Multiplex PCR and minisequencing of SNPs—

a model with 35 Y chromosome SNPs

Juan J. Sanchez

a,*

, Claus Børsting

a

, Charlotte Hallenberg

a

, Anders Buchard

a

,

Alexis Hernandez

b

, Niels Morling

a

a

Department of Forensic Genetics, Institute of Forensic Medicine, University of Copenhagen, 11 Frederik V’s Vej,

DK-2100 Copenhagen, Denmark

b

Departamento de Canarias, Instituto Nacional de Toxicologı´a, Campus de Ciencias de la Salud, 38320 La Laguna, Tenerife, Spain

Received 22 January 2003; received in revised form 2 July 2003; accepted 7 July 2003

Abstract

We have developed a robust single nucleotide polymorphism (SNPs) typing assay with co-amplification of 25 DNA-fragments

and the detection of 35 human Y chromosome SNPs. The sizes of the PCR products ranged from 79 to 186 base pairs. PCR
primers were designed to have a theoretical T

m

of 60

5 8C at a salt concentration of 180 mM. The sizes of the primers ranged

from 19 to 34 nucleotides. The concentration of amplification primers was adjusted to obtain balanced amounts of PCR products
in 8 mM MgCl

2

. For routine purposes, 1 ng of genomic DNA was amplified and the lower limit was approximately 100 pg DNA.

The minisequencing reactions were performed simultaneously for all 35 SNPs with fluorescently labelled dideoxynucleotides.
The size of the minisequencing primers ranged from 19 to 106 nucleotides. The minisequencing reactions were analysed by
capillary electrophoresis and multicolour fluorescence detection. Female DNA did not influence the results of Y chromosome
SNP typing when added in concentrations more than 300 times the concentrations of male DNA. The frequencies of the 35 SNPs
were determined in 194 male Danes. The gene diversity of the SNPs ranged from 0.01 to 0.5.
# 2003 Elsevier Ireland Ltd. All rights reserved.

Keywords: Y chromosome; Single nucleotide polymorphism; Multiplex PCR; Minisequencing; Genotyping

1. Introduction

A large number of single nucleotide polymorphisms

(SNPs) have been identified

[1]

. Investigations of SNPs

on the Y chromosome in various populations have given
us important information on the history of the human male
populations (e.g.

[2–8]

). Due to the low mutation rates of

SNPs, the information relates to longer periods of time
compared to the information obtained with e.g. short tandem
repeat (STR)

[9–11]

and minisatellite markers as, for exam-

ple MSY1

[12,13]

.

Presently, typing of selected short tandem repeat (STR)

systems is the state of the art in forensic routine casework. It
is, however, anticipated that SNP typing will be used for

parentage testing and forensic casework in the future. The
advantage of SNPs in forensic casework is that small DNA
fragments of 40–50 bps from e.g. heavily degraded DNA can
be SNP typed. Furthermore, the SNP technology has a high
potential for automation. Although the genetic information
obtained by a SNP, in average, is much lower than that
obtained by an STR system, typing of 50–100 selected SNPs
would be sufficient for forensic casework

[14]

. The low

mutation rate of SNPs

[15,16]

makes these markers an

attractive tool for parentage testing.

Genetic markers on the Y chromosome are valuable tools

in forensic casework in special situations, e.g. in cases with
mixtures of DNA with a dominant amount of female DNA
and a very small amount of male DNA. In such cases, the
DNA profile of the autosomes of the male cannot be
obtained, but the Y chromosome markers can usually be
typed, even in situations with a very large relative amount of
female DNA

[17]

. In special cases of parentage testing, e.g.

Forensic Science International 137 (2003) 74–84

*

Corresponding author. Tel.:

þ45-35-32-62-25;

fax:

þ45-35-32-61-20.

E-mail address: [e-mail] (J.J. Sanchez).

0379-0738/$ – see front matter # 2003 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/S0379-0738(03)00299-8

if the alleged father is unavailable for testing while close
male relatives are available, investigation of genetic markers
on the Y chromosome are valuable.

