Forensic Science International86 (1997) 25–33

Report of the European DNA profiling group(EDNAP): an investigation of the complex STR loci

D21S11 and HUMFIBRA (FGA)

a , b c d e*Peter Gill , E. d’Aloja , J. Andersen , B. Dupuy , M. Jangblad ,f g h iV. Johnsson , A.D. Kloosterman , A. Kratzer , M.V. Lareu ,

j k l m nM. Meldegaard , C. Phillips , H. Pfitzinger , S. Rand , M. Sabatier ,o p q rR. Scheithauer , H. Schmitter , P. Schneider , M.C. Vide

aService Development, Forensic Science Service, Birmingham, UKbCatholic University, Rome, Italy

cForensic Science Service, London, UKdRettsmedisinsk Institut, Ricshospitalet 0027, Oslo, Norway

eSKL — National Laboratory of Forensic Science, Stockholm, SwedenfNational Bureau of Investigation, Helsinki, Finland

gNetherlands Forensic Science Instiiute, Volmerrlanan 17, Rijswijk, The NetherlandshInstitute of Legal Medicine, University of Zurich, Zurich, Switzerland

iInstitute of Legal Medicine, Faculty of Medicine, University of Santiago de Compostela, Santiago deCompostela, Spain

jDepartment of Forensic Genetics, Institute of Forensic Medicine, University of Copenhagen, Copenhagen,Denmark

kDepartment of Haemotology, London Hospital Medical College, London, UKlCodgene, University of Strasbourg, Strasbourg, France

mInstitute of Legal Medicine, University of Munster, Munster, GermanynLaboratorie de Police Scientifique, Toulouse, France

oInstitute of Forensic Medicine, University of Innsbruck, Innsbruck, AustriapBundeskriminalamt, Wiesbaden, Germany

qInstitute of Legal Medicine, University of Mainz, Mainz, GermanyrInstituto de Medicina Legal, Coimbra, Portugal

Received 28 November 1996; accepted 6 January 1997

*Corresponding author. Tel.: 144 121 6076871; fax: 144 121 6225889.

0379-0738/97/$17.00 1997 Elsevier Science Ireland Ltd. All rights reservedPII S0379-0738( 97 )02108-7

26 P. Gill et al. / Forensic Science International 86 (1997) 25 –33

Abstract

This paper describes a collaborative exercise which was intended to demonstrate whetheruniformity of DNA profiling results could be achieved between European laboratories using twocomplex short tandem repeat (STR) loci. The loci D21S11 and HUMFIBRA (FGA) were chosenbecause they are commonly used by different European laboratories. D21S11 has approximately14 common alleles ( f .0.001), whereas HUMFIBRA has 19 common alleles. Laboratories wereasked to test seven blood stains, one of which was a known control, and to report the results to thecoordinating laboratory. The exercise demonstrated that complex STRs were amenable tostandardisation. Copyright 1997 Elsevier Science Ireland Ltd

Keywords: DNA profiling; Uniformity; Short tandem repeat (STR) loci; D21S11 and HUMFIBRA(FGA); European laboratories; Standardisation

1. Introduction

As part of a collaborative research programme dedicated to standardising DNAprofiling techniques throughout Europe, the European DNA profiling Group (EDNAP) isundertaking a programme to test the reproducibility of identifying short tandem repeat(STR) alleles between different laboratories.

Previous exercises demonstrated the reproducibility of simple tetrameric STR systemssuch as HUMTH01 and HUMVWFA31 [1,4,6] across a wide range of differentelectrophoretic systems. Subsequently, these two STRs have been adopted as Europeanstandards. However, the utility of simple STRs is restricted by their relatively lowdiscriminating powers. Complex STRs (defined by Urquhart [12]) have the advantage ofgreater DPs but have the disadvantage that the resolution of the electrophoretic systemmust distinguish alleles which may differ by only one or two bases. The purpose of thisexercise was to determine if it was possible to standardise the results of complex STRloci HUMFIBRA [2,7] and D21S11 [8,11,12] across laboratories which use a range oftechniques which include automated sequencer to silver staining systems.

