STR analysis of artificially degraded DNA—results ofa collaborative European exercise

Peter M. Schneidera,*, Klaus Bendera, Wolfgang R. Mayrb, Walther Parsonc,Bernadette Hosted, Ronny Decortee, Jan Cordonnierf, Daniel Vanekg, Niels Morlingh,

Matti Karjalaineni, C. Marie-Paule Carlottij, Myriam Sabatierk, Carsten Hohoffl,Hermann Schmitterm, Werner Pflugn, Rainer Wenzelo, Dieter Patzeltp, Rudiger Lessigq,

Peter Dobrowolskir, Geraldine O’Donnells, Luciano Garafanot, Marina Doboszu,Peter de Knijffv, Bente Mevagw, Ryszard Pawlowskix, Leonor Gusmaoy,

Maria Conceicao Videz, Antonio Alonso Alonsoa1, Oscar Garcıa Fernandeza2,Pilar Sanz Nicolasa3, Ann Kihlgreena4, Walter Bara5, Verena Meiera6,

Anne Teyssiera7, Raphael Coquoza8, Conxita Brandta9, Ursula Germanna10,Peter Gilla11, Justine Halletta12, Matthew Greenhalgha13

aInstitut fur Rechtsmedizin, Universitat Mainz, Mainz, GermanybKlinische Abteilung fur Blutgruppenserologie, Universitat Wien, Wien, Austria

cInstitut fur Gerichtliche Medizin, Universitat Innsbruck, Innsbruck, AustriadInstitut National de Criminalistique, Bruxelles, Belgium

eLaboratory for Forensic Genetics and Molecular Archaeology, Leuven, BelgiumfChemiphar, Brugge, Belgium

gInstitute of Criminalistics, Prague, Czech RepublichDepartment of Forensic Genetics, Institute of Forensic Medicine,

University of Copenhagen, Copenhagen, DenmarkiNational Bureau of Investigation, Vantaa, Finland

jInstitut de Recherche Criminelle, Rosny-Sous-Bois, FrancekLaboratoire de Police Scientifique, Toulouse, France

lInstitut fur Rechtsmedizin, Universitat Munster, GermanymBundeskriminalamt, Wiesbaden, Germany

nLKA Baden-Wurttemberg, Stuttgart, GermanyoLKA Rheinland-Pfalz, Mainz, Germany

pInstitut fur Rechtsmedizin, Universitat Wurzburg, Wurzburg, GermanyqInstitut fur Rechtsmedizin, Universitat Leipzig, Leipzig, Germany

rLKA Sachsen, Dez. DNA-Analytik, Dresden, GermanysForensic Science Laboratory, Dublin, Ireland

tReparto Carabinieri Investigazioni Scientifiche, Parma, ItalyuIstituto di Medicina Legale Universita Cattolica, Roma, Italy

vForensics Laboratory, University of Leiden, Leiden, The NetherlandswRettsmedisinsk Institutt, University of Oslo, Oslo, Norway

xInstitute of Forensic Medicine, Medical University of Gdansk, Gdansk, PolandyIPATIMUP, Universidade do Oporto, Oporto, Portugal

zInstituto de Medicina Legal, Universidade de Coimbra, Coimbra, Portugala1Instituto National de Toxicologia, Madrid, Spain

a2Area de Laboratorio Ertzaina, Bilbao, Spaina3Instituto National de Toxicologia, Sevilla, Spain

Forensic Science International 139 (2004) 123–134

* Corresponding author. Present address: Institute of Legal Medicine, Am Pulverturm 3, D-55131 Mainz, Germany.

Tel.: þ49-6131-3932687; fax: þ49-6131-3933183.

E-mail address: pschneid@uni-mainz.de (P.M. Schneider).

