Studies and Issues Reported in the Literature:
Reference Listing of Validation Studies
Summary of EDNAP Collaborative STR Exercises
Validation Studies on STR Systems
Before a new STR system or STR multiplex may be routinely employed in human identity testing it should be extensively validated to insure reliability of results. We have included below many of the validation studies which have appeared in the literature. We hope this material will help future researchers design their validation studies and allow those who use common STR systems to review what studies have been performed previously.
Crouse and Schumm {73} examined the species specificity of nine STR systems (CSF1PO, TPOX, TH01, HPRTB, FES/FPS, VWA, F13A1, and CSF1PO/TPOX/TH01 and HPRTB/FESFPS/VWA triplexes). They reported that no STR PCR products were obtained for 17 of the 23 species tested. Human, gorilla, chimpanzee, and orangutan DNAs were amplified with 8 of the 9 STR systems. Only FES/FPS primers failed to amplify DNA fragments from non-human primates. Most of the STR PCR products migrated outside of the human allelic ladder fragment range and could not be typed with the PAGE silver stain method employed.
The British Forensic Science Service {332} studied six STR loci and the amelogenin sex-test. Among the non-primates no STR loci were amplified. However, a DNA fragment 2 bp shorter than the X allele was observed for 5 species (pig, cattle, sheep, fox, and badger). The STR loci were observed to amplify quite well among primates, particularly the chimpanzees. However, the allele sizes often fell outside those normally observed for humans {332}.
Microbial DNA templates from 30 microorganisms that may be found in forensic samples were examined with the STR systems HUMTH01, TPOX, and CSF1PO {238}. No PCR products were observed at these STR loci. On the other hand, D1S80 amplifications from six of the bacterial DNAs analyzed produced some nonspecific PCR products that were located within the range of human D1S80 alleles {238}.
DNA mixtures originating from multiple donors may exist in forensic samples, particularly from rape cases where the perpetrator=s DNA will be mixed with the victim=s. Usually, a mixture is identified by the presence of 3 or more bands at one or more STR loci {346}. If the components of the mixture have identical alleles at a particular locus, then the peaks will be higher (with a system that records peak height or area) as a result. The quantitative nature of allele identification with fluorescence detection facilitates the identification of mixed body samples which may occur in forensic cases {121}.
Useful studies to evaluate the ability of a detection system to recognize mixtures include {363}:
examining mixtures of purified DNA in defined ratios
examining peak height ratios between heterozygous alleles within an STR locus
determining the range of stutter band percentage for each allele of each locus
A null allele is a term for the failure to amplify DNA sequences which are present in a sample. Allele amplification failure may result if polymorphisms occur in the primer binding site. This condition was first predicted by Caskey and Edwards {121} (U.S. Patent #5,364,759, column 19) and was recently reported for the MBP-B {379} and the D19S253 {332} STR systems. The problem of failed PCR amplifications was resolved by redesigning the primer sets to avoid the polymorphism in the former primer annealing region. In the case of D19S253, it was dropped from consideration in future multiplex STR systems {332}.
The higher sensitivity of PCR-based DNA typing assays make it imperative to separate laboratory procedures, such as DNA extraction and PCR amplification, into designated working areas. Potential sources of DNA contamination include: sample contamination with genomic DNA from the environment, contamination between samples during preparation, and contamination of a sample with amplified DNA from a previous PCR reaction {346}. The first source of contamination has been discussed in detail by Peter Gill {419} where he examines the utility of substrate controls (Forensic Sci. Int. (1997) 85:105-111). The last two may be controlled by appropriate laboratory procedures.
Materials which fluoresce in the visible region of the spectrum (~500-600 nm) may also interfere with DNA typing when using fluorescent scanners or one of the ABI PRISM systems by appearing as identifiable peaks in the electropherogram. Urquhart and coworkers {332} examined a number of fluorescent compounds and their apparent mobility when electrophoresed in a polyacrylamide gel. All of the compounds studied, which included antibiotics, vitamins, polycyclic aromatics, fluorescent brighteners, and various dyes, could be removed with an organic extraction (i.e., phenol/chloroform, as is commonly used to extract DNA from cells). The Chelex method of DNA extraction (Walsh, P.S., et al. (1991) BioTechniques 10:506-518) failed to remove all of the contaminating fluorescent peaks. However, these interfering peaks were usually wide and possessed a broader fluorescent spectrum which made it fairly easy to distinguish them from the fluorescent dye-labelled PCR products. Urquhart et al. {332} recommends comparing unamplified material to PCR-amplified sample when appropriate substrate controls are not present. They also describe four possible forensic scenarios where contaminants would be possible: a) body fluid stains on dyes materials from which the dye may leach during extraction; b) body fluid stains on plant material, from which chlorophyll may co-extract with DNA; c) blood or tissue samples from individuals with some pathological conditions, e.g., lead poisoning or some forms of porphyria, in which blood porphyrin levels are greatly elevated; d) bone or tooth samples from individuals who were treated with tetracycline-group antibiotics in their youth (growing bones and teeth are known to incorporate and accumulate these antibiotics) {332}.
