4
ORIGINAL ARTICLE Abstract In recent years there has been considerable improvement in short-tandem repeat (STR) investigations of hair, which were previously marred by small amounts of nuclear DNA and its degradation. This study examined the suitability of two STR kits with shortened amplicons for the investigation of hairs from routine casework. The overall success rate was more than 20%. Furthermore, the usefulness of quan- tification with real-time ploymerase chain reaction as a screening method was demonstrated. Key Words: Forensic identification; hair roots; genRES ® MPX-SP1 and 2; genRES MPX-2; Quantifiler Human. (DOI: 10.1385/Forensic Sci. Med. Pathol.:3:1:41) Comparative Investigation of Hair With the genRES ® MPX-SP1, genRES MPX-SP2, and genRES MPX-2 Kits K. Anslinger, B. Bayer, and B. Rolf Institute of Legal Medicine, Ludwig-Maximilians-University, Munich Address for correspondence and reprints: K. Anslinger Institute of Legal Medicine Ludwig-Maximilians-University of Munich Frauenlobstrasse 7a 80337 München, Germany E-mail: katja.anslinger@med. uni-muenchen.de Accepted for publication: July 23, 2006 Forensic Science, Medicine, and Pathology Copyright © 2007 Humana Press Inc. All rights of any nature whatsoever are reserved. ISSN 1547-769X/07/3:41–44/$30.00 (Online) 1556-2891 DOI: 10.1385/Forensic Sci. Med. Pathol.:3:1:41 INTRODUCTION Investigation of telogen hairs from crime scenes is often pertinent to forensic casework. Until recently,however,results of short-tandem repeat (STR) profiling were often insufficient because of the small amounts of nuclear DNA and its degra- dation. In most cases, successful investigation of the mito- chondrial DNA (mtDNA) of telogen hairs as well as hair shafts is possible (1–3). However, sequencing results cannot be com- pared with national DNA databases and the use of these inves- tigations is thus limited to cases with known suspects. In recent years, there has been considerable improvement in STR inves- tigations of hair. New approaches,which include better extrac- tion methods as well as new polymerase chain reaction (PCR) set-ups with shortened amplicons for degraded DNA samples, have been published (4–9). In this current study, the suitabil- ity of two kits with shortened amplicons (genRES ® MPX-SP1 and MPX-SP1 2 kits, Serac, Bad Homburg, Germany) were tested. MPX-SP1 allows the simultaneous amplification of the four STR-loci VWA, D3S1358, TH01, and D8S1179 as well as the sex-determining amelogenin system. MPX-SP2 amplifies the FGA, TH01, D18S51, D21S11, and SE33 loci. The length of amplicons ranges between 64 and 150 for MPX- SP1 and 290 bp for MPX-SP2 (Table 1). The results were com- pared with profiles obtained from the genRES MPX-2 amplification kit (Serac GmbH, Bad Homburg, Germany), which allows the simultaneous amplification of all eight STR- loci plus amelogenin in one single reaction (10). Corresponding amplicon sizes were listed in Table 1. Furthermore, the use of quantification with real-time PCR as a screening method was tested. MATERIALS AND METHODS Ninety-six hair roots taken from our routine casework (from head or body with telogen as well as roots with sheath cells) were incubated in 100 µL TN CA buffer for 2 h at 56°C, accord- ing to the method described by Hellmann et al. (4). Following digestion, DNA was extracted as described by Meissner et al. (11): 200 µL 5% Chelex (Bio-Rad, Hercules, CA) was added, incubated for 8 min at 56°C and boiled for 8 min at 100°C. After centrifugation for 3 min at 14,000 rpm, the supernatant was removed and 20 µL 3 M Na-acetate (pH 4.8), 8 µL 0.23% linear polyacrylamide (GenEluteTm LPA, Sigma, Deisenhofen, Germany) and 2.5 vol ethanol (preincubated at –25°C) were added. After mixing, DNA was precipitated by overnight incubation at –20°C and centrifugation for 30 min at 20,000g and 4°C. After discarding the supernatant, the pellet was washed with 1 mL 70% ethanol and dissolved in 40 µL water. 41

Comparative investigation of hair with the genRES® MPX-SP1, genRES MPX-SP2, and genRES MPX-2 kits

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ORIGINAL ARTICLE

Abstract

In recent years there has been considerable improvement in short-tandem repeat(STR) investigations of hair, which were previously marred by small amounts ofnuclear DNA and its degradation. This study examined the suitability of two STRkits with shortened amplicons for the investigation of hairs from routine casework.The overall success rate was more than 20%. Furthermore, the usefulness of quan-tification with real-time ploymerase chain reaction as a screening method wasdemonstrated.