0379-0738/$ – see front matter # 2003 Elsevier Ireland Ltd. All rights reserved.

doi:10.1016/j.forsciint.2003.10.002

a4National Laboratory of Forensic Science, Linkoping, Swedena5Institut fur Rechtsmedizin, Universitat Zurich, Zurich, Switzerland

a6Instiut fur Rechtsmedizin, Universitat Basel, Basel, Switzerlanda7Institut Universitaire de Medecine Legale, Universite de Geneve, Geneve, Switzerland

a8Laboratoire AMS, Lausanne, Switzerlanda9Institut Universitaire de Medecine Legale, Lausanne, Switzerland

a10Institut fur Rechtsmedizin, Universitat St. Gallen, St. Gallen, Switzerlanda11Forensic Science Service Headquarters, Birmingham, UK

a12LGC, Teddington Middlesex, UKa13Orchid Bioscience Europe, Abingdon, UK

Received 21 May 2003; received in revised form 2 October 2003; accepted 3 October 2003

Abstract

Degradation of human DNA extracted from forensic stains is, in most cases, the result of a natural process due to the exposure of

the stain samples to the environment. Experiences with degraded DNA from casework samples show that every sample may exhibit

different properties in this respect, and that it is difficult to systematically assess the performance of routinely used typing systems

for the analysis of degraded DNA samples. Using a batch of artificially degraded DNA with an average fragment size of approx.

200 bp a collaborative exercise was carried out among 38 forensic laboratories from 17 European countries. The results were

assessed according to correct allele detection, peak height and balance as well as the occurrence of artefacts. A number of common

problems were identified based on these results such as strong peak imbalance in heterozygous genotypes for the larger short

tandem repeat (STR) fragments after increased PCR cycle numbers, artefact signals and allelic drop-out. Based on the observations,

strategies are discussed to overcome these problems. The strategies include careful balancing of the amount of template DNA and

the PCR cycle numbers, the reaction volume and the amount of Taq polymerase. Furthermore, a careful evaluation of the results of

the fragment analysis and of automated allele calling is necessary to identify the correct alleles and avoid artefacts.

# 2003 Elsevier Ireland Ltd. All rights reserved.

Keywords: Short tandem repeat; Alleles; PCR degradation

1. Introduction

Genetic analysis of short tandem repeat (STR) loci is

currently the most powerful and widely used method to

identify the contributor of biological samples collected in the

context of criminal investigations, and the identification of

human remains. The successful application includes a vari-

ety of substrates such as blood, saliva, epithelial cells, hair

roots and even compact bone samples [1,2]. However, when

more problematic samples such as old stains or decomposed

tissue are subjected to STR typing, failure to obtain repro-

ducible results may occur due to degradation of high mole-

cular weight DNA [3].

In crime case investigations, degradation of human

DNA usually is the result of a natural process resulting

from the exposure of the stain or tissue samples to the

environment. Light, humidity, elevated temperatures as

well as bacterial and fungal contaminations followed by

the growth of these microorganisms lead to physical,

chemical and biochemical degradation of genomic

DNA. Once the average DNA fragment length is reduced

to sizes smaller than 300 bp, loss of genetic information

may occur due to the lack of suitable template DNA and

the subsequent failure of frequently used STR typing

methods [4].

Experience with degraded DNA from casework samples

shows that every sample may exhibit different properties in

this respect, and that it is difficult to systematically assess the

performance of routinely used typing systems to analyze

degraded DNA samples. To learn more about the efficiency

of STR systems, a standardized reference sample of

degraded DNA in sufficient amounts would be quite helpful,

and could also be used for validation studies of new STR

typing systems [5–7]. Therefore, a collaborative exercise on

degraded DNA was planned in the process of the ‘‘Standar-

dization of DNA Profiling Methods in the European Union’’

(STADNAP) network, and participation was offered to 50

forensic DNA laboratories across Europe. The intention of

this exercise was to better understand the different para-

meters influencing STR typing results obtained from

degraded DNA as well as to learn more about the strategies

applied by the participating laboratories to optimize the

typing of problematic samples.