If SNP typing is going to be used in forensic casework, it

is essential that the investigations can be performed on small
amounts of DNA, if possible, <1 ng DNA. If the polymerase
chain reaction (PCR) is used, the amplifications of all DNA
fragments to be investigated must be done in one or very few
amplification reactions.

We decided to explore a SNP typing method that is based on

multiplex PCR and multiplex minisequencing. We chose SNP
markers on the Y chromosome because these markers, in
forensic genetics, offer additional information to the informa-
tion obtained by STR typing. Furthermore, the Y chromosome
SNPs are useful tools for the study of genetics of populations.

In the last years, a number of multiplex PCR Y chromo-

some SNP analyses have been reported. Most of them
included a limited amount of SNPs (often 3–10 SNPs) in
each PCR (e.g.

[2,3,18]

) although larger multiplexes have

been reported

[19,20]

.

We selected Y chromosome SNPs that were reported to be

polymorphic in European and other populations

[4,21,22]

.

However, the main purpose of the study was to explore the
technical issues related to multiplexing a larger number of
DNA fragments and simultaneous detection of a large
number of SNPs. The intention was not to make a final
panel for typing of major Y chromosome haplogroups. In
order to assess the technical performance of the SNP typing
system, we included four pairs of SNPs each of which pair
was expected to give concordant results (e.g. M40 and M96).

Here, we describe a method for typing 35 SNPs on the Y

chromosome. The typing was performed by (1) multiplex
PCR amplification of 25 Y chromosome DNA fragments, (2)
multiplex primer extension reactions of 35 SNPs with
fluorescence labelled nucleotides, and (3) detection of the
35 SNPs by capillary electrophoresis and multicolour fluor-
escence detection.

2. Materials and methods

2.1. Donors and DNA preparations

A total of 194 unrelated males and 15 unrelated female

Danes donated blood samples or buccal cells. DNA was
isolated from 200 ml of peripheral blood using QIAamp
DNA Blood Mini Kit according to the manufacturer’s pro-
tocol (Qiagen, Hagen, Germany). Alternatively, 1.2 mm
(diameter) FTA

1

paper (Whatman International, Cam-

bridge, UK) soaked with blood or buccal cells was used.

Mixtures of DNA from males and females were prepared in

checker board with three concentrations of male DNA (0.16,
0.8 and 1.6 ng) and female DNA ranging from 0 to 60 ng.

Fluorometric measurement of DNA concentration was

done by SYBR Green I and analysed in a LightCycler
instrument (Roche Diagnostics GmbH, Germany) and

Hoechst 33258 (Molecular Probes Inc., Eugene, OR) using
a Hoefer DyNA Quant 200 instrument (Molecular Vision).

Calibration reference curves were established using a calf

thymus DNA standard (Sigma–Aldrich, Missouri, USA).

2.2. Selection of PCR amplification primers

The Y chromosome SNPs selected (

Table 1

) included

those used by Semino et al.

[21]

for a study of the distribu-

tion of Y chromosome SNPs in European populations. In
addition, we included SNPs that were reported to be poly-
morphic in other ethnic groups.

DNA segments including the SNPs selected were identi-

fied and complementary primers were designed so that the
lengths of the amplified genomic Y chromosome DNA
fragments would range from 79 to 186 nucleotides. Some
SNPs were situated very closely to each other and it was
decided to include a number of amplification targets with
two or three SNPs (

Table 1

).