Key Words: Forensic identification; hair roots; genRES® MPX-SP1 and 2; genRESMPX-2; Quantifiler™ Human.

(DOI: 10.1385/Forensic Sci. Med. Pathol.:3:1:41)

Comparative Investigation of Hair With the genRES® MPX-SP1, genRES MPX-SP2, and genRES MPX-2 KitsK. Anslinger, B. Bayer, and B. Rolf

Institute of Legal Medicine, Ludwig-Maximilians-University, Munich

Address for correspondence and reprints:

K. AnslingerInstitute of Legal MedicineLudwig-Maximilians-University of MunichFrauenlobstrasse 7a80337 München, GermanyE-mail: [email protected]

Accepted for publication:July 23, 2006

Forensic Science, Medicine, and PathologyCopyright © 2007 Humana Press Inc. All rights of any nature whatsoever are reserved.ISSN 1547-769X/07/3:41–44/$30.00 (Online) 1556-2891DOI: 10.1385/Forensic Sci. Med. Pathol.:3:1:41

INTRODUCTIONInvestigation of telogen hairs from crime scenes is often

pertinent to forensic casework. Until recently, however, resultsof short-tandem repeat (STR) profiling were often insufficientbecause of the small amounts of nuclear DNA and its degra-dation. In most cases, successful investigation of the mito-chondrial DNA (mtDNA) of telogen hairs as well as hair shaftsis possible (1–3). However, sequencing results cannot be com-pared with national DNA databases and the use of these inves-tigations is thus limited to cases with known suspects. In recentyears, there has been considerable improvement in STR inves-tigations of hair. New approaches, which include better extrac-tion methods as well as new polymerase chain reaction (PCR)set-ups with shortened amplicons for degraded DNA samples,have been published (4–9). In this current study, the suitabil-ity of two kits with shortened amplicons (genRES® MPX-SP1and MPX-SP1 2 kits, Serac, Bad Homburg, Germany) weretested. MPX-SP1 allows the simultaneous amplification ofthe four STR-loci VWA, D3S1358, TH01, and D8S1179 aswell as the sex-determining amelogenin system. MPX-SP2amplifies the FGA, TH01, D18S51, D21S11, and SE33 loci.The length of amplicons ranges between 64 and 150 for MPX-SP1 and 290 bp for MPX-SP2 (Table 1). The results were com-pared with profiles obtained from the genRES MPX-2

amplification kit (Serac GmbH, Bad Homburg, Germany),which allows the simultaneous amplification of all eight STR-loci plus amelogenin in one single reaction (10). Correspondingamplicon sizes were listed in Table 1. Furthermore, the use of quantification with real-time PCR as a screening methodwas tested.

MATERIALS AND METHODSNinety-six hair roots taken from our routine casework (from

head or body with telogen as well as roots with sheath cells)were incubated in 100 µL TNCA buffer for 2 h at 56°C, accord-ing to the method described by Hellmann et al. (4). Followingdigestion, DNA was extracted as described by Meissner et al.(11): 200 µL 5% Chelex (Bio-Rad, Hercules, CA) was added,incubated for 8 min at 56°C and boiled for 8 min at 100°C.After centrifugation for 3 min at 14,000 rpm, the supernatantwas removed and 20 µL 3 M Na-acetate (pH 4.8), 8 µL 0.23%linear polyacrylamide (GenEluteTm LPA, Sigma,Deisenhofen, Germany) and 2.5 vol ethanol (preincubated at–25°C) were added. After mixing, DNA was precipitated byovernight incubation at –20°C and centrifugation for 30 minat 20,000g and 4°C. After discarding the supernatant, thepellet was washed with 1 mL 70% ethanol and dissolved in40 µL water.

41

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42 _____________________________________________________________________________________Anslinger et al.