2. Materials and methods

2.1. Production of degraded DNA samples

Batches of high molecular weight genomic DNA were

prepared from two human cell lines, HepG2 and P118, with

124 P.M. Schneider et al. / Forensic Science International 139 (2004) 123–134

male and female genotypes, respectively. The DNA samples

were degraded under standardized conditions to an average

fragment length of less than 200 bp using a combination of

physical and biochemical methods, i.e. sonication and treat-

ment with DNAse I. Based on previous experiences from a

smaller exercise among the STADNAP partner laboratories

using a single degraded DNA sample (data not shown) it was

intended to achieve a slightly higher degree of degradation in

sample B compared to sample A. The degradation process was

closely monitored to control the resulting fragment sizes as

well as the suitability to obtain partial results by multiplex

STR typing. It was observed that the final fragment size

distribution heavily depends on the DNA quality and con-

centration, the reaction conditions and volumes, as well as the

properties and the concentration of the DNAse I digestion.

Batch to batch reproducibility could only be achieved by

repeatedly testing the degraded DNA using STR analysis [8].

2.2. Exercise design

Two aliquots of 20 ml each were shipped to the 50

participating laboratories with the following instructions:

� Apply your standard combination of forensic STR typing

systems using regular PCR conditions.

� If your first amplification fails, try to modify these con-

ditions as you would do in casework, or use singleplex

systems.

� Record the results for each locus with successful ampli-

fication by allele designation and peak height in a tabu-

lated form, and enclose prints showing the DNA profiles

using the GeneScan or GenoTyper format.

� If you routinely perform mtDNA typing, please try to

sequence HV-I and HV-II.

In addition, the participating laboratories were asked to

provide further information about equipment, routine typing

procedures, PCR conditions and rules for data analysis, e.g.

the threshold for allele calling.

2.3. Data analysis

The submitted data were recorded for each STR locus

based on the peak height (in relative fluorescence units, rfu)

for each allele observed. To allow a comparison between

laboratories, the peak heights were grouped into three

categories: strong signal: >150 rfu, low signal: 150–

30 rfu, very low or no signal: <30 rfu. Furthermore, two

categories of data were defined based on the exercise

instructions (see above): ‘‘standard’’ PCR conditions using

0.25–2.5 ng degraded DNA and 28 PCR cycles, and

‘‘enhanced’’ PCR conditions using 0.5–5 ng DNA and

28–36 PCR cycles. Unexpected results, artefacts or incorrect

alleles were recorded separately for each locus and were not

included in the peak height analysis.

3. Results and discussion

Results were received from 38 laboratories from 17

European countries (Fig. 1). All laboratories submitted data

for at least one multiplex STR kit. The total number of

datasets for each STR kit included in the study are summar-

ized in Fig. 2. The majority of laboratories (63%) used the

Profiler SGM Plus kit from Applied Biosystems (ABI). A

total of 26% of the laboratories used the PowerPlex 16 kit

from Promega, and another 29% used the Profiler, Profiler

Plus and Cofiler multiplexes from ABI. In addition, 29% of

the laboratories submitted data for other commercial kits or

‘‘home made’’ singleplex STR systems, and 21% of the

laboratories performed a DNA sequence analysis of the

hypervariable D-loop regions of mitochondrial DNA. The

majority of laboratories (58%) carried out capillary electro-

phoresis using the ABI Prism 310 Analyzer (Fig. 2, columns

on the right).

The correct typing results for the DNA samples A and B

are listed in Table 1 for all STR loci comprised in the SGM

Plus, Profiler Plus and PowerPlex 16 multiplexes, including

Fig. 1. Number and country of origin of the participating laboratories.

P.M. Schneider et al. / Forensic Science International 139 (2004) 123–134 125

data on the maximal fragment size and fluorescent dye label

color for each system. Fragment sizes for each locus are

consistent among the kits manufactured by ABI, although

some have been labeled with different colors as shown. In

the Promega multiplex kit, some STR systems are located in

a different size range in comparison to the ABI kits.

The multiplex typing results based on the recorded peak

heights (in rfu) are depicted for the three most commonly

used multiplexes SGM Plus (Fig. 3), Profiler Plus including

the blue and yellow systems from Profiler (Fig. 4), and

PowerPlex 16 (Fig. 5). In each histogram, the STR systems

are arranged with increasing fragment size from left to right

(see also Table 1). In each figure, the results for sample A (a,

b) and sample B (c, d) are shown for standard (a, c) and

enhanced (b, d) PCR conditions.