2. Materials and methods

The format of the exercise followed that previously described by Gill et al. [4].A total of seven bloodstains, including a control sample, were distributed to

participants. Allelic ladders, described by Gill et al. [5] for D21S11 and HUMFIBRAwere supplied. Laboratories were requested to use their own primers and protocols(Table 1). Primers used by the coordinating laboratory are described by [10] unlessotherwise indicated in Table 1. In addition three laboratories made comparisons using aD21S11 ladder donated from Professor Brinkmann.

Thirteen laboratories used ABD 373 or 377 automated sequencers; two used thePharmacia ALF sequencer and one laboratory used silver staining. All laboratories useddenaturing 4–6.5% acrylamide gels. Gel run/ read distances were between 12 and 36 cm.

P. Gill et al. / Forensic Science International 86 (1997) 25 –33 27

Annealing temperatures ranged between 55 and 638C with 26–35 cycles of amplifica-tion. All laboratories except lab 16 (Table 1) used the local Elder and Southern methodto determine the sizes of STR fragments.

In this exercise, a total of 16 laboratories participated. The aim was to establishwhether it was possible to reproducibly analyse complex repeat STRs between differentlaboratories employing a variety of different techniques ranging from automatedsequencers to silver staining.

3. Results and discussion

Some laboratories had little or no previous experience of typing HUMFIBRA andD21S11 loci, hence this exercise must be viewed in the context of the EDNAPexperimental programme. It was not a proficiency test and has no relevance to separateand independent quality assurance programmes carried out by the participating lab-oratories.

3.1. Importance of dye-labelling homologous strands of ladders and primers

Some laboratories had problems comparing samples with the allelic ladders supplied.There were two reasons for this:

1. Taking HUMFIBRA as an example, allelic ladders were supplied with the CTTTstrand labelled with HEX dye [10]. One laboratory used the ladder supplied, butamplified samples using a primer labelled on the complementary strand such that theAAAG strand was labelled. Because complementary strands migrate at differentrates, the resulting sample fragments were shifted in relation to the ladder by morethan 2 bases.

2. If allelic ladder is supplied from an external source, then this can be used to createindefinite in-house supplies, but it is important to ensure that the primers used areeither the same as those of the original manufacturer, or if different primers are used,then they must not lie outside the supplied DNA fragment, otherwise amplificationwill be unsuccessful.

3.2. The D21S11 and HUMFIBRA analysis

Results are compiled in Table 2 and Table 3.Mis-typing occurred in the following samples: D21S11 — Laboratory 13, samples 4

and 7 attributed to sample leakage from adjacent lanes. HUMFIBRA — Laboratory 1,sample 2 mistyping attributed to contamination of the extract. Laboratory 11, samples 3and 7. In both of these samples alleles are separated by just 2 bp. The resolution of thesystem used was insufficient to distinguish the two peaks separately, hence both sampleswere scored as homozygous. Laboratory 13, samples 4 and 6 were mis-typed because ofthe poor resolution of the internal size standard.

28 P. Gill et al. / Forensic Science International 86 (1997) 25 –33T

able

1D

etai

lsof

diff

eren

tla

bora

tory

met

hodo

logi

es

Lab

orat

ory

code

12

34

56

78

Met

hod

373A

AB

D37

3AA

BD

377

AB

D37

3AA

BD

Phar

mac

iaA

LF

373A

AB

D37

3AA

BD

373A

AB

DIn

tern

alsi

zest

anda

rd35

0R

OX

2500

Rox

2500

RO

X35

0R

OX

AL

FSi

zer

2500

Rox

350

Tam

ra35

0T

AM

RA

500

Tam

raR

ead

dist

ance

24cm

12cm

;24

cm24

cm24

cm24

cm24

cm24

cm24

cm%

Acr

ylam

ide

6%de

nat

6%de

nat

6%de

nat

6%de

nat

6%de

nat

6%de

nat

6%de

nat

6%de

nat

Am

plifi

catio

nco

nditi

ons

Den

atur

atio

n94

8C;4

5s

948C

;45

s94

8C;