Quantification of DNA was carried out using theQuantifiler™ Human DNA Quantification kit (AppliedBiosystems, PE Corporation, Foster City, CA) according tothe user’s manual and a 7300 real-time PCR sequence detec-tion system from Applied Biosystems. Each sample was quan-tified twice, and the mean was then calculated.

For STR profiling, multiplex PCRs were performed usingthe genRES MPX-SP1, MPX-SP 2 and genRES MPX-2 ampli-fication kits (Serac GmbH, Bad Homburg, Germany) by adding10 µL DNA to a total reaction volume of 25 µL and 32 (genRESMPX-SP1) and 34 (genRES MPX-SP2 and genRES MPX-2)cycles were performed respectively. The PCR products wereanalysed on an ABI PRISM 3100 Avant capillary elec-trophoresis system (Applied Biosystems).

The profiles were analyzed with the genotyper software(Applied Biosystems). Furthermore, the quality of the pro-files was classified by (a) the number of STR systems typedcompletely (all alleles detectable and above 50 rfu); (b) thenumber of STR systems that showed allelic drop-out (thresh-old 50 rfu) and (c) the number of STR systems that showedlocus drop-out (threshold 50 rfu). The values for all three arelisted in a table using an A_B_C format. Samples, whichshowed less then three of the five MPX-SP1 STR systemstyped completely, were not tested with MPX-SP2. Similarly,only samples showing three or more completely typed STRsystems with MPX-SP2 were amplified with MPX-2.

RESULTS AND DISCUSSIONIn 65 out of the 96 investigated hair roots (exclusively

telogen hairs), no DNA was detectable in the two TaqManquantification reactions. STR profiling with genRES MPX-SP1 revealed no alleles (data not shown). Based on the resultsof the internal positive control (IPC), which was amplified

simultaneously in each sample tested with the Quantifilerhuman DNA quantification kit, PCR inhibition owing to, forexample, melanin, could be excluded. Therefore, quantifi-cation with the Quantifiler human DNA quantification kitcan be seen as a reliable screening method. Duplicate test-ing minimizes the risk of false-negative results. Becausehairs classified as telogen by visual investigation showedgreat variation in the DNA yield (Table 2), histological resultswere inconclusive for predicting the success rate of ensuingDNA profiling.

Partial genRES MPX-SP1 profiles could be detected downto the 11 pg/µL sample (Table 2). For the 185 pg/µL sampleand above all samples revealed full genRES MPX-SP1 pro-files. Results obtained from samples showing DNA yieldsbetween 11 and 110 pg/µL DNA varied. No correlationbetween the quality of the obtained profile and the DNA yieldcould be detected, which probably reflects the amount of DNAdegradation in the sample. Of the 17 samples in this DNArange, 15 showing full genRES MPX-SP1 profiles, revealeda complete genRES MPX-SP2 profile (up to a DNA yield of37 pg/µL), whereas the remaining showed partial profiles,lacking some alleles from the larger STR fragment systems.Whenever partial profiles were obtained with both kits for onesample, genRES MPX-SP1 profiles were mostly better thanthose of the corresponding genRES MPX-SP2 kit. ComparinggenRES MPX-2 and the two shortplex kits, in 7 out of 19 sam-ples better results were obtained with genRES MPX-SP1 andMPX-SP 2. In the remaining samples, full profiles could beobtained from the two shortplex kits as well as from genRESMPX-2. Once again no correlation between the quality of theobtained profile and the DNA yield could be detected. Thus,investigation of hair with the two shortplex kits appears to bemore promising.

Forensic Science, Medicine, and PathologyV3–1

Table 1Comparison of the Amplicon Size Ranges for Each Locus and Multiplex Kit Used

in This Study Given in Base Pairs (bp)

genRES® MPX-SP1 LengthLocus Ladder-alleles and MPX-SP2 of amplicons in bp

Amelogenin X and Y 70 and 76 103 and 109D3S1358 11 to 20 84 to 120 109 to 145VWA 11 to 22 102 to 146 128 to 172FIBRA 16 to 29a/45.2b 134 to 252 159 to 211TH01 5 to 11 64 to 88 122 to 146SE33 6.3 to 37 171 to 292 212 to 333D8S1179 8 to 18 87 to 127 258 to 298D21S11 26 to 36 165 to 205 206 to 246D18S51 7 to 24 110 to 178 243 to 311

aAllele range for the genRES MPX-2 kit, which does not include the larger alleles. ballele range for the genRES MPX-SP2 kit.

