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Solid-Phase Minisequencing 361 53 Analysis of Nucleotide Sequence Variations by Solid-Phase Minisequencing Anu Suomalainen and Ann-Christine Syvänen 1. Introduction The Sanger dideoxynucleotide sequencing method has been simplified by a number of methodological improvements, such as the use of the polymerase chain reaction (PCR) technique for generating DNA templates in sufficient quantities followed by affinity-capture techniques for convenient and efficient purification of the PCR fragments for sequencing, or altenatively the use of cyclic Sanger sequencing reactions that are easy to automate with laboratory robots, and the development of instruments for automatic on-line analysis of fluorescent products of the sequencing reactions (references to this book, solid phase sequencing, cyclic sequencing). Despite these technical improvements, the requirement for gel electrophoretic separation remains an obstacle when sequence analysis of large numbers of samples are needed, as in DNA diagnosis, or in the analysis of sequence variation for genetic, evolutionary, or epidemiological studies. We have developed a method for analyzing DNA fragments that differ from each other in one or a few nucleotide positions (1) denoted solid-phase minisequencing, in which gel electrophoretic separation is avoided. Analogous to the methods for solidphase sequencing of PCR products, the solid-phase minisequencing method is based on PCR amplification using one biotinylated and one unbiotinylated primer, followed by affinity-capture of the biotinylated PCR product on an avidin- or streptavidincoated solid support. The nucleotide at the variable site is detected in the immobilized DNA fragment by a primer extension reaction: A detection step primer that anneals immediately adjacent to the nucleotide to be analyzed is extended by a DNA polymerase with a single labeled nucleotide complementary to the nucleotide at the variable site (Fig. 1). The amount of the incorporated label is measured, and it serves as a specific indicator of the nucleotide present at the variable site. We have used the solid-phase minisequencing method for detecting numerous mutations causing human genetic disorders, for analyzing allelic variation in genetic linkage studies, and for identifying individuals (2–4). The protocol described below is generally applicable for detecting any variable nucleotide. The method suits well for analyzing large numbers of samples because it comprises simple manipulations in 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 361
362 Suomalainen and Syvänen Fig. 1. Steps of the solid-phase minisequencing method. 1. PCR with one biotinylated (black ball) and one unbiotinylated primer. 2. Affinity-capture of the biotinylated PCR product in streptavidin-coated microtiter wells. 3. Washing and denaturation. 4. The minisequencing primer extension reaction. 5. Measurement of the incorporated label. 6. Calculation of the result.
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82 Hyndman and Mitsuhashi 3.1. Effi
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106 Wang and Young full-length cDNA
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124 Iannone et al. Fig. 1. Diagram
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128 Iannone et al. 5. For microsphe
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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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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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198 McDonagh the dilution method is
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200 McDonagh 2.2. Basic PCR 1. dNTP
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206 Bartlett Table 1 Example Assay
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282 Oien 8. PCR. With SAGE, achievi
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452 Gilchrist and Befus References
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468 Pearson and Stirling into PCR p
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472 Wang Fig. 1. A brief outline of
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474 Wang
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484 Horton, Raju, and Conti-Fine
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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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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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530 Burke and Barik purified mutant
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532 Burke and Barik
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