The sequence of each locus was obtained from GenBank

1

(

http://www.ncbi.nlm.nih.gov

) using a nucleotide basic local

alignment search tool (BLAST). Published PCR primers
were initially used as the reference sequence for each Y
SNP locus, but all of them needed to be redesigned.
The primers for the genomic segments spanning one or more
Y chromosome markers were designed with the Primer
3.0 program v. 0.2 (

http://www-genome.wi.mit.edu/cgi-bin/

primer/primer3_www.cgi

). All primers were selected to have

theoretical melting temperatures of 60

5 8C at a salt con-

centration of 180 mM and a purine:pyrimidine content close
to 1:1, when possible. The lengths of the primers ranged
between 19 and 34 nt. Primers with four or more bases at the
3

0

end complementary to another part of the primer were

discarded or redesigned to avoid artefacts due to hairpin
formation. Each primer pair was tested for primer–primer
interactions, and the primer sequences were checked to avoid
similarities with repetitive sequences or with other loci in the
genome. The primers were checked for homology to other
amplicons in the pool of 25 primer pairs.

Table 1

shows the

sequences of the amplification primers selected.

2.3. PCR conditions

HPLC purified primers for amplification were purchased

from TAG A/S (Copenhagen, Denmark). A primer stock
solution was prepared by dissolving the lyophilized primers
in Tris/EDTA buffer (10 mM Tris, 100 mM EDTA, pH 7.5;
Sigma–Aldrich) to a final DNA concentration of 100 pmol/ml.

Each primer pair was tested in singleplex PCR. Ten ng

template was amplified by PCR in a 25 ml reaction volume
containing 1

PCR buffer, 1.5 mM MgCl

2

, 200 mM of each

dNTP, 0.4 mM of each primer, and 0.6 units of AmpliTaq
Gold DNA polymerase at 94 8C for 5 min followed by 30
cycles of 30 s at 95 8C, 30 s at 60 8C, 30 s at 72 8C, and a
final extension for 5 min at 72 8C. The products were
analysed by electrophoresis in 11% polyacrylamide gels.

J.J. Sanchez et al. / Forensic Science International 137 (2003) 74–84

75

TBE (1

) (89 mmol/l Tris base, 89 mmol/l boric acid,

2 mmol/l EDTA, pH 8.3) was used as electrophoresis buffer.
The gels were stained with 0.5 mg/ml ethidium bromide. The
10 bp ladder from invitrogen (Groningen, The Netherlands)
was used to assign the sizes of the fragments.

The final setup of the PCR amplification included 1 ng

DNA in a 50 ml reaction volume containing 1

PCR buffer,

8 mM MgCl

2

, 400 mM of each dNTP, 0.01–0.42 mM of each

primer, and 2.5 units of AmpliTaq Gold DNA polymerase
(AB, Foster City, CA).

All DNA amplifications were performed in a GeneAmp

9600 thermal cycler (Perkin-Elmer, Wellesley, USA) using
the following programme: denaturation at 94 8C for 5 min
followed by 33 cycles for 30 s at 95 8C, 30 s at 60 8C, and 30 s
at 65 8C, followed by a final extension for 7 min at 65 8C.

The concentrations of the primers in the multiplex reac-

tion were adjusted in order to obtain equal amount of each
PCR product. The primer concentrations ranged from 0.01 to
0.42 mM (

Table 1

).

The PCR products were analysed on 11% polyacrylamide

gels as described later (

Fig. 1

).

In order to eliminate the excess of primers and dNTPs, the

PCR products was purified on a MinElute PCR purification
spin column (Qiagen, Hagen, Germany) following the man-
ufacturer’s protocol. The DNA was eluted in 30 ml of Milli-Q
water.

E. coli exonuclease I (Exo I) and shrimp alkaline phos-

phatase (SAP) was also used to remove primers and unin-
corporated dNTPs (USB Corporation, Cleveland, USA). Six
microliters ExoSAP-IT kit or 5 units of SAP and 2 units of

Table 1
Y chromosome SNPs and primer sequences for PCR amplification of 25 Y chromosome DNA fragments with SNPs

Locus

GenBank or
dbSNPs accesion

Mutation

PCR primers (5

0

! 3

0

)

mM

Amplicon
size (bp)

