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Reduction of Shadow Band Synthesis During PCR Amplification of Repetitive Sequences from Modern and Ancient DNA

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PCR Protocols

Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 226))

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Abstract

Repetitive sequences like short tandem repeat (STR) loci are generally referred to as slippery DNA (1). They owe this nickname to a characteristic leading to slippage within the primer-template complex during PCR elongation of the new strand (2,3), resulting in the synthesis of byproducts shortened by one repeat unit compared with the original sequence. The generation of these so-called shadow bands (4) is a well-known problem connected with the amplification of repetitive DNA, complicating the genotype analysis of modern (e.g., ref. 5), forensic (6), and ancient (7,8) specimens. In some applications, the occurrence of this artifact makes it necessary to develop guidelines for allele designation (6,9).

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References

  1. Kunkel, T. A. (1993) Nucleotide repeats. Slippery DNA and diseases. Nature 365, 207–208.

    Article  PubMed  CAS  Google Scholar 

  2. Levison, G. and Gutman, G. A. (1987) Slipped-strand mispairing: a mayor mechanism for DNA sequence evolution. Mol. Biol. Evol. 4, 203–221.

    Google Scholar 

  3. Schlötterer, C. and Tautz, D. (1992) Slippage synthesis of simple sequence DNA. Nucleic Acids Res. 20, 211–215.

    Article  PubMed  Google Scholar 

  4. Weber, J. L. and May, P. E. (1989) Abundant class of human polymorphisms which can be typed using the polymerase chain reaction. Am. J. Hum. Genet. 44, 388–396.

    PubMed  CAS  Google Scholar 

  5. Bluteau, O., Legoix, P., Laurent-Puig, P., and Zucman-Rossi, J. (1999) PCR-based genotyping can generate artifacts in LOH analyses. BioTechniques 27, 1100–1102.

    PubMed  CAS  Google Scholar 

  6. Gill, P., Sparkes, R., and Kimpton, C. (1997) Development of guidelines to designate alleles using a STR multiplex system. Forensic Sci. Int. 89, 185–197.

    Article  PubMed  CAS  Google Scholar 

  7. Schmerer, W. M., Hummel, S., and Herrmann, B. (1997) Reproduzierbarkeit von aDNA-typing. Anthrop. Anz. 55, 199–206.

    CAS  Google Scholar 

  8. Ivanov, P. L. and Isaenko, M. V. (1999) Identification of human decomposed remains using the STR systems: effect on typing results, in Proceedings of the second European symposium on human identification 1998. Promega Corporation, Innsbruck, Austria.

    Google Scholar 

  9. Fourney, R. M., Fregau, C. J., Bowen, J. H., Bowen, K. L., Shutler, G. G., Elliot, J. C., et al. (1995) Interpretation guidelines for fuorescent automated detection of STRs: Defining the allele and the limits of detection, in Proceedings from the Sixth International Symposium on Human Identification 1995. Promega Corporation, Innsbruck, Austria.

    Google Scholar 

  10. Murray, V., Monchawin, C., and England, P. R. (1993) The determination of the sequences present in the shadow bands of a dinucleotide repeat PCR. Nucleic Acids Res. 21, 2395–2398.

    Article  PubMed  CAS  Google Scholar 

  11. Kimpton, C., Fisher, D., Watson, S., Adams, M., Urquhard, A., Lygo, J., et al. (1994) Evaluation of an automated DNA profiling system employing multiplex amplification of four tetrameric STR loci. Int. J. Leg. Med. 106, 302–311.

    Article  CAS  Google Scholar 

  12. Lygo, J. E., Johnson, P. E., Holaway, D. J., Woodroffe, S., Whitaker, J. P., Clayton, T. M., et al. (1994) The validation of short tandem repeat (STR) loci for use in forensic casework. Int. J. Leg. Med. 107, 77–89.