20s

948C

;60

s94

8C;

45s

948C

;45

s94

8C;

60s

948C

;45

sA

nnea

ling

608C

;30

s60

C;

60se

c58

8C;

45s

588C

;60

s60

8C;

60s

558C

;30

s54

8C;

60s

548C

;60

s64

8C;

30s

608C

;60

sE

xten

sion

728C

;40

s72

8C;

60s

728C

;60

s72

8C;1

20s

728C

;60

s72

8C;

30s

728C

;90

s72

8C;

60s

728C

;30

s72

8C;

60s

No

cycl

es35

3028

3030

3030

26Po

wer

25W

30W

26W

30W

45W

36W

30W

30W

Tim

eof

run

14h

6an

d8

h8

h8

h3

h7

h6

h3

hM

odel

ofth

erm

ocyc

ler

480P

E48

0PE

9600

PET

C1

PEM

JRB

iom

etra

9600

PEM

JRPT

C10

0Si

zing

met

hod

Loc

alSo

uthe

rnL

ocal

Loc

alL

ocal

Loc

alL

ocal

Loc

alL

ocal

Sout

hern

Sout

hern

Sout

hern

Sout

hern

Sout

hern

Sout

hern

Sout

hern

Prim

ers

(if

diff

eren

tfr

omR

ef.

[10]

)D

21S1

159

–GT

GA

GT

CA

AT

TC

CC

CA

AG

D21

S11

59–G

TG

AG

TC

AA

TT

CC

CC

(lab

3)A

AG

(lab

6)D

21S1

159

–GT

TG

TA

TT

AG

TC

AA

TG

TT

CT

CC

(lab

3)D

21S1

159

–GT

TG

TA

TT

AG

TC

AA

TG

TT

CT

CC

(lab

6)

P. Gill et al. / Forensic Science International 86 (1997) 25 –33 29L

abor

ator

yco

de9

1011

1213

1415

16

Met

hod

377A

BD

377A

BD

Phar

mac

iaA

LF

373A

AB

D37

3AA

BD

377A

BD

373A

AB

DV

ertic

alel

ectr

opho

resi

sSA

-32

cham

ber

Inte

rnal

size

stan

dard

350

Rox

350

Tam

raN

one

350

Rox

2500

TA

MR

A35

0T

amra

2500

RO

XSi

lver

stai

ning

Rea

ddi

stan

ce36

cm36

cm20

cm24

cm12

cm36

cm27

.5cm

32cm

%A

cryl

amid

e4%

dena

t4%

dena

t6.

5%de

nat

6%de

nat

6%de

nat

4%de

nat

6%de

nat

4%de

nat

Am

plifi

catio

nco

nditi

ons

Den

atur

atio

n94

8C;

60s

908C

;2

min

948C

;30

s94

8C;

60s

938C

;30

s90

8C;2

min

958C

;30

s94

8C;

30s

948C

;15

s94

8C;

60s

Ann

ealin

g60

8C;

60s

588C

;75

s55

8C;

60s

588C

;60

s58

8C;

60s

588C

;75

s60

8C;

60s

558C

;30

s57

8C;

25s

608C

;60

s60

8C;

60s

638C

;60

sE

xten

sion

728C

;60

s72

8C;

15s

728C

;60

s72

8C;1

20s

728C

;15

s72

8C;

15s

728C

;60

s72

8C;

90s

728C

;50

s72

8C;

60s

No

cycl

es28

3027

3028

3026

30Po

wer

200

mW

200

mW

27W

30W

30W

200

mW

40W

30W

Tim

eof

run

2h

2h

4h

83

h2

h8

h2–

3h

Mod

elof

ther

moc

ycle

r96

00PE

9600

PE96

00PE

TC

1PE

9600

PE96

00PE

9600

PET

C-1

PE24

00P

ESi

zing

met

hod

Loc

alL

ocal

Loc

alL

ocal

Loc

alL

ocal

Loc

alN

one

Sout

hern

Sout

hern

Sout

hern

Sout

hern

Sout

hern

Sout

hern

Sout

hern

(com

pare

with

ladd

er)

Add

ition

alco

mm

ents

FA

Mdy

eSi

lver

used

for

stai

ning

both

loci

used

IfH

UM

FIB

RA

and

D21

S11

met

hods

diff

erfr

omea

chot

her

then

the

latte

ris

give

nin

bold

.

30 P. Gill et al. / Forensic Science International 86 (1997) 25 –33

Tab

le2

HU

MFI

BR

Are

sults

Sam

ple

Lab

s2–

10;1

2;14

–16

Lab

s2–

10;1

2;14

–16

Lab

1L

ab1

Lab

11L

ab11

Lab

13L

ab13

Mol

ecul

arw

t.of

alle

leL

owH

igh

Low

Hig

hL

owH

igh

Low

Hig

h1

2125

2125

2125

2125

223

2422

2623

243

3

322

22.2

2222

.222

2222

22.2

423

2323

2323

2321

215

1823

1823

1823

1823

619

2219

2219

2217

19.2

723

.224

23.2

2423

233

3

The

corr

ect

resu

ltsar

egi

ven

inth

efir

sttw

oco

lum

ns.D

iscr

epan

cies

obse

rved

from

two

labo

rato

ries

are

high

light

edin

bold

type

.‘3

’in

dica

tes

nore

sult

was

obta

ined

.T

heno

men

clat

ure

desc

ribe

din

Ref

.[1

2]is

used

.T

his

can

beco

nver

ted

into

the

nom

encl

atur

eof

Ref

.[8

]by

appl

ying

the

form

ula

M5

(U/2

)-5

asde

scri

bed

inth

ete

xt.

P. Gill et al. / Forensic Science International 86 (1997) 25 –33 31

Table 3D21S11 results

Sample Molecular weight of allele

Low High Low High

1 59 63 59 632 63 67 3 3

3 63 65 63 654 61 65 61 635 61 70 61 706 61 63 61 637 59 65 61 63

The correct results (obtained from all but one laboratory) are given in the first two columns. Discrepant resultsfrom laboratory 13 are given in the second two columns. Nomenclature from Ref. [12] is used.

3.3. D21S11 allelic ladders

A comparison was made of two allelic ladders which were independently constructedand sequenced described by Gill et al. [5]; the second ladder was supplied by ProfessorBrinkmann. Two different nomenclatures have been described to accompany each allelicladder [8,12]. The former is based upon the number of dimeric repeats, whereas thelatter is based upon tetramers. The Moller notation [9] is recommended for general usesince it is close to the original ISFH [3].

To assess whether the two allelic ladders were comparable, they were co-electrophor-esed (Fig. 1). Taking allele 59 (Urquhart designation) as an example, this corresponds toMoller’s allele 27; the size of the allele can be calculated as 59325118 bases and27345108 bases respectively, i.e. the Moller notation gives rise to a fragment which is

Fig. 1. A comparison of co-electrophoresed D21S11 ladders and their respective nomenclatures. The higherpeaks correspond to the allelic ladder described by [5] and nomenclature from [12]; the lower peaks utilise theladder and nomenclature of [8].

32 P. Gill et al. / Forensic Science International 86 (1997) 25 –33

10 bases (2.5 tetramers) smaller. This is because the starting point of the Moller D21S11sequence designation is 10 bp closer to the 39 end of the DNA fragment. A simpleformula can be used to convert the Moller (M) designation into the Urquhart (U)equivalent:

(U 2 5)]]]M 5 2

To summarise, the impetus of the EDNAP collaborative experimentation, has focusedupon the scope of standardisation of STR loci. To succeed, it is important that thesystem works under diverse experimental conditions. Although the majority of lab-oratories use ABD 373 and 377 or Pharmacia ALF automated sequencers, one laboratoryused silver staining. Nevertheless, this exercise demonstrated comparability withoutspecifying any of the experimental parameters (including the primers used). Thisprovides a firm foundation for different laboratories to compare the complex lociHUMFIBRA and D21S11. The key to standardisation is use of a common laddermarker. Even here there is flexibility. It is possible for laboratories to use differentladders provided that they are properly compared (preferably by co-electrophoresis or bysequencing) as demonstrated by D21S11. Different nomenclatures can also be convertedusing simple formulae, although in principle this should be avoided by adopting acommon nomenclature for each STR system.

References

[1] J. Andersen, P. Martin, E. D’Aloja, A. Carracedo, B. Eriksen, V. Johnsson, C. Kimpton, A. Kloosterman,C. Konialis, A. Kratzer, P. Lincoln, B. Mevag, H. Pfitzinger, S. Rand, B. Rosen, H. Schmitter, P.Schneider, M.Vide. Report on the third EDNAP collaborative STR exercise. Forensic Sci. Int., 78 (1996)83–93.

[2] M.D. Barber, B.J. McKeown and B.H. Parkin, Structural variation in the alleles of a short tandem repeatsystem at the human alpha fibrinogen locus. Int. J. Leg Med., 108 (1996) 180–185.

[3] DNA Commission of the International Society for Forensic Haemogenetics, DNA recommendations —1994 report concerning further recommendations of the DNA commission of the ISFH regardingPCR-based polymorphisms in STR (short tandem repeat) systems. Int. J. Leg. Med., 107 (1994)159–160.

[4] P. Gill, C. Kimpton, E. D’Aloja, J.F. Andersen, W. Bar, B. Brinkmann, S. Holgersen, V. Johnsson, A.D.Kloosterman, M.V. Lareu, L. Nelleman, H. Pfitzinger, C.P. Phillips, H. Schmitter, P.M. Schneider and M.Stenersen, Report of the European DNA profiling group (EDNAP) — towards standardisation of shorttandem repeat (STR) loci. Forensic Sci. Int., 65 (1994) 51–59.

[5] P. Gill, A. Urquhart, E. Millican, N. Oldroyd, S. Watson, R. Sparkes, C.P. Kimpton, A new method ofSTR interpretation using inferential logic — development of a criminal intelligence database. Int. J. Leg.Med., 109 (1996) 14–22.

[6] C. Kimpton, P. Gill, E. D’Aloja, J.F. Andersen, W. Bar, S. Holgersson, S. Jacobsen, V. Johnsson, A.D.Kloosterman, M.V. Lareu, L. Nelleman, H. Pfitzinger, C.P. Phillips, S. Rand, H. Schmitter, P.M.Schneider, M. Stenersen and M.C. Vide, Report on the second EDNAP collaborative STR exercise.Forensic Sci. Int., 71 (1995) 137–152.

[7] K.A. Mills, D. Even and J.C. Murray, Tetranucleotide repeat polymorphism at the human alphafibrinogen locus (FGA). Hum. Mol. Genet., 1 (1992) 779.

[8] A. Moller, E. Meyer and B. Brinkmann (1994) Different types of structural variation in STRs:HumFES/FPS, HumVWA and HumD21S11. Int. J. Leg. Med., 106 (1994) 319–323.

P. Gill et al. / Forensic Science International 86 (1997) 25 –33 33

[9] A. Moller, M. Schurenkamp and B. Brinkmann, Evaluation of an ACTBP2 ladder composed of 26sequenced alleles. Int. J. Leg. Med., 108 (1995) 75–78.

[10] N.J. Oldroyd, A.J. Urquhart, C.P. Kimpton, E.S. Millican, S.K. Watson, T. Downes, and P.D. Gill, Ahighly discriminating octoplex short tandem repeat PCR system suitable for human individual identifica-tion. Electrophoresis 16 (1995) 334–337.

[11] V. Sharmer, and M. Litt, Tetranucleotide repeat polymorphism at the D21S11 locus. Hum. Mol. Genet. 1(1992) 67.

[12] A.J. Urquhart, C.P. Kimpton, T.J. Downes and P. Gill, Variation in short tandem repeat sequences — asurvey of twelve microsatellite loci for use as forensic indentification markers. Int. J. Leg. Med., 107(1994) 13–20.