Forward primer

Reverse primer

M2/sY81

Rs3893

A/G

acggaaggagttctaaaattcagg

aaaatacagctccccctttatcct

0.15

128

M9

a

Rs3900

C/G

aggaccctgaaatacagaactg

aaatatttcaacatttcacaaaggaa

0.36

186

M17

a

Rs3908

4G/3G

cctggtcataacactggaaatc

agctgaccacaaactgatgtaga

0.09

170

M18

a

Rs3909

2 bp insertion

cctggtcataacactggaaatc

agctgaccacaaactgatgtaga

0.09

170

M19

a

Rs3010

T/A

cctggtcataacactggaaatc

agctgaccacaaactgatgtaga

0.09

170

M32

a

AC009977

T/C

tgaccgtcataggctgagaca

ttgaagcccccaagagagac

0.07

160

M33

a

AC009977

A/C

tgaccgtcataggctgagaca

ttgaagcccccaagagagac

0.07

160

M35

Rs1179188

G/C

agggcatggtccctttctat

tccatgcagactttcggagt

0.42

96

M40/SRY

4064

AC006040

G/A

tggtctcaatctcttcaccctgt

catttcagtaaatgccacacaaga

0.18

119

M45

a

Rs2032631

G/A

gagagaggatatcaaaaattggcagt

tgacagtggcaccaaaggtc

0.03

138

M46/Tat

AC002531

T/C

tatatggactctgagtgtagacttgtga

ggtgccgtaaaagtgtgaaataatc

0.46

115

M52

AC009977

A/C

cctcaacttcccagagtgttg

gacgaagcaaacatttcaagagag

0.03

152

M78

a

AC010889

C/T

tgcattactccgtatgttcgac

tggaagcttaccatctttttatga

0.08

132

M81

a

Rs2032640

C/T

catctcttaacaaaagaggtaaattttgtcc

cattgtgttacatggcctataatattcagt

0.24

179

M89

Rs2032652

C/T

tggattcagctctcttcctaaggttat

ctgctcaggtacacacagagtatca

0.03

135

M96

AC010889

G/C

tgccctctcacagagcactt

ccacccactttgttgctttg

0.27

143

M123

AC010889

G/A

gttgcccaggaatttgcat

cacagagcaagtgactctcaaag

0.02

88

M139

a

AC010137

5G/4G

ccccgaaagttttattttattcca

ttctcagacaccaatggtcctatc

0.06

113

M151

a

AC010889

G/A

catctcttaacaaaagaggtaaattttgtcc

cattgtgttacatggcctataatattcagt

0.24

179

M153

a

AC010137

T/A

ccccgaaagttttattttattcca

ttctcagacaccaatggtcctatc

0.06

113

M154

a

AC010889

T/C

catctcttaacaaaagaggtaaattttgtcc

cattgtgttacatggcctataatattcagt

0.24

179

M157

a

AC010889

A/C

gagagaggatatcaaaaattggcagt

tgacagtggcaccaaaggtc

0.03

138

M163

a

AC009977

A/C

aggaccctgaaatacagaactg

aaatatttcaacatttcacaaaggaa

0.36

186

M167/SRY

2627

AC006040

C/T

cggaaccactaccagcttca

agttaaggccccacgcagt

0.03

113

M170

Rs2032597

A/C

cagctcttattaagttatgttttcatattctgtg gtcctcattttacagtgagacacaac

0.07

119

M172

Rs2032604

T/G

tgagccctctccatcagaag

gccaggtacagagaaagtttgg

0.16

179

M173

Rs2032624

A/C

ttttcttacaattcaagggcatttag

ctgaaaacaaaacactggcttatca

0.10

81

M175

Rs2032678

5 bp

gatttaaactctctgaatcaggcacat

ttctactgatacctttgtttctgttcattc

0.02

79

M212

a

Rs2032664

C/A

ccatataaaaacgcagcattctgtt

tggagagaacttgagaaaaagtagagaa

0.12

176

M213

a

Rs2032665

T/C

ccatataaaaacgcagcattctgtt

tggagagaacttgagaaaaagtagagaa

0.12

176

M224

a

AC010889

T/C

tgcattactccgtatgttcgac

tggaagcttaccatctttttatga

0.08

132

SRY

10831

/SRY

1532

Rs2534636

A/G

tcatccagtccttagcaaccatta

ccacataggtgaaccttgaaaatg

0.06

150

12f2

AC005820

Present/absent

cactgactgatcaaaatgcttacagat

ggatcccttccttacaccttataca

0.06

90

92R7

Rs2535813

GA/A

ttaaatccctcctatttgtgctaacc

aatgcatgaacacaaaagacgtaga

0.04

89

P25

Rs150173

C/CA

tggaccatcacctgggtaaagt

ggcagtataaggttgtcacatcacat

0.01

109

a

SNP markers on the same DNA fragment: (M9 and M163), (M17, M18 and M19), (M32 and M33), (M45 and M157), (M78 and M224),