The amount of genomic DNA and the number of cycles

varied significantly between laboratories. Therefore, the

decision to assign the results into the two categories

Fig. 2. STR multiplex kits and sequencing equipment used by exercise participants.

Table 1

STR loci, fragment sizes and dye label colors of three multiplex kits used by the exercise participants

STR system Genotypes SGM Plus Profiler/Plus Power Plex

Sample A Sample B Allele size

range (bp)

Ba Ga Ya Allele size

range (bp)

B G Y Allele size

range (bp)

B G Y

D3S1358 15, 16 16, 17 <150 þ <150 þ <150 þVWA 17 16, 18 <210 þ <210 þ <170 þFGA 22, 25 21, 22 <360 þ <360 þ <450 þAmelogenin XY XX <112 þ <112 þ <112 þD8S1179 15, 16 14 <170 þ <170 þ <250 þD21S11 29, 31 30 <250 þ <250 þ <260 þD18S51 13, 14 13 <350 þ <350 þ <370 þD5S818 11, 12 12, 14 <180 þ <160 þD13S317 9, 13 11, 12 <240 þ <200 þD7S820 10 8, 10 <300 þ <250 þD19S433 15.2 14, 15 <140 þTHO1 9 6 <200 þ <200 þTPOX 8, 9 8, 12 <290 þCSF1PO 10, 11 10, 11 <360 þD16S539 12, 13 9, 11 <270 þ <305 þD2S1338 19, 20 16, 17 <350 þPenta E 15, 20 5 <480 þPenta D 9, 13 11, 13 <440 þ

a B: blue; G: green; Y: yellow.

126 P.M. Schneider et al. / Forensic Science International 139 (2004) 123–134

‘‘standard’’ and ‘‘enhanced’’ PCR conditions could not be

based on clear-cut rules. In general, results were assigned to

the ‘‘standard’’ category when 28 cycles were performed,

and to the ‘‘enhanced’’ category for 30 cycles or more.

However, some laboratories either did not use more than 28

cycles, but rather increased the amount of DNA for enhance-

ment, or used 30 cycles already as their standard procedure.

Furthermore, other laboratories provided only one result for

each sample, which had then to be assigned to one of the two

categories for analysis. Thus, the ranges for the number of

Fig. 3. Results of STR typing for the SGM Plus multiplex kit. For each locus, the data are summarized for the three peak height categories:

>150 rfu (black columns), 150–30 rfu (grey columns) and <30 rfu (white columns). The results for all STR loci are arranged in the order of

observed fragment sizes from left (small fragments) to right (large fragments) for the three categories. (a) Results for degraded DNA sample A

using standard PCR conditions, i.e. 0.25–2 ng of DNA and 28–30 PCR cycles according to manufacturer or established operational procedures

(n ¼ 15 laboratories); (b) results for degraded DNA sample A using enhanced PCR conditions, i.e. up to 5 ng of DNA and up to 35 PCR cycles

according to established operational procedures (n ¼ 19 laboratories); (c) results for degraded DNA sample B using standard PCR conditions,

i.e. 0.5–2 ng of DNA and 28 PCR cycles according to manufacturer or established operational procedures (n ¼ 12 laboratories); (d) results for

degraded DNA sample B using enhanced PCR conditions, i.e. up to 10 ng of DNA and up to 35 PCR cycles according to established

operational procedures (n ¼ 17 laboratories).

P.M. Schneider et al. / Forensic Science International 139 (2004) 123–134 127

cycles and the amount of template DNA given in the figure

legends may overlap, and the number of laboratories con-

tributing to the ‘‘standard’’ and ‘‘enhanced’’ category are not

identical.