Spurious PCR products may arise from non-templated nucleotide addition of adenine or from stutter bands and thus provide 'contaminating' peaks in an electropherogram.
Non-template Addition of Nucleotides
An extra adenine is often included at the 3' end of a PCR product resulting in what is commonly referred to as non-template addition (Clark, J.M. (1988) Nucleic Acids Res. 16:9677-9686). Oldroyd and coworkers {8} found that inconsistent addition of an extra base by Taq DNA polymerase following extension resulted in double peaks differing in size by 1 base for each allele at the D21S11 locus. The problem was eliminated by switching the fluorescent label to the reverse primer. Caskey and Edwards {121} resolved this problem by digesting the PCR product with the restriction enzyme MluI after incorporating a restriction site through the reverse primer (U.S. patent, column 19-20).
Stutter Bands
Stutter bands or 'shadow bands' were first reported with dinucleotide repeat polymorphisms by Hauge and Litt {85}. This phenomenon results in minor peaks which appear one repeat unit shorter than the major allele peak. Hauge and Litt {85} proposed slipped strand mispairing during PCR as the major mechanism for generation of these shadow bands. Several commonly used STR systems exhibit stutter bands including VWA {106}. Using Taq DNA polymerase, stutter bands are typically less than 10% of the allele band {106}.
Linkage of STR Systems to Genetic Disease
From C. Kimpton, et al. Forensic Sci. Int. 71 (1995) 137-152:
It is likely that many or possibly most STRs will eventually be shown to be useful in following a genetic disease or other genetic trait within a family and therefore this possibility must be recognized at the outset of the use of such systems {66}.
First one (P. Gill, et al. Forensic Sci. Int. 1994, 65, 51-59)
examined HUMTH01 and SE33 (ACTBP2)
Result: good agreement with HUMTH01 but a diversity of responses for the more complex SE33 locus
Second one (C. Kimpton, et al. Forensic Sci. Int. 1995, 71, 137-152)
examined HUMTH01, VWA, F13A1, FES/FPS
Result: robust analysis with ABI 373 detection of dye-labelled primers but problems with allele designation at F13A1 and FES/FPS when alternative detection methods were used
Third one (J. Andersen, et al. Forensic Sci. Int. 1996, 78, 83-93)
examined HUMTH01 and VWA with DNA mixtures
Result: all participating laboratories successfully typed the DNA from five stains, including two of mixed origin; these results were obtained for HUMTH01 and VWA despite diverse amplification, electrophoresis, and detection conditions (no amplification reagents or protocols were provided or specified as part of the study)
References Regarding Validation Studies
(Alphabetical order; see complete STR reference listing)
363. AmpFlSTR BlueTM PCR Amplification Kit: User's Manual (1996) The Perkin-Elmer Corporation.
423. Andersen, J.F. and Bramble, S. (1997) The effects of fingermark enhancement light sources on subsequent PCR-STR DNA analysis of fresh bloodstains. J. Forensic Sci. 42: 303-306.
91. Andersen, J.F., Greenhalgh, M.J., Butler, H.R., Kilpatrick, S.R., Piercy, R.C., Way, K.A., Myhill, H.S., Wright, J.C., Hallett, R. and Parkin, B.H. (1996) Further validation of a multiplex STR system for use in routine forensic identity testing. Forensic Sci. Int. 78: 47-64.
71. Andersen, J.F., Martin, P., Carracedo, A., Dobosz, M., Eriksen, B., Johnsson, V., Kimpton, C.P., Kloosterman, A.D., Konialis, C., Kratzer, A., Phillips, P., Mevag, B., Pfitzinger, H., Rand, S., Rosen, B., Schmitter, H., Schneider, P.M. and Vide, M.C. (1996) Report on the third EDNAP collaborative STR exercise. Forensic Sci. Int. 78: 83-93.