(M81, M151 and M154), (M139 and M153), (M212 and M213). All primers were redesigned compared to previously published primers.

76

J.J. Sanchez et al. / Forensic Science International 137 (2003) 74–84

Exo I were added to 15 ml of PCR product, mixed, and
incubated at 37 8C for 1 h. The enzymes were inactivated at
75 8C for 15 min.

2.4. Design of PCR minisequencing primers

Table 2

shows the genotyping primers designed for each

SNP. Primers for detection of deletions and insertions were
designed with the 3

0

, base corresponding to the last base

before the possible deletion or insertion. For each SNP
system investigated in the present study, the following base
would identify the polymorphism. The sequences of the
primers were checked for the possibility of primer–dimer
and hairpin formation and investigated in PCR without
template (‘self-extension reaction’). In order to distinguish
between the sizes of the detection primers, the primers
were synthesized with lengths between 19 and 106 nucleo-
tides with intervals of four nucleotides for the great major-
ity of the primers (

Table 2

). The lengths of the template

specific parts of the primers ranged from 16 to 29 nucleo-
tides. The desired length of a primer was adjusted at the
5

0

end by addition of a piece of a ‘neutral’ sequence

and, if necessary, a poly-C tail. The neutral sequence,

5

0

-AACTGACTAAACTAGGTGCCACGTCGTGAAAGT-

CTGACAA-3

0

, is a random sequence that did not match

with any human sequence in the NCBI non-redundant
database

[19]

.

For each 4 bp DNA fragment size interval of the detection

primers, two SNP loci were detected. This was done by
selecting two SNP loci with different nucleotide polymorph-
ism. One SNP could be, e.g. an A/T SNP and the other a C/G
SNP. Thus, the minisequencing primers for the two SNPs
could have the same length and the two polymorphisms
would still be detectable. Primers for minisequencing were
HPLC purified (DNA-Technology A/S, Aarhus, Denmark
and Proligo France SAS, Paris, France).

2.5. Minisequencing reaction and capillary
electrophoresis

Multiplex PCR minisequencing was performed in 8 ml

reactions with 0.2 ml purified PCR product (6–10 ng equiva-
lent to 5–8 fmol of each fragment), 4 ml of SNaPshot

TM

reaction mix and 0.01–0.5 mM of the primers (

Table 2

). The

thermal cycling was performed with a rapid thermal ramp to
96 8C for 10 s, 50 8C for 5 s, and 60 8C for 30 s for 25 cycles.

Fig. 1. Multiplex PCR products of 25 Y chromosome DNA fragments. Ethidium bromide stained polyacrylamide gel with
PCR products obtained from various sources of blood. A negative control with DNA from a female was included. (L) 10 bp ladder
from invitrogene.

J.J. Sanchez et al. / Forensic Science International 137 (2003) 74–84

77

A positive control (provided with the kit) and negative
control (sterile water or PCR product from a female), was
performed for each batch of 44 samples.

The homogeneity of each primer was checked in single-

plex minisequencing. The occurrence of extra peaks one or
more nucleotides smaller than the expected size indicated
heterogeneity of the minisequencing primer.