    Article  CAS  Google Scholar 

  13. Ramos, M. D., Lalueza, C., Girbau, E., Perez-Perez, A., Quevedo, S., Turbon, D., et al. (1995) Amplifying dinucleotide microsatellite loci from bone and tooth samples of up to 5000 years of age: More inconsistency than usefulness. Hum. Genet. 96, 205–212.

    Article  PubMed  CAS  Google Scholar 

  14. Schultes, T., Hummel, S., and Herrmann, B. (1997) Recognizing and overcoming inconsistencies in microsatellite typing of ancient DNA samples. Ancient Biomol. 1, 227–233.

    CAS  Google Scholar 

  15. Schmerer, W. M. (1996) Reproduzierbarkeit von Mikrosatelliten-DNA-Amplifkation und Alleldetermination aus bodengelagertem Skelettmaterial. Unpublished diploma-thesis, Göttingen.

    Google Scholar 

  16. Taberlet, P., Griffin, S., Goossens, B., Questiau, S., Manceau, V., Escaravage, N., et al. (1996) Reliable genotyping of samples with very low DNA quantities using PCR. Nucleic Acids Res. 24, 3189–3194.

    Article  PubMed  CAS  Google Scholar 

  17. Schmerer, W. M., Hummel, S., and Herrmann, B. (1999) Optimized DNA extraction to improve reproducibility of short tandem repeat genotyping with highly degraded DNA as target. Electrophoresis 20, 1712–1716.

    Article  PubMed  CAS  Google Scholar 

  18. Schmerer, W. M., Hummel, S., and Herrmann, B. (2000) STR-genotyping of archaeological human bone: Experimental design to improve reproducibility by optimisation of DNA extraction. Anthrop. Anz. 58, 29–35.

    CAS  Google Scholar 

  19. Schmerer, W. M. (2000) Optimierung der STR-Genotypenanalyse an Extrakten alter DNA aus bodengelagertem menschlichen Skelettmaterial. Cuvillier Verlag, Göttingen.

    Google Scholar 

  20. Kimpton, C. P., Walton, A., and Gill, P. (1992) A further tetranucleotide repeat polymorphism in the vWF gene. Hum. Mol. Genet. 1, 287.

    Article  PubMed  CAS  Google Scholar 

  21. Bacher, J. and Schumm, J. W. (1998) Development of highly polymorphic pentanucleotide tandem repeat loci with low stutter. Profiles DNA 2, 3–6.

    Google Scholar 

  22. Cheng, S., Fockler, C., Barnes, W. M., and Higuchi, R. (1994) Effective amplification of long targets from cloned inserts and human genomic DNA. Proc. Natl. Acad. Sci. USA 91, 5695–5699.

    Article  PubMed  CAS  Google Scholar 

  23. Baskaran, N., Kandpal, R. P., Bhargava, A. K., Glynn, M. W., Bale, A., and Weissman, S. M. (1996) Uniform amplification of a mixture of deoxyribonucleic acids with varying GC content. Genome Res. 6, 633–638.

    Article  PubMed  CAS  Google Scholar 

  24. Weissensteiner, T. and Lanchbury, J. S. (1996) Strategy for controlling preferentiual amplification and avoiding false negatives in PCR Typing. Biotechniques 21, 1102–1108.

    PubMed  CAS  Google Scholar 

  25. Pääbo, S., Gifford, J. A., and Wilson, A. C. (1988) Mitochondrial DNA sequences from a 7000-year old brain. Nucleic Acids Res. 20, 9775–9787.

    Article  Google Scholar 

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Schmerer, W.M. (2003). Reduction of Shadow Band Synthesis During PCR Amplification of Repetitive Sequences from Modern and Ancient DNA. In: Bartlett, J.M.S., Stirling, D. (eds) PCR Protocols. Methods in Molecular Biology™, vol 226. Humana Press. https://doi.org/10.1385/1-59259-384-4:309

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  • DOI: https://doi.org/10.1385/1-59259-384-4:309

  • Publisher Name: Humana Press

  • Print ISBN: 978-0-89603-642-0

  • Online ISBN: 978-1-59259-384-2

  • eBook Packages: Springer Protocols

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