331. Bever, R.A. and Creacy, S. (1995) Validation and utilization of commercially available STR multiplexes for parentage analysis. Proceedings from the 5th International Symposium on Human Identification (1994). pp. 61-63.
600. Budowle, B., Koons, B.W., and Moretti, T.R. (1998) Subtyping of the HLA-DQA1 locus and independence testing with PM and STR/VNTR loci. J. Forensic Sci. 43(3):657-660.
536. Budowle, B., Moretti, T.R., Keys, K.M., Koons, B.W., and Smerick, J.B. (1997) Validation studies of the CTT STR multiplex system. J. Forensic Sci. 42(4):701-707.
70. Clayton, T.M., Whitaker, J.P., Fisher, D.L., Lee, D.A., Holland, M.M., Weedn, V.W., Maguire, C.N., DiZinno, J.A., Kimpton, C.P. and Gill, P. (1995) Further validation of a quadruplex STR DNA typing system: a collaborative effort to identify victims of a mass disaster. Forensic Sci. Int. 76: 17-25.
61. Clayton, T.M., Whitaker, J.P. and Maguire, C.N. (1995) Identification of bodies from the scene of a mass disaster using DNA amplification of short tandem repeat (STR) loci. Forensic Sci. Int. 76: 7-15.
337. Creacy, S., Kelly, C.M. and Bever, R.A. (1996) Application and utilization of STR multiplexes for parentage analysis. Proceedings from the 6th International Symposium on Human Identification (1995). pp. 28-31.
73. Crouse, C. and Schumm, J.W. (1995) Investigation of species specificity using nine PCR-based human STR systems. J. Forensic Sci. 40: 952-956.
483. Crouse, C. and Glidewell, D. (1996) Practical application of STRs to casework and acceptance in the courtroom. Proceedings from the Seventh International Symposium on Human Identification. Promega Corp. :97-100, 1997.
323. d'Aloja, E. and Domenici, R. (1996) HumTH01 allele frequencies in Italy--report of the GEFI collaborative study. Advances in Forensic Haemogenetics. Volume 6, pp. 692-694.
183. Eisenberg, M., Guerrieri, R., Maha, G.C., Mason, J.M., Heine, U., Burkhart, B. and Geyer, J. (1994) PCR-based analyses for identity testing. Advances in Forensic Haemogenetics. Volume 5, pp. 357-359.
64. Evett, I.W., Lambert, J.A., Knight, S.D., Fairley, M. and Lee, L.D. (1996) A study of independence between STR and conventional blood type loci. Forensic Sci. Int. 79: 163-166.
238. Fernandez-Rodriguez, A., Alonso, A., Albarran, C., Martin, P., Iturralde, M.J., Montesino, M. and Sancho, M. (1996) Microbial DNA challenge studies of PCR-based systems used in forensic genetics. Advances in Forensic Haemogenetics. Volume 6, pp. 177-179.
826. Foreman, L.A., Smith, A.F.M., and Evett, I.W. (1999) Bayesian validation of a quadruplex STR profiling system for identification purposes. J. Forensic Sci. 44(3):478-486.
134. Frazier, R.R.E., Millican, E.S., Watson, S.K., Oldroyd, N.J., Sparkes, R.L., Taylor, K.M., Panchal, S., Bark, L., Kimpton, C.P. and Gill, P.D. (1996) Validation of the Applied Biosystems PrismTM 377 automated sequencer for forensic short tandem repeat analysis. Electrophoresis 17: 1550-1552.
758. Fregeau, C.J., Bowen, K.L., and Fourney, R.M. (1999) Validation of highly polymorphic fluoresceht multiplex short tandem repeat systems using two generations of DNA sequencers. J. Forensic Sci. 44(1):133-166.
419. Gill, P. (1997) The utility of 'substrate controls' in relation to 'contamination'. Forensic Sci. Int. 85: 105-111.
69. Gill, P., Kimpton, C.P., d'Aloja, E., Andersen, J.F., Bar, W., Brinkmann, B., Holgersson, S., Johnsson, V., Kloosterman, A.D., Lareu, M.V., Nellemann, L., Pfitzinger, H., Phillips, C.P., Schmitter, H., Schneider, P.M. and Stenersen, M. (1994) Report of the European DNA profiling group (EDNAP)--towards standardisation of short tandem repeat (STR) loci. Forensic Sci. Int. 65: 51-59.
