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Sequencing 337 49 Sequencing A Technical Overview David Stirling 1. Introduction The huge advances that have been made in human (and other species) genome projects have been lead by, and have themselves fueled, tremendous innovations in the field of sequencing. These innovations have lead to a specialization, with very expensive equipment, often in core sequencing centers or services. Until the cost of automated sequencing equipment falls considerably, the economics of scale mean that unless an investigator has a huge amount of sequencing to perform, they are most likely to be best served by using these services rather than by investing in the technology themselves. There is always a temptation in such situations to treat the sequencing as a black box, without worrying too much how the data are generated. This is a mistake. A clear understanding of the principals involved will not only aid the design of sequencing strategies but will also help in the interpretation of less than perfect data. DNA/RNA sequence analysis has become fundamental to the understanding of biological processes. The foundations of this science were established in 1977 when Maxam and Gilbert (1) described a method for sequencing by base-specific chemical cleavage. Subsequently, Sanger and co-workers (2) developed a method for enzymatic sequencing using chain terminators. Both techniques produce populations of labeled DNA fragments that can be electrophoretically resolved to reveal the base sequence. Many different strategies have been developed to improve on these initial approaches and to make genome-sequencing projects feasible; all owe a great debt to those original protocols. 2. Chemical DNA Sequencing In the original Maxam-Gilbert method of DNA sequencing, target DNA is radioactively labeled at one end (3′or 5′ end), which acts as the reference point for determining the positions of the remaining bases. This labeled DNA is processed with four basespecific reactions. First, a base-specific chemical modification, then a chain cleavage of modified bases. These methods have fallen from popularity, both because of the toxicity of the reagents (dimethyl sulphate, hydrazine, potassium permanganate) and the development From: Methods in Molecular <strong>Bio</strong>logy, Vol. 226: PCR Protocols, Second Edition Edited by: J. M. S. <strong>Bartlett</strong> and D. Stirling © Humana Press Inc., Totowa, NJ 337
338 Stirling of simple and improved methods for enzymatic DNA. Although the chemical method is not widely used as the enzymatic method, it has some advantages and can be very useful in certain situations. Because it does not rely on the hybridization of a primer, very short sequences, such as oligonucleotides, can be analyzed. It is also useful for analyses of DNA modifications, such as methylation, and to study DNA–protein interactions (footprinting). 3. Enzymatic DNA Sequencing Dideoxy sequencing reactions (Sanger method) are essentially primer extension reactions where ddNTPs are included in the mix. In the conventional dideoxy sequencing reaction, an oligo primer is annealed to a single-stranded DNA template and extended by DNA polymerase to synthesize a complementary copy of single-stranded DNA in the presence of four dNTPs, one of which is 35 S-labeled. Chain growth involves the formation of a phosphodiester bridge between the 3′-OH at the growing end of the primer and the 5′-phosphate group of the incorporated dNTP. Thus, overall chain growth is in the 5′→3′ direction. The reaction also contains one of four ddNTPs that terminate elongation when incorporated into the growing DNA chain. When a ddNTP is incorporated at the 3′ end of the growing primer chain, the elongation is terminated selectively at A, C, G, or T owing to the missing 3′ OH group of the primer chain. The enzymatic method is based on the ability of DNA polymerase to use both 2′ dNTPs and 2′,3′ ddNTPs as substrates. After completion of the sequencing reactions, the products are subjected to electrophoresis on a high-resolution denaturing polyacrylamide gel and then autoradiographed to visualize the DNA sequence. For this form of sequencing, the purity and concentration of the template has to be very carefully controlled, and the template has to be rendered single stranded before the reaction will proceed. To reliably obtain good quality sequence from PCR products, these would first have to be cloned into a suitable vector. Any clone obtained is the product of a single molecule of PCR product, and so the likelihood of that sequence containing misincorporated bases is relatively high. Multiple clones have therefore to be sequenced to produce reliable data. 4. Cycle Sequencing The introduction of thermostable DNA polymerase had the same revolutionary impact on sequencing protocols as in other areas (3). The ability to function at higher temperature resulted in template remaining single stranded for longer periods, overcame many problems of secondary structure, and allowed more stringent primer annealing conditions resulting on less background noise in sequencing data. In addition, repeated cycles of primer annealing and extension amplifies (linearly rather than exponentially as for PCR) even small amounts of sample DNA to generate more template. The ability to sequence very low template concentrations means that even relatively impure material, such as PCR products, can be used, simply by diluting out the impurities. 5. Automation One of the major advances in sequencing technology has been the development of automated DNA sequencers, which automate the gel electrophoresis step, detection of band pattern, and analysis of bands. These machines are based on the enzymatic, cycle
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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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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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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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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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Page 309 and 310: 316 Aubele and Smida 2.2. Chemicals
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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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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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444 Wiedorn and Goldmann 25. Kommin
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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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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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