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STR Kits ___________________________________________________________________________________________43

The authors have stated that they do not have a significantfinancial interest or other relationship with any product man-ufacturer or provider of services discussed in this article.

REFERENCES1. Wilson MR, Polanskey D, Butler J, DiZinno JA, Replogle J,

Budowle B. Extraction, PCR amplification and sequencing ofmitochondrial DNA from human hair shafts. Biotechniques1995;18:662–669.

2. Pfeiffer H, Huhne J, Ortmann C, Waterkamp K, Brinkmann B.Mitochondrial DNA typing from human axillary, pubic and headhair shafts—success rates and sequence comparisons. Int J LegalMed 1999;112:287–290.

Forensic Science, Medicine, and PathologyV3–1

1. Quantification with TaqMan followed by profiling withshortplex kits provides a useful strategy for the investi-gation of hairs in forensic casework.

2. Screening with TaqMan enables hairs with sufficientDNA yield to be selected. Duplicate testing minimizesthe risk of false-negative results.

3. The use of shortplex kits appears to provide an advan-tage over the use of a kit with normal amplicon size.

4. The overall success rate was more than 20%.

Educational Message

Table 2Genotyping Results

DNA yield genRES genRES genRESSample Root pg/µL MPX-SP1 MPX-SP2 MPX-2

Hair 62 T 2 0_0_5 n.t. n.t.Hair 59 T 3 0_0_5 n.t. n.t.Hair 71 T 3 0_0_5 n.t. n.t.Hair 30 T 4 0_0_5 n.t. n.t.Hair 50 T 5 0_0_5 n.t. n.t.Hair 1 T 8 0_0_5 n.t. n.t.Hair 51 T 9 0_0_5 n.t. n.t.Hair 83 T 11 4_1_0 3_2_0 2_5_2Hair 14 T 15 2_0_3 n.t. n.t.Hair 11 T 18 2_0_3 n.t. n.t.Hair 6 T 27 5_0_0 2_3_0 n.t.Hair 27 T 33 5_0_0 3_0_0 4_2_3Hair 29 T 37 5_0_0 5_0_0 9_0_0Hair 42 T 59 5_0_0 5_0_0 9_0_0Hair 24 T 72 3_1_1 2_1_2 n.t.Hair 20 T 78 4_1_0 5_0_0 2_4_3Hair 22 T 103 5_0_0 5_0_0 2_2_5Hair 19 T 108 3_1_1 1_1_3 n.t.Hair 44 T 110 5_0_0 5_0_0 9_0_0Hair 21 T 142 4_1_0 4_0_1 3_4_2Hair 10 T 185 5_0_0 5_0_0 9_0_0Hair 85 T 194 5_0_0 5_0_0 9_0_0Hair 25 T 234 5_0_0 5_0_0 7_2_0Hair 92 T 251 5_0_0 5_0_0 9_0_0Hair 5 ? 350 5_0_0 5_0_0 9_0_0Hair 88 S 536 5_0_0 5_0_0 9_0_0Hair 94 T 555 5_0_0 5_0_0 9_0_0Hair 23 ? 827 5_0_0 5_0_0 7_2_0Hair 89 S 895 5_0_0 5_0_0 9_0_0Hair 82 S 1176 5_0_0 5_0_0 9_0_0Hair 95 T 2798 5_0_0 5_0_0 9_0_0

Samples with DNA yields > 0, sorted ascending by pg/µL DNA. Qualities of profiles were classified by (a) thenumber of STR systems typed completely (all alleles detectable and > 50 rfu); (b) the number of STR systems thatshowed allelic dropout (threshold 50 rfu); (c) the number of STR systems that showed locus dropout (threshold 50rfu) using an A_B_C format.

n.t., not tested; T, telogen hair; S, hair with sheath cells; ?, no microscopic evaluation.

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Part 2. An optimised genomic DNA extraction procedure revealsdonor dependence of STR profiles. Forensic Sci Int 2005;153:247–259.

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