92. Gill, P., Kimpton, C.P., Urquhart, A., Oldroyd, N.J., Millican, E.S., Watson, S.K. and Downes, T.J. (1995) Automated short tandem repeat (STR) analysis in forensic casework--a strategy for the future. Electrophoresis 16: 1543-1552.
252. Greenhalgh, M.J. (1996) Casework experiences with a multiplex STR system. Advances in Forensic Haemogenetics. Volume 6, pp. 249-251.
594. Gross, A.M., Carmody, G., and Guerrieri, R. (1997) Validation studies for the genetic typing of the D1S80 locus for implementation into forensic casework. J. Forensic Sci. 42(6):1140-1146.
424. Hochmeister, M.N., Whelan, M., Borer, U.V., Gehrig, C., Binda, S., Berzlanovich, A., Rauch, E. and Dirnhofer, R. (1997) Effects of toluidine blue and destaining reagents used in sexual assault examinations on the ability to obtain DNA profiles from postcoital vaginal swabs. J. Forensic Sci. 42: 316-319.
705. Junge, A. and Madea, B. (1998) Validation studies and characterization of variant alleles at the short tandem repeat locus D12S391. Int. J. Legal Med. 112(1):67-69.
19. Kimpton, C.P., Fisher, D., Watson, S., Adams, M., Urquhart, A., Lygo, J. and Gill, P. (1994) Evaluation of an automated DNA profiling system employing multiplex amplification of four tetrameric STR loci. Int. J. Leg. Med. 106: 302-311.
66. Kimpton, C.P., Gill, P., d'Aloja, E., Andersen, J.F., Bar, W., Holgersson, S., Jacobsen, S., Johnsson, V., Kloosterman, A.D., Lareu, M.V., Nellemann, L., Pfitzinger, H., Phillips, C.P., Rand, S., Schmitter, H., Schneider, P.M., Sternersen, M. and Vide, M.C. (1995) Report on the second EDNAP collaborative STR exercise. Forensic Sci. Int. 71: 137-152.
62. Kimpton, C.P., Oldroyd, N.J., Watson, S.K., Frazier, R.R.E., Johnson, P.E., Millican, E.S., Urquhart, A., Sparkes, B.L. and Gill, P. (1996) Validation of highly discriminating multiplex short tandem repeat amplification systems for individual identification. Electrophoresis 17: 1283-1293.
63. Klintschar, M. and Crevenna, R. (1996) Validation of the STR system FXIIIB for forensic purposes in an Austrian population sample. Forensic Sci. Int. 81: 35-42.
352. Klintschar, M. (1995) Validation of the STR system FES/FPS for forensic purposes in an Austrian population sample. Int. J. Leg. Med. 108: 162-164.
519. Klintschar, M. and Crevenna, R. (1997) HUMCD4 - Validation of a STR system for forensic purposes in an Austrian caucasian population sample. J. Forensic Sci. 42(5):907-910.
723. LaFountain, M.J., Schwartz, M., Cormier, J., and Buel, E. (1998) Validation of capillary electrophoresis for analysis of the X-Y homologous amelogenin gene. J. Forensic Sci. 43(6):1188-1194.
65. Lee, L.D., Fairley, M., Lambert, J.A. and Evett, I.W. (1996) Validation of a frequency database for four STR loci for use in casework in the Strathclyde Police Forensic Science Laboratory. Forensic Sci. Int. 79: 43-48.
346. Lygo, J.E., Johnson, P.E., Holdaway, D.J., Woodroffe, S., Whitaker, J.P., Clayton, T.M., Kimpton, C.P. and Gill, P. (1994) The validation of short tandem repeat (STR) loci for use in forensic casework. Int. J. Leg. Med. 107: 77-89.
97. Mannucci, A., Sullivan, K.M., Ivanov, P.L. and Gill, P. (1994) Forensic application of a rapid and quantitative DNA sex test by amplification of the X-Y homologous gene amelogenin. Int. J. Leg. Med. 106: 190-193.
482. McElfresh, K., DiPierro, D., DelRio, S., Hayden, A., Jarvis, D., Chinsee, M., and Tracey, M. (1996) A comprehensive analysis of short tandem repeat polymorphisms: detection methods, population genetics, NRC II, and proficiency tests. Proceedings from the Seventh International Symposium on Human Identification. Promega Corp. :89-95.
