Skip to main content
Log in

Improved analysis of long STR amplicons from degraded single source and mixed DNA

  • Technical Note
  • Published:
International Journal of Legal Medicine Aims and scope Submit manuscript

Abstract

DNA profiles from degraded samples often suffer from information loss at the longer short tandem repeat (STR) loci. Sensitising the reactions, either by performing additional PCR cycles or increasing the capillary electrophoresis injection settings, carries the risk of over-amplifying or overloading the shorter fragments. We explored whether profiling of degraded DNA can be improved by preferential capturing of the longer amplified fragments. To this aim, a post-PCR purification protocol was developed that is based on AMPure XP beads that have size-selective properties. A comparison was made with an unselective post-PCR purification system (DTR gel filtration) and no purification of the PCR products. Besides a set of differently and serially degraded single source samples, unequal mixtures of degraded DNAs were analysed, in order to extract more genotyping information for the minor contributor without overloading the major component at the shorter amplicons. Purification by the AMPure protocol resulted in higher peak heights especially for the longer amplicons, while DTR gel filtration gave higher peaks for all amplicon sizes. Both purification methods presented more detected alleles, with the AMPure protocol performing slightly better, on average. In conclusion, the in-house developed AMPure protocol can be employed to improve STR profile analysis of degraded single source and (unequally) mixed DNA samples.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3

References

  1. Alaeddini R, Walsh SJ, Abbas A (2010) Forensic implications of genetic analyses from degraded DNA—a review. Forensic Sci Int Genet 4:148–157

    Article  PubMed  CAS  Google Scholar 

  2. Butler JM, Shen Y, Mccord BR (2003) The development of reduced size STR amplicons as tools for analysis of degraded DNA. J Forensic Sci 48:1054–1064

    PubMed  CAS  Google Scholar 

  3. Wiegand P, Kleiber M (2001) Less is more—length reduction of STR amplicons using redesigned primers. Int J Leg Med 114:285–287

    Article  CAS  Google Scholar 

  4. Hill CR, Kline MC, Coble MD, Butler JM (2008) Characterization of 26 miniSTR loci for improved analysis of degraded DNA samples. J Forensic Sci 53:73–80

    Article  PubMed  CAS  Google Scholar 

  5. Westen AA, Sijen T (2009) Degraded DNA sample analysis using DNA repair enzymes, mini-STRs and (tri-allelic) SNPs. Forensic Sci Int Genet Suppl Ser 2:505–509

    Article  Google Scholar 

  6. Dixon LA, Dobbins AE, Pulker HK, Butler JM, Vallone PM, Coble MD, Parson W, Berger B, Grubwieser P, Mogensen HS, Morling N, Nielsen K, Sanchez JJ, Petkovski E, Carracedo A, Sanchez-Diz P, Ramos-Luis E, Brion M, Irwin JA, Just RS, Loreille O, Parsons TJ, Syndercombe-Court SH, Stradmann-Bellinghausen B, Bender K, Gill P (2006) Analysis of artificially degraded DNA using STRs and SNPs—results of a collaborative European (EDNAP) exercise. Forensic Sci Int 164:33–44

    Article  PubMed  CAS  Google Scholar 

  7. Tucker VC, Hopwood AJ, Sprecher CJ, McLaren RS, Rabbach DR, Ensenberger MG, Thompson JM, Storts DR (2012) Developmental validation of the PowerPlex(R) ESX 16 and PowerPlex(R) ESX 17 Systems. Forensic Sci Int Genet 6:124–131

    Article  PubMed  CAS  Google Scholar 

  8. Kloosterman AD, Kersbergen P (2003) Efficacy and limits of genotyping low copy number (LCN) DNA samples by multiplex PCR of STR loci. J Soc Biol 197:351–359

    PubMed  CAS  Google Scholar 

  9. Westen AA, Nagel JH, Benschop CC, Weiler NE, de Jong BJ, Sijen T (2009) Higher capillary electrophoresis injection settings as an efficient approach to increase the sensitivity of STR typing. J Forensic Sci 54:591–598

    Article  PubMed  CAS  Google Scholar 

  10. Forster L, Thomson J, Kutranov S (2008) Direct comparison of post-28-cycle PCR purification and modified capillary electrophoresis methods with the 34-cycle "low copy number" (LCN) method for analysis of trace forensic DNA samples. Forensic Sci Int Genet 2:318–328

    Article  PubMed  Google Scholar 

  11. Lederer T, Braunschweiger G, Betz P, Seidl S (2002) Purification of STR-multiplex-amplified microsamples can enhance signal intensity in capillary electrophoresis. Int J Leg Med 116:165–169

    Article  CAS  Google Scholar 

  12. Butler JM (2011) Advanced topics in forensic DNA typing: methodology. Academic, Amsterdam

    Google Scholar 

  13. Hill CR, Butler JM, Vallone PM (2009) A 26plex autosomal STR assay to aid human identity testing. J Forensic Sci 54:1008–1015

    Article  PubMed  CAS  Google Scholar 

  14. Weiler NE, Matai AS, Sijen T (2012) Extended PCR conditions to reduce drop-out frequencies in low template STR typing including unequal mixtures. Forensic Sci Int Genet 6:102–107

    Article  PubMed  CAS  Google Scholar 

  15. Benschop C, Haned H, Sijen T (2011) Consensus and pool profiles to assist in the analysis and interpretation of complex low template DNA mixtures. Int J Leg Med. doi:10.1007/s00414-011-0647-5

