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Purification of PCR Products from Agarose Gels for Direct Sequencing

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PCR Sequencing Protocols

Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 65))

Abstract

The advent of direct sequencing of polymerase chain reaction (PCR) products has permitted extremely rapid analysis of DNA mutants and cDNA clones. However, direct PCR sequencing has been problematic for a number of technical reasons, including the presence of impurities and excess oligonucleotide primers used for the PCR amplifications (14). Therefore, a number of protocols have been devised that address these technical issues, and allow efficient sequencing of either conventional double-stranded PCR products or asymmetrically amplified single-stranded products (e.g., 14). Many of these protocols are described in detail in this volume.

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References

  1. Dorit, R. L., Ohara, O., and Hwang, C. B.-C. (1991) Direct DNA sequencing of PCR products, in Current Protocols in Molecular Biology, vol. II (Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., and Struhl, K. S., eds.), John Wiley, New York, pp 15.2.1–15.2.11.

    Google Scholar 

  2. Phear, G. A. and Harwood, J. (1994) Direct sequencing of PCR products. Methods Mol. Biol. 31, 247–256.

    PubMed  CAS  Google Scholar 

  3. Cassanova, J.-L., Pannetier, C., Jaulin, C., and Kourilsky, P. (1990) Optimal conditions for directly sequencing double-stranded PCR products with Sequenase. Nucleic Acids Res. 18, 4028.

    Article  Google Scholar 

  4. Rao, V. B. (1994) Direct sequencing of polymerase chain reaction-amplified DNA. Anal. Biochem. 216, 1–14.

    Article  PubMed  CAS  Google Scholar 

  5. Cao, X., and Brosius, F. C, III. (1993) Direct sequencing of double-stranded PCR products isolated from conventional agarose gels. BioTechniques 15, 384–386.

    PubMed  CAS  Google Scholar 

  6. Heery, D. M., Gannon, F., and Powell, R. (1990) A simple method for subcloning DNA fragments from gel slices. Trends Genet. 6, 173.

    Article  PubMed  CAS  Google Scholar 

  7. Chomczynski, P. and Sacchi, N. (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162, 156–159.

    Article  PubMed  CAS  Google Scholar 

  8. United States Biochemical Corporation (1992) Step-by-Step Protocols for DNA Sequencing with Sequenase version 2.0 T7 DNA Polymerase, 6th ed., Cleveland, OH.

    Google Scholar 

  9. Chomczynski, P. (1993) A reagent for the single-step simultaneous isolation of RNA, DNA and proteins from cell and tissue samples. BioTechniques 15, 532–534.

    PubMed  CAS  Google Scholar 

  10. Coen, D. M. (1992) Quantitation of rare DNAs by PCR., in Current Protocols in Molecular Biology, vol. II (Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., and Struhl, K. S., eds.), John Wiley, New York, pp. 15.3.1–15.3.6.

    Google Scholar 

  11. Khorana, S., Gagel, R. F., and Cote, G. J. (1994) Direct sequencing of PCR products in agarose gel slices. Nucleic Acids Res. 22(16), 3425–3426.

    Article  PubMed  CAS  Google Scholar 

  12. Trewick, S. A. and Dearden, P. (1994) A rapid protocol for DNA extraction and primer annealing for PCR sequencing. BioTechniques 17, 842–844.

    PubMed  CAS  Google Scholar 

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© 1996 Humana Press Inc., Totowa, NJ

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Brosius, F.C., Holzman, L.B., Cao, X. (1996). Purification of PCR Products from Agarose Gels for Direct Sequencing. In: Rapley, R. (eds) PCR Sequencing Protocols. Methods in Molecular Biology™, vol 65. Springer, Totowa, NJ. https://doi.org/10.1385/0-89603-344-9:11

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  • DOI: https://doi.org/10.1385/0-89603-344-9:11

  • Publisher Name: Springer, Totowa, NJ

  • Print ISBN: 978-0-89603-344-3

  • Online ISBN: 978-1-59259-551-8

  • eBook Packages: Springer Protocols

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