Using standard conditions for the SGM Plus kit, a sig-

nificant loss of information with 40% of negative results

(<30 rfu) occurred for sample A around 180 bp (the size of

the VWA alleles; Fig. 3a), whereas more than 60% success-

ful amplification (>30 rfu) was achieved for the majority of

laboratories up to approx. 320 bp (FGA/FIBRA system;

Fig. 3b) using enhanced conditions. For the supposedly

more degraded sample B, the 40% failure rate was reached

as well using VWA and FGA for standard and enhanced

conditions, respectively, although the pattern was not so

straightforward (Fig. 3c and d). Some loci such as D8S1179,

THO1 and D21S11 actually performed better with sample B

than with sample A. Also, the proportion of laboratories

reporting successful amplification for all loci from Amelo-

genin to D2S1338 using enhanced conditions was almost

50% for sample B (Fig. 3d), but only 25% for sample A

(Fig. 3b). However, it has to be kept in mind that successful

amplification does not necessarily mean that the typing

Fig. 3. (Continued ).

128 P.M. Schneider et al. / Forensic Science International 139 (2004) 123–134

results were correct for all loci. This aspect will be discussed

in the context of the results from all three multiplexes.

The results for the Profiler Plus kit were supplemented

with data from the ‘‘blue’’ and ‘‘yellow’’ systems from the

Profiler kit as these have an identical locus composition in

both kits. When compared to the SGM Plus results, the

Profiler Plus data exhibited a higher proportion of low

molecular weight alleles with peak heights >150 rfu both

Fig. 4. Results of STR typing for the Profiler/Profiler-Plus multiplex kit. For each locus, the data are summarized for the three peak height

categories: >150 rfu (black columns), 150–30 rfu (grey columns) and <30 rfu (white columns). The results for all STR loci are arranged in the

order of observed fragment sizes from left (small fragments) to right (large fragments) for the three categories. (a) Results for degraded DNA

sample A using standard PCR conditions, i.e. 0.25–1.25 ng of DNA and 28–32 PCR cycles according to manufacturer or established

operational procedures (n ¼ 8 laboratories); (b) results for degraded DNA sample A using enhanced PCR conditions, i.e. up to 2.5 ng of DNA

and up to 34 PCR cycles according to established operational procedures (n ¼ 5 laboratories); (c) results for degraded DNA sample B using

standard PCR conditions, i.e. 0.25–1.25 ng of DNA and 28–32 PCR cycles according to manufacturer or established operational procedures

(n ¼ 6 laboratories); (d) results for degraded DNA sample B using enhanced PCR conditions, i.e. up to 2.5 ng of DNA and up to 34 PCR

cycles according to established operational procedures (n ¼ 5 laboratories).

P.M. Schneider et al. / Forensic Science International 139 (2004) 123–134 129

for samples A and B. A loss of information with >40%

failure rate begins with D21S11 around 220 bp for standard

conditions (Fig. 4a and c), and, using enhanced conditions,

with D7S820 for sample A (approx. 280 bp, Fig. 4b) and

D13S317 for sample B which has a similar size as D21S11

(Fig. 4d). It might be speculated that some of these differ-

ences can be related to the fact that some loci have changed

colors when the SGM Plus kit was designed to accommodate

the additional STR loci (Table 1). In particular, using

standard conditions, FGA performed significantly better

in the Profiler Plus kit carrying a blue dye (30–50% success-

ful amplification) compared to the SGM Plus kit with a

yellow dye (complete failure; Fig. 4a and c). Similar obser-

vations were made in a recent study on the interpretation of

mixtures from forensic casework. It was found that the

detection of mixed profiles was more successful using

blue-labeled STR systems [9]. On the other hand, successful

amplification across all loci (>30 rfu) using enhanced con-

ditions was not better than for the SGM Plus kit, i.e. 25% for

both samples due to the low values for D18S51, and although

the equally large D2S1338 system is present in the SGM

Plus kit.

Fig. 4. (Continued ).