After the minisequencing reaction, 1 Unit of SAP was

added and the tube was incubated at 37 8C for 1 h in order to
remove the 5

0

phosphoryl groups of the unincorporated

[F]ddNTPs. SAP was inactivated by incubation at 75 8C
for 15 min.

One ml of the purified minisequencing PCR product was

analysed on an AB Prism 3100 Genetic Analyser with a
36 cm capillary array, POP-4 polymer and 10 s at 3000 V

injections. GeneScan-120 LIZ

TM

was used as internal size

standard. The data were analysed using GeneScan Analysis
software v. 3.7 (Applied Biosystems). After background
substraction and colour separation, peaks were sorted
into bins according to sizes by comparison to the internal
size standard. Peaks above 400 relative fluorescence units
were considered positive signals and a SNP type was
assigned.

2.6. Reproducibility studies

DNA samples from 194 unrelated male Danes were typed

twice with the minisequencing technique and assigned SNP
types for the 35 SNP systems. The assignments of SNP types
of the duplicate testing were compared.

Table 2
Minisequencing primer sequences for typing of 35 Y chromosome SNP markers

Locus

Poly
(dC)

Neutral Sequence
(5

0

! 3

0

)

Target specific sequence
(5

0

! 3

0

)

Orientation

a

mM

Primer
size (nt)

M170

None

None

caacccacactgaaaaaaa

Reverse

0.02

19

M45

None

caa

ctcagaaggagctttttgc

Reverse

0.02

22

M139

None

aa

taatctgacttggaaagggg

Forward

0.01

22

M2/sY81

None

gacaa

ctttatcctccacagatctca

Reverse

0.28

26

M46/Tat

None

None

gctctgaaatattaaattaaaacaac

Reverse

0.25

26

M167/SRY

2627

None

tgaaagtctgacaa

aagccccacagggtgc

Forward

0.35

30

M213

None

tgacaa

tcagaacttaaaacatctcgttac

Reverse

0.02

30

M52

None

tctgacaa

aatatcaagaaacctatcaaacatcc

Reverse

0.02

34

P25

None

tcgtgaaagtctgacaa

tgcctgaaacctgcctg

Forward

0.04

34

M78

None

gaaagtctgacaa

cttattttgaaatatttggaagggc

Reverse

0.02

38

92R7

None

gtgaaagtctgacaa

catgaacacaaaagacgtagaag

Reverse

0.01

38

M89

None

cacgtcgtgaaagtctgacaa

aactcaggcaaagtgagagat

Reverse

0.09

42

M123

None

acgtcgtgaaagtctgacaa

atttctaggtattcaggcgatg

Reverse

0.03

42

M35

None

ggtgccacgtcgtgaaagtctgacaa

tcggagtctctgcctgtgtc

Reverse

0.25

46

M153

None

ggtgccacgtcgtgaaagtctgacaa

gctcaaagggtatgtgaaca

Forward

0.02

46

M40/SRY

4064

None

aaactaggtgccacgtcgtgaaagtctgacaa

tccaccctgtgatccgct

Reverse

0.08

50

M154

None

gccacgtcgtgaaagtctgacaa

gttacatggcctataatattcagtaca

Reverse

0.03

50

M32

None

taggtgccacgtcgtgaaagtctgacaa

agacaagatctgttcagtttatctca

Forward

0.50

54

M151

None

aggtgccacgtcgtgaaagtctgacaa

caatctactacatacctacgctatatg

Forward

0.02

54

M17

None

actaaactaggtgccacgtcgtgaaagtctgacaa

ccaaaattcacttaaaaaaaccc

Reverse

0.02

58

M96

None

aactgactaaactaggtgccacgtcgtgaaagtctgacaa

ggaaaacaggtctctcataata

Forward

0.15

62

M172

7

aactgactaaactaggtgccacgtcgtgaaagtctgacaa

caaacccattttgatgctt

Forward

0.10

66

M173

3

aactgactaaactaggtgccacgtcgtgaaagtctgacaa

tacaattcaagggcatttagaac

Forward

0.03

66

M19

4

aactgactaaactaggtgccacgtcgtgