These data are intended to benefit research and application of short tandem repeat DNA markers to aid human identity testing. The authors are solely responsible for the information herein. [Purpose of Database]
This database has been accessed I www.digits.com - see disclaimer.)
times since 10/02/97. (Counter courtesy
Created by John M. Butler and Dennis J. Reeder (NIST Biotechnology Division). with invaluable help from Christian Ruitberg and Michael Tung
Site creators' curriculum vitaes available using links above.
*Partial support for the design and maintenance of this website is being provided by The National Institute of Justice through the NIST Office of Law Enforcement Standards. *
o Background information on STRs
» Description of each STR system (STR Fact Sheets)
° Sequence Information o Chromosomal Locations o Non-published Variant Allele Reports^pdatea o Allele Frequency Distribution Tables » Sex-typing markers ° Technology for resolving STR alleles ° Y-chromosome STRs o Population data o Validation studies « Multiplex STR sets o PCR primers o FBI Core STR Loci o NIST Standard Reference Material for PCR-Based Testing o DNA Advisory Board Quality Assurance Standards
« Reference List o Original papers describing common STR systems o Addresses for scientists working with STRs o Links to other web sites
» Glossary of commonly used terms the following URL: http://www.cstl.nist.gov/biotech/strbase. The home page for STRBase is shown in Figure 5.12.
STRBase contains a number of useful elements. Continually updated information includes the listing of references related to STRs and DNA typing (over 2000 references), addresses for scientists working in the field, and new microvariant or 'off-ladder' STR alleles. Other information that is updated less frequently includes STR fact sheets (with allele information similar to Appendix I), links to other web pages, a review of technology used for DNA typing as well as published primer sequence information, and population data for STR markers.
STR markers have become important tools for human identity testing. Commercially available STR kits are now widely used in forensic and paternity testing laboratories. The adoption of the 13 CODIS core loci for the U.S. national
DNA database ensures that these STR markers will be used for many years to come. However, as we will see in the next two chapters, results from STR
markers require careful interpretation in order to be effective tools for law enforcement.
REFERENCES AND ADDITIONAL READING
Applied Biosystems (2001) AmpF/STR® Identifiler™ PCR Amplification Kit User's Manual. Foster City, CA.
Applied Biosystems (2002) AmpF/STR® SEfiler™ PCR Amplification Kit User's Manual. Foster City, CA.
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Barber, M.D. and Parkin, B.H. (1996) Internationa/ Journa/ of Lega/ Medicine, 109, 62-65.
Bacher, J.W., Hennes, L.F., Gu, T., Tereba, A., Micka, K.A., Sprecher, C.J., Lins, A.M., Amiott, E.A., Rabbach, D.R., Taylor, J.A., Helms, C., Donis-Keller, H. and Schumm, J.W. (1999) Proceedings of the Ninth Internationa/ Symposium on Human Identification, pp. 24-37. Madison, Wisconsin: Promega Corporation.
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Broman, K.W., Murray, J.C., Sheffield, V.C., White, R.L. and Weber, J.L. (1998) American Journa/ of Human Genetics, 63, 861-869.
Budowle, B., Moretti, T.R., Niezgoda, S.J. and Brown, B.L. (1998) Proceedings of the Second European Symposium on Human Identification, pp. 73-88. Madison, Wisconsin: Promega Corporation.
Budowle, B., Masibay, A., Anderson, S.J., Barna, C., Biega, L., Brenneke, S., Brown, B.L., Cramer, J., DeGroot, G.A., Douglas, D., Duceman, B., Eastman, A., Giles, R., Hamill, J., Haase, D.J., Janssen, D.W., Kupferschmid, T.D., Lawton, T., Lemire, C., Llewellyn, B., Moretti, T., Neves, J., Palaski, C., Schueler, S., Sgueglia, J., Sprecher, C., Tomsey, C. and Yet, D. (2001) Forensic Science Internationa/, 124, 47-54.
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Butler, J.M., Devaney, J.M., Mario, M.A. and Vallone, P.M. (2001) Comparison of primer sequences used in commercial STR kits. Proceedings of the 53rd American Academy of Forensic Sciences (Seattle, Washington); Presentation available at: http://www.cstl.nist.gov/biotech/strbase/NISTpub.htm.
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Carracedo, A. and Lareu, M.V. (1998) Proceedings from the Ninth International Symposium on Human Identification, pp. 89-107. Madison, Wisconsin: Promega Corporation.
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BIOLOGY OF STRs: STUTTER PRODUCTS, NON-TEMPLATE ADDITION, M ICRO VARIANTS, NULL ALLELES AND MUTATION RATES
The most humbling aspect of the Human Genome Project so far has been the realization that we know remarkably little about what the vast majority of human genes do.
(James Watson, DNA: The Secret of Life, 2003, p. 217)
During polymerase chain reaction (PCR) amplification of short tandem repeat (STR) alleles, a number of artifacts can arise that may interfere with the clear interpretation and genotyping of the alleles present in the DNA template. In this chapter, we will focus on those PCR products that give rise to additional peaks besides the true, major allele peak(s). These artifacts include stutter products and nontemplate nucleotide addition. Other factors that impact STR typing, including microvariants, tri-allelic patterns, allele dropout, and mutations will also be covered.
A close examination of electropherograms containing STR data typically reveals the presence of small peaks several bases shorter than each STR allele peak (Figure 6.1). These 'stutter product' peaks result from the PCR process when STR loci are copied by a DNA polymerase. In the literature, this stutter product has also been referred to as a shadow band or a DNA polymerase slippage product (Hauge and Litt 1993).
Sequence analysis of stutter products from the tetranucleotide repeat locus VWA has shown that they contain one repeat unit less than the corresponding main allele peak (Walsh et al. 1996). Stutter products that are larger in size by one repeat unit than the corresponding alleles are only rarely observed in commonly used tetranucleotide repeat STR loci.
Stutter products have been reported in the literature since STRs (microsatellites) were first described. The primary mechanism that has been proposed to explain the existence of stutter products is slipped-strand mispairing (Hauge and Litt 1993, Walsh et al. 1996). In the slipped-strand mispairing model, a region of primer-template complex becomes unpaired during primer extension allowing slippage of either primer or template strand such that one repeat forms a non-base-paired loop (Hauge and Litt 1993). The consequence of this
STR alleles shown with stutter products (indicated by arrows). Only the stutter percentage for the first allele from each locus is noted.
Illustration of slipped-strand mispairing process that is thought to give rise to stutter products. (a) During replication the two DNA strands can easily come apart in the repeat region and since each repeat unit is the same, the two strands can re-anneal out of register such that the two strands are off-set by a single repeat unit. (b) If a repeat unit bulges out on the new synthesized strand during extension then an insertion results in the next round of amplification. (c) If on the other hand, the repeat unit bulge occurs in the template strand, then the resulting synthesized strand is one repeat unit shorter than the full length STR allele. The frequency at which this process occurs is related to the flanking sequence, the repeat unit, and the length of the allele being amplified. Generally for tetranu-cleotide STR loci, stutter occurs less than 15% of the time and is observed as a small peak one repeat shorter than the STR allele.
200 225 250 275
e c 3000
Al uv one repeat loop is a shortened PCR product that is less than the primary ampli-con (STR allele) by a single repeat unit (Figure 6.2).
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