single copy of the template of the malignant clone. 3.7. Optimization of PCR Conditions for ASO Primers 1. As a starting point, use PCRs of 50 µL containing 5 µL of 10× PCR buffer, 2 mM MgCl 2 , each deoxynucleotide at 0.1 mM, the ASO primer pair or the CDR3-specific primer plus LJH at 0.8 µM each, 2.5 U Taq-DNA-polymerase, and a maximum of 1000 ng of DNA. Amplify with a program consisting of 7 min preheating at 94°C, 60 cycles of 1 min of denaturation at 94°C and 1 min of annealing and extension at 63°C, ending with a final extension at 63°C for 5 min. 2. If these amplification conditions lead to unsatisfactory results, for example, too many nonspecific products or low intensity of the specific product, optimize the reaction conditions. Always amplify a negative control without added DNA, a negative control with buffy coat DNA from healthy donors, and a positive control with DNA from a BM sample of the patient. 3. Vary the annealing temperature in steps of 1°C. Vary the MgCl 2 concentration in steps of 0.25 mM. These parameters work synergistically. 4. Vary the concentration of primers in 0.2-µM steps in a range of 0.4 to 1.2 µM. Higher concentrations of primers can lead to a higher sensitivity; however, this is hampered by the occurrence of more nonspecific products. 5. Increase the number of amplification cycles to improve sensitivity. Decrease the number of cycles to reduce nonspecific products. At least 50 cycles should be performed to allow detection of single copies of template, which is a prerequisite for the statistical analysis of limiting dilution series by likelihood maximization. 4. Notes 1. No distinct band or multiple bands are visible after consensus PCR: Some BM samples will not lead to a single distinct band of the expected size after consensus PCR. Either only a polyclonal smear is visible or, in rare cases, more than one band of the expected size. (1) Change the strategy used for consensus PCR (FR3 or FR1). Try DNA instead of RNA. Prepare a PCR mixture containing 10 µL of 10× PCR buffer, 2 mM MgCl 2 , each deoxynucleotide at 0.05 mM, primers FR1C or FR3A plus LJH at 0.4 µM each, and 2.5 U Taq- DNA-polymerase. Add 500 to 1000 ng of DNA and distilled water to a final volume of 100 µL. Amplify with a program consisting of 7 min denaturation and enzyme activation at 94°C, followed by 50 cycles of denaturation for 1 min at 94°C and combined annealing and extension at 63°C (for primer FR3A) or 65°C (for primer FR1C), followed by a final extension step at 65°C for 5 min. If possible, collect another BM aspirate and start again. 2. Sequence has no resemblance to known CDR regions: This is most probably caused by a nonspecific product of the consensus PCR. Perform consensus RT-PCR again. 3. Sequence of a CDR-region is unsuitable for primer design: Sometimes the sequence of a CDR region is too short, has too many base repeats, or has too many long stretches rich in G and C, which makes designing primers difficult. Because CDR1 and CDR2 region are shorter and less variable than the CDR3-region, one should never do without a CDR3-specific primer. If segments suitable for primer design are not long enough, identify at least a short segment that is apt. Devise primers complementary to this stretch, and elongate in 5′-end direction (never in 3′-direction, because this end is more important for specific annealing of the primer to the template). Use the CDR1/2- plus CDR3-specific primer strategy, if the CDR3 region is the one that is difficult for primer design.
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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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Page 161 and 162: 162 Abe 5. Moloney murine leukemia
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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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