130 P.M. Schneider et al. / Forensic Science International 139 (2004) 123–134

The PowerPlex 16 multiplex comprises the largest num-

ber of loci and covers the widest fragment size range up to

approx. 480 bp for the Penta E system. Therefore, it is no

surprise that the systems with fragment sizes >300 bp per-

formed only poorly, and the overall pattern of successfully

amplified loci showed more variation. Using standard con-

ditions, successful amplification of >60% was achieved for

D16S539 for sample A and D8S1179 for sample B, which

Fig. 5. Results of STR typing for the Powerplex 16 multiplex kit. For each locus, the data are summarized for the three peak height categories:

>150 rfu (black columns), 150–30 rfu (grey columns) and <30 rfu (white columns). The results for all STR loci are arranged in the order of

observed fragment sizes from left (small fragments) to right (large fragments) for the three categories. (a) Results for degraded DNA sample A

using standard PCR conditions, i.e. 0.5–1.25 ng of DNA and 30–32 PCR cycles according to manufacturer or established operational

procedures (n ¼ 8 laboratories); (b) results for degraded DNA sample A using enhanced PCR conditions, i.e. up to 2.5 ng of DNA and up to 36

PCR cycles according to established operational procedures (n ¼ 4 laboratories); (c) results for degraded DNA sample B using standard PCR

conditions, i.e. 0.5–1.25 ng of DNA and 30–32 PCR cycles according to manufacturer or established operational procedures (n ¼ 8

laboratories); (d) results for degraded DNA sample B using enhanced PCR conditions, i.e. up to 2.5 ng of DNA and up to 36 PCR cycles

according to established operational procedures (n ¼ 4 laboratories).

P.M. Schneider et al. / Forensic Science International 139 (2004) 123–134 131

have sizes up to 270 bp (Fig. 5a and c). The size range of

successful amplification was extended using enhanced con-

ditions to CSF1PO for sample A and to D18S51 for sample B

(both approx. 340 bp). Using enhanced conditions, the pro-

portion of systems exhibiting peak heights of >150 rfu was

clearly improved. In general, the performance of the Power-

Plex 16 kit was comparable to those of the ABI kits based on

fragment length assessment, although the locus composition

is quite different regarding STR size ranges and dye colors

(Table 1).

As already mentioned, not all alleles could be identified

correctly for all loci. A number of common problems were

observed. However, due to the heterogeneity of the submitted

data regarding the amount of DNA used, the PCR parameters

and other conditions it was not possible to compile a locus-by-

locus statistical analysis. Therefore, typical problems are

summarized. A strong peak imbalance was found in hetero-

zygous genotypes in particular for the larger STR systems, and

for enhanced conditions applying more than 30 cycles. Arte-

fact signals (‘‘pull-ups’’) occurred due to overamplification

mimicking alleles not present in the sample. Allelic drop-out

(i.e. the complete loss of one allele in a heterozygous geno-

type) was observed more frequently, and sometimes the

smaller of two alleles at a given locus was affected.

Fig. 5. (Continued ).

132 P.M. Schneider et al. / Forensic Science International 139 (2004) 123–134

Some STR loci repeatedly exhibited problems. FGA,

D21S11 and D18S51 were most frequently affected by

allelic dropout in the ABI multiplexes. This was more

prominent using enhanced conditions for loci that did not

amplify at all under standard conditions. A similar pattern

was found for the D21S11, D8S1179 and D16S539 systems

for the PowerPlex 16. The fragment sizes of these loci are

around or slightly above the fragment size threshold of

successful amplification, and stochastic effects most prob-

ably generate the ambiguous and unreliable results. A

special situation was found in the D19S433 system of the

SGM Plus kit. Although this locus generates very small

fragments <140 bp, two laboratories reported the presence

of a 14.2 allele, and another two laboratories a 15 allele for

sample A which was homozygous for the 15.2 allele. The

peak heights of these extra alleles had almost the same

height as the correct allele. Whereas the presence of the 14.2

allele could be explained by an extreme stutter effect, this

cannot be the case for the false 15 allele, as it is separated by

only 2 bp from the true allele.

Most laboratories apply a lower threshold for allele

detection to prevent the inclusion of false alleles or stutter

bands. A total of 60% of the laboratories use a peak height

threshold of 50 rfu (see Table 2). Thus, the use of the

GenoTyper software for automatic data analysis some-

times prevented the detection of a regular allele due to

the threshold settings of peak height detection, although

this threshold also prevented the inclusion of artefact bands

in some cases. As we have included a peak height category

of 30–150 rfu for our analysis, we have of course picked up

more alleles than the majority of participating laboratories

would do using their standard threshold. This was done

deliberately to learn more about the detection limits and

the occurrence of artefacts using the various multiplexes.

