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PCR Detection of Tumor Cells 195 4. The ASO primer does not generate specific PCR product or is not specific or sensitive enough: Optimize PCR conditions and test primer for specificity or sensitivity. Devise new primers. Change primer strategy (CDR3 or CDR1/ plus CDR3). If no specific product can be generated at all, it is most probable that the sequence of the CDR-regions is not that of the malignant clone. 5. qPCR results are implausible: This is most probably caused by a faulty dilution series. Prepare dilution series anew and reanalyze the sample by qPCR. 5. Discussion The described qPCR assay with ASO primers complementary to CDR regions of the malignant clone can be used to quantitate the proportion of malignant cells in peripheral blood (PB) and BM, aliquots of leukapheresis products, sorted cell fractions, or in cultured cells. It is possible to use this method for quantitative follow up in the course of therapy (16), to determine the tumor load of different stem cell sources (17), to determine the effect of different mobilization regimens and the duration of collection on the number of malignant cells in leukapheresis products (18,19) or to assess the extent of involvement in the malignant process of different cell fractions, for example, in the CD19+ or CD20+ cells of PB (20,21). The qPCR assay has evolved into an accurate and very sensitive tool for the detection of malignant cells in MM. References 1. Cremer, F. W., Kiel, K., Wallmeier, M., Goldschmidt, H., and Moos, M. (1997) A quantitative PCR assay for the detection of low amounts of malignant cells in multiple myeloma. Ann. Oncol. 8, 633–636. 2. Tonegawa, S. (1983) Somatic generation of antibody diversity. Nature 302, 575–581. 3. Walter, M. A., Surti, U., Hofker, M. H., and Cox, D. W. (1990) The physical organisation of the human immunoglobulin heavy chain gene complex. Eur. Mol. <strong>Bio</strong>l. Org. J. 9, 3303–3313. 4. Bakkus, M. H., Heirman, C., Van Riet, I., Van Camp, B., and Thielemans, K. (1992) Evidence that multiple myeloma Ig heavy chain VDJ genes contain somatic mutations but show no intraclonal variation. Blood 80, 2326–2335. 5. Aubin, J., Davi, F., Nguyen-Salomon, F., Leboeuf, D., Debert, C., Taher, M., et al. (1995) Description of a novel FR1 IgH PCR strategy and its comparison with three other strategies for the detection of clonality in B cell malignancies. Leukemia 9, 471– 479. 6. Brisco, M. J., Tan, L. W., Orsborn, A. M., and Morley, A. A. (1990) Development of a highly sensitive assay, based on the polymerase chain reaction, for rare B-lymphocyte clones in a polyclonal population. Br. J. Haematol. 75, 163–167. 7. Campbell, M. J., Zelenetz, A. D., Levy, S., Levy, R. (1992) Use of family specific leader region primers for PCR amplification of the human heavy chain variable region gene repertoire. Mol. Immunol. 29, 193–203. 8. Deane, M., McCarthy, K. P., Wiedemann, I. M., and Norton, J. D. (1991) An improved method for detection of B-lymphoid clonality by polymerase chain reaction. Br. J. Haematol. 5, 726–730. 9. Diss, T. C., Peng, H., Wotherspoon, A. C., Isaacson, P. G., and Pan, L. (1993) Detection of monoclonality in low-grade β-cell lymphoma using the polymerase chain reaction is dependent on primer selection and lymphoma type. J. Pathol. 169, 291–295. 10. Moore, G. E. and Kitamura, H. (1968) Cell line derived from patient with myeloma. N. Y. State J. Med. 68, 2054–2060.
196 Cremer and Moos 11. Klein, E., Klein, G., Nadkarmi, J. F., Nadkarmi, J. J., Vigzell, H., and Clifford, P. (1968) Surface IgM-k specificity on a Burkitt lymphoma cell in vivo and in derived culture lines. Cancer Res. 28, 1300–1310. 12. Yamada, M., Wasserman, R., Reichard, B. A., Shane, S., Caton, A. J., and Rovera, G. (1991) Preferential utilization of specific immunoglobulin heavy chain diversity and joining segments in adult human peripheral blood B lymphocytes. J. Exp. Med. 173, 395– 407. 13. Brisco, M. J., Condon, J., Sykes, P. J., Neoh, S. H., and Morley, A. A. (1991) Detection and quantitation of neoplastic cells in acute lymphoblastic leukaemia, by use of the polymerase chain reaction. Br. J. Haematol. 79, 211–217. 14. Wiesner, R. J. (1992) Direct quantitation of picomolar concentrations of mRNAs by mathematical analysis of a reverse transcription/exponential polymerase chain reaction assay. Nucleic Acids Res. 51, 5863–5864. 15. Taswell, C. (1981) Limiting dilution assays for the determination of immunocompetent cell frequencies. J. Immunol. 126, 1614–1619. 16. Cremer, F. W., Dada, R., Kiel, K., Ehrbrecht, E., Goldschmidt, H., and Moos, M. (1998) Quantitative PCR monitoring of the effect of double high-dose therapy on the tumor load of patients with multiple myeloma. Ann. Hematol. 77 (Suppl. II), 182 (abstract 722). 17. Vescio. R. A., Han, E. J., Schiller, G. J., Lee, J. C., Wu, C. H., Cao, J., et al. (1996) Quantitative comparison of multiple myeloma tumor contamination in bone marrow harvest and leukapheresis autografts. Bone Marrow Transplant. 18, 103–110. 18. Cremer, F. W., Kiel, K., Wallmeier, M., Haas, R., Goldschmidt, H., and Moos, M. (1998) Leukapheresis products in multiple myeloma: lower tumor load after mobilization with cyclophosphamide plus granulocyte colony-stimulating factor (G-CSF) compared with G-CSF alone. Exp. Hematol. 26, 969–975. 19. Kiel, K., Cremer, F. W., Ehrbrecht, E., Wallmeier, M., Hegenbart, U., Goldschmidt, H., aet al. (1998) First and second apheresis in patients with multiple myeloma: no differences in tumor load and hematopoietic stem cell yield. Bone Marrow Transplant. 21, 1109–1115. 20. Kiel, K., Cremer, F. W., Rottenburger, C., Kallmeyer, C., Ehrbrecht, E., Atzberger, A., et al. (1998) Circulating tumor cells in patients with multiple myeloma in the course of high-dose therapy. Ann. Hematol. 77 (Suppl. II), 182 (abstract 724). 21. Rottenburger, C., Kiel, K., Cremer, F. W., B′′sing, T., Atzberger, A., Moldenhauer, G., et al. (1998) No differences in the tumor load in the CD19+ and CD20+ cell fractions of peripheral blood from patients with multiple myeloma post high-dose therapy and with peripheral blood stem cell support. Ann. Hematol. 77 (Suppl. II), 184 (abstract 731).
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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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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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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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Page 157 and 158: 158 Olmos et al. 8. The sensitivity
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Page 161 and 162: 162 Abe 5. Moloney murine leukemia
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Page 221 and 222: 226 Dominguez et al. Table 1 Primer
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248 Ringquist et al. 5. Total RNA s
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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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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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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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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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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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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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524 Jones and Winistorfer 7. Goulde
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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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