72. Micka, K.A., Sprecher, C.J., Lins, A.M., Comey, C.T., Koons, B.W., Crouse, C., Endean, D., Pirelli, K., Lee, S.B., Duda, N., Ma, M. and Schumm, J.W. (1996) Validation of multiplex polymorphic STR amplification sets developed for personal identification applications. J. Forensic Sci. 41: 582-590.
137. Moller, A., Schurenkamp, M. and Brinkmann, B. (1995) Evaluation of an ACTBP2 ladder composed of 26 sequenced alleles. Int. J. Leg. Med. 108: 75-78.
113. Moller, A., Wiegand, P., Gruschow, C., Seuchter, S.A., Baur, M.P. and Brinkmann, B. (1994) Population data and forensic efficiency values for the STR systems HumVWA, HumMBP and HumFABP. Int. J. Leg. Med. 106: 183-189.
140. Pestoni, C., Lareu, M.V., Rodriguez, M.S., Munoz, I., Barros, F. and Carracedo, A. (1995) The use of the STRs HUMTH01, HUMVWA31/A, HUMF13A1, HUMFES/FPS, HUMLPL in forensic application: validation studies and population data for Galicia (NW Spain). Int. J. Leg. Med. 107: 283-290.
334. Robertson, J.M., Badger, C.A. and Buoncristiani, M.R. (1995) Design of short tandem repeat systems suitable for human identification. Proceedings from the 5th International Symposium on Human Identification (1994). pp. 95-102.
209. Schmitt, C., Schmutzler, A., Prinz, M. and Staak, M. (1994) High sensitive DNA typing approaches for the analysis of forensic evidence: comparison of nested variable number of tandem repeats (VNTR) amplification and a short tandem repeats (STR) polymorphism. Forensic Sci. Int. 66: 129-141.
420. Sparkes, R., Kimpton, C.P., Watson, S., Oldroyd, N.J., Clayton, T.M., Barnett, L., Arnold, J., Thompson, C., Hale, R., Chapman, J., Urquhart, A. and Gill, P. (1996) The validation of a 7-locus multiplex STR test for use in forensic casework: (I) Mixtures, ageing, degradation and species studies. Int. J. Legal Med. 109: 186-194.
431. Sparkes, R., Kimpton, C.P., Watson, S., Oldroyd, N.J., Urquhart, A. and Gill, P. (1997) The forensic validation of a 7-locus multiplex STR test. Proceedings from the First European Symposium on Human Identification (1996). pp. 82-89.
470. Sparkes, R., Kimpton, C., Gibard, S., Carne, P., Andersen, J., Oldroyd, N., Thomas, D., Urquhart, A., and Gill, P. (1996) The validation of a 7-locus multiplex STR test for use in forensic casework. Int. J. Legal Med. 109:195-204.
799. Thomson, J.A., Pilotti, V., Stevens, P., Ayres, K.L., and Debenham, P.G. (1999) Validation of short tandem repeat analysis for the investigation of cases of disputed paternity. Forensic Sci. Int. 100(1-2):1-16.
332. Urquhart, A., Chiu, C.T., Clayton, T.M., Downes, T., Frazier, R.R.E., Jones, S., Kimpton, C.P., Lareu, M.V., Millican, E.S., Oldroyd, N.J., Thompson, C., Watson, S., Whitaker, J.P. and Gill, P. (1995) Multiplex STR systems with fluorescent detection as human identification markers. Proceedings from the 5th International Symposium on Human Identification (1994). pp. 73-83.
67. van Oorschot, R.A.H., Gutowski, S.J., Robinson, S.L., Hedley, J.A. and Andrew, I.R. (1996) HUMTH01 validation studies: Effect of substrate, environment, and mixtures. J. Forensic Sci. 41: 142-145.
680. Wallin, J.M., Buoncristiani, M.R., Lazaruk, K., Fildes, N., Holt, C., and Walsh, P.S. (1998) TWGDAM validation of the AmpFlSTRTM Blue PCR Amplification Kit for forensic casework analysis. J. Forensic Sci. 43(4):854-870.
74. Walsh, P.S., Erlich, H.A. and Higuchi, R. (1992) Preferential PCR amplification of alleles: Mechanisms and solutions. PCR Meth. Appl. 1: 241-250.
68. Wiegand, P., Budowle, B. and Brinkmann, B. (1993) Forensic validation of the STR systems SE33 and TC11. Int. J. Leg. Med. 105: 315-320.
52 References as of 06/16/00