In a real casework scenario, some of the wrongly recorded

low level alleles as well as low level correct alleles would

have been left out in favor of a more reliable analysis. Only

two laboratories have reported results using a flexible

threshold for allele calling based on the overall quality

of the results (Table 2).

A number of laboratories have used ‘‘home-made’’ sin-

gleplex STR systems such as THO1, VWA, FGA, D2S1338,

SE33, D8S1179, D18S51, and others. As these results are

quite diverse regarding the loci studied, they could not be

subjected to a comparative analysis. However, all results

submitted were correct. Most laboratories typically use these

singleplex systems for difficult samples such as degraded

DNA. In particular, several STR systems with redesigned

primer sequences to generate short amplicons gave complete

and correct results for both samples [10,11].

Eight laboratories reported their results for mitochon-

drial DNA sequence analysis (Table 3). Of these, four

laboratories failed to obtain results due to poor amplifica-

tion. The other four laboratories reported either complete or

partial results, as the HV-II region was not analyzed by all

participants. The two laboratories with complete results

applied an overlapping PCR amplification strategy with

short fragments of approx. 200 bp. Our control experiments

after preparation of the degraded DNA indicated that such

fragments can be amplified reliably [8]. This has also been

shown in recent studies on the genetic identity of historical

samples [12].

From these results, some conclusions can be drawn:

� To overcome the effects of template DNA degradation, a

PCR protocol optimized for all parameters such as the

Table 2

Allele calling thresholds for STR systems based on peak heights in

relative fluorescence units (rfu)

Threshold (rfu) No. of labs

n %

30 4 10.5

50 23 60.5

60–100 3 7.9

>100 3 7.9

Flexible 2 5.3

n.d. 3 7.9

Total 38 100

n.d.: not defined.

Table 3

Results of HV-I and HV-II mitochondrial DNA sequence analysis

Sample HV-I Resultsa

16126 16182 16183 16189 16217 16294 16295 16304 Pos. Neg. n.t.

A – C C C C – T – 4 4

B C – – – – T – C 3 4 1

HV-II

73 146 152 263 309.1 315.1

A G C – G – C 2 5 1

B G – C G C C 2 4 2

a Pos.: correct result; Neg.: no result; n.t.: not tested.

P.M. Schneider et al. / Forensic Science International 139 (2004) 123–134 133

amount of template DNA and Taq polymerase concentra-

tion relative to the number of PCR cycles has to be

established.

� Flexible guidelines for the interpretation of the observed

DNA profiles have to be applied. The guidelines should

include strategies to reanalyze the sample if evidence for

low-level peak signals is obtained. To allow for this, a

careful human evaluation of all results in addition to a

computer-based analysis (e.g. using the GenoTyper soft-

ware) is recommended.

� For mtDNA analysis of heavily degraded DNA, PCR

primers covering short overlapping DNA regions (as

described e.g. in [13]) should be applied.

Some of the characteristics of STR typing results from

degraded DNA samples from this study have also been

observed under ‘‘low copy number’’ (LCN) conditions,

where stochastic effects strongly influence the outcome of

PCR amplification. Thus, some of the guidelines developed

for interpretation of LCN STR profiles [14] may also be

applicable when highly degraded DNA has to be analyzed.

The use of standardized degraded control DNA is helpful

to understand and optimize the parameters affecting the

success of STR typing. The availability of such a degraded

control DNA in sufficient amounts would enable the forensic

laboratories to simulate a situation of heavily decomposed

DNA, and thus allow studies to enhance the efficiency of

their typing methods.

Acknowledgements

The present study was supported by contract SMT 97-

7506 from the European Commission in the context of the

‘‘Standardization of DNA Profiling Methods in the European

Union’’ (STADNAP) network initiative.

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