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Digoxigenin 347 51 Nonradioactive PCR Sequencing Using Digoxigenin Siegfried Kösel, Christoph B. Lücking, Rupert Egensperger, and Manuel B. Graeber 1. Introduction Techniques for direct sequencing of polymerase chain reaction (PCR) products are of central importance to contemporary research in molecular biology and genetics. The rapidly growing number of cloned human disease genes increasingly allows sequencing of PCR amplicons for diagnostic purposes. Nonradioactive sequencing protocols are of particular use because health, environmental, and administrative risks are minimized compared with conventional isotopic methods. The PCR-based nonradioactive cycle sequencing protocol described in this chapter has been successfully used to sequence mitochondrial and nuclear genes in Parkinson’s and Alzheimer’s disease brains using DNA extracted from formalin-fixed and paraffin-embedded neuropathological material (1–3). This method, which allows sequence information of PCR products to be obtained within a single day, can be performed in a research or clinical laboratory using relatively inexpensive equipment.After initial PCR amplification, amplicons are purified using spin columns for affinity chromatography or ultrafiltration. Subsequently, cycle sequencing (4) is performed using 5′-digoxigenin end-labeled oligonucleotide primers. Because the nucleotide sequences of the PCR and sequencing primers can be identical, both reactions may be performed using the same thermalcycling protocol. This obviates the need for time-consuming optimization procedures. For visualization of sequencing results, sequencing reactions are separated on a standard sequencing gel, the gel is contact-blotted to a nylon membrane, and sequencing bands are visualized using alkaline phosphatase-conjugated antibodies (Fig. 1). Potential pitfalls of our method are primarily related to the extreme sensitivity of PCR. The need for positive and negative sample controls cannot be overemphasized. We use different rooms and different pipets for setting up PCRs, pipetting sequencing templates, and thermal cycling (5). In addition, aerosol-resistant pipet tips are always used. 2. Materials 2.1. Purification of Sequencing Templates 1. 10× TNE: 100 mM Tris-HCl, pH 7.4, 1.0 M NaCl, 10 mM EDTA (see Note 1). 2. Wizard PCR Preps DNA Purification System containing affinity chromatography spin columns, purification buffer, and resin (A7170, Promega, Madison, WI). Not contained From: Methods in Molecular Biology, Vol. 226: PCR Protocols, Second Edition Edited by: J. M. S. Bartlett and D. Stirling © Humana Press Inc., Totowa, NJ 347
348 Kösel et al. Fig. 1. Schematic drawing summarizing the essential steps of nonradioactive PCR sequencing using digoxigenin. in the kit are 1× TE: 10 mM Tris-HCl, pH 8.0, 1 mM EDTA (6); 80% isopropanol; and 2-mL disposable syringes (one per reaction) or, alternatively, Microcon-30 Concentrators (#42410, Amicon, Beverly, MA). 3. Eppendorf tubes (15 mL). 4. Eppendorf centrifuge (#5415C Eppendorf, Hamburg, Germany). 5. Fluorometer (TKO 100, Hoefer, San Francisco, CA) for quantification of template concentrations using DNA dye Hoechst No. 33258 and calf thymus standard DNA (supplied with the fluorometer). 2.2. Sequencing Reactions 1. Sequencing primer, 5′digoxigenin end-labeled (1 pmol/µL), desalted, and HPLC-purified (see Note 2). 2. Mineral oil (M-5904, Sigma, St. Louis, MO). 3. Dig Taq DNA Sequencing Kit (#1449443, Boehringer Mannheim, Mannheim, Germany), containing Taq DNA polymerase (3 U/µL); sequencing buffer (250 mM Tris-HCl,
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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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8 Carroll and Casimir of PCR. Such
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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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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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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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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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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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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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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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136 Benjamin, Smith, and Waites amp
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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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184 Stirling
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186 Cremer and Moos Fig. 1. Rearran
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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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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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224 Bartlett
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226 Dominguez et al. Table 1 Primer
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236 Dominguez et al. 11. Joshi, C.
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252 Ringquist et al. 2. The present
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278 Oien 50-mL conical tubes. Resus
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292 Edwards and Bartlett Stepwise m
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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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418 Speel, Ramaekers, and Hopman 3.
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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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436 Wiedorn and Goldmann 41. Protei
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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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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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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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530 Burke and Barik purified mutant
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532 Burke and Barik
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