  16. Edwards D (2012) PCR purification: AMPure and simple. http://www.keatslab.org/blog/pcrpurificationampureandsimple

  17. Rodrigue S, Materna AC, Timberlake SC, Blackburn MC, Malmstrom RR, Alm EJ, Chisholm SW (2010) Unlocking short read sequencing for metagenomics. PLoS One 5:e11840

    Article  PubMed  Google Scholar 

  18. Westen AA, Groen WJ and Maat GJR (2013) Human remains from the cloister garth of the 'Koningsveld' priory near the Medieval city of Delft - Ca. 1450–1572 AD. Barge's Anthropologica, Amsterdam

  19. Westen AA, Matai AS, Laros JF, Meiland HC, Jasper M, de Leeuw WJ, de Knijff P, Sijen T (2009) Tri-allelic SNP markers enable analysis of mixed and degraded DNA samples. Forensic Sci Int Genet 3:233–241

    Article  PubMed  CAS  Google Scholar 

  20. Nicklas JA, Buel E (2006) Simultaneous determination of total human and male DNA using a duplex real-time PCR assay. J Forensic Sci 51:1005–1015

    Article  PubMed  CAS  Google Scholar 

  21. Scientific Working Group on DNA Analysis Methods (2010) SWGDAM Interpretation Guidelines for Autosomal STR Typing by Forensic DNA Testing Laboratories. http://www.fbi.gov/about-us/lab/codis/swgdam.pdf

  22. Westen AA, Grol LJ, Harteveld J, Matai AS, de Knijff P, Sijen T (2012) Assessment of the stochastic threshold, back- and forward stutter filters and low template techniques for NGM. Forensic Sci Int Genet 6:708–715

    Article  PubMed  Google Scholar 

  23. Benschop CC, van der Beek CP, Meiland HC, van Gorp AG, Westen AA, Sijen T (2011) Low template STR typing: effect of replicate number and consensus method on genotyping reliability and DNA database search results. Forensic Sci Int Genet 5:316–328

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

This study was supported by a grant from the Netherlands Genomics Initiative/Netherlands Organization for Scientific Research (NWO) within the framework of the Forensic Genomics Consortium Netherlands. The authors would like to thank Arnoud Kal for critically reading the manuscript.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Titia Sijen.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary Fig. 1

DNA integrity of Covaris-degraded samples. Three DNA samples were subjected to seven Covaris settings (Supplementary Table 1) to fragment the DNA. DNA quality is visualised on a QIAxcel system (Qiagen, Venlo, The Netherlands) using DNA size markers of 250 bp–8 kb (four fragments can be seen: 250 bp, 500 bp, 750 bp and 1 kb) and 25 bp–450 bp (fragments at 25 bp intervals) (JPEG 29 kb)

High resolution image (TIFF 8337 kb)

Supplementary Fig. 2

Post-PCR purification by AMPure XP magnetic beads (according to the manufacturer’s protocol) in various volume ratios of PCR product and beads (1:1.8 down to 1:0.4). These experiments are all based on 500 pg pristine DNA007 NGM PCR amplifications of which 24 μL was used during the AMPure purification and 1 μL purified PCR product was analysed by CE. A non-purified control sample is shown in the bottom row. On each row, the first (D10S1248; 70–125 bp) and the last locus (D2S1338; 280–360 bp) of the blue (6-FAM) channel are shown. The peaks of the control sample are labelled both with the allele call and the peak height; the other samples are labelled with the peak height only. The Y-axis is scaled to 6,000 rfu in all panels (JPEG 150 kb)

High resolution image (TIFF 4500 kb)

Supplementary Fig. 3

Average ratio of the fold increase in peak heights of the AMPure protocol and DTR gel filtration for a no or little degraded samples and b severely degraded samples. The loci are ordered by fragment length (from shorter ones on the left-hand to longer fragments on the right-hand side). The error bars represent the standard deviation. The horizontal line at ratio 1 represents an equal fold increase in peak height after DTR gel filtration and AMPure purification. a For the no or little degraded samples 224 ratios could be calculated, varying between 9 (for D8S1179 and vWA) and 18 (for D22S1045 and D12S391) ratios per locus. b For the severely degraded samples 157 ratios could be calculated, varying between 3 (for D12S391) and 18 (for D2S441 and D3S1358) ratios per locus. No ratios could be determined for the longer loci, due to the lack of genotyping information in the profiles of non-purified PCR products (JPEG 65 kb)

High resolution image (TIFF 6340 kb)

Supplementary Fig. 4

The effect of post-PCR purification by DTR gel filtration or AMPure purification on the number of donor alleles detected for non-purified PCR products. Gained alleles are shown as a positive number, saturated alleles as a negative value. Sample degradation methods are abbreviated as: C = Covaris, UV = UV-light, B = bone sample, FT = freeze/thaw cycles and S = swab overgrown with microbes. Alleles on short loci are shown in red, on mid-range loci in grey and on long loci in green (JPEG 84 kb)

High resolution image (TIFF 4509 kb)

Table S1

(DOC 33 kb)

ESM 1

(DOC 37 kb)

ESM 2

(DOC 29 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Westen, A.A., van der Gaag, K.J., de Knijff, P. et al. Improved analysis of long STR amplicons from degraded single source and mixed DNA. Int J Legal Med 127, 741–747 (2013). https://doi.org/10.1007/s00414-012-0816-1

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00414-012-0816-1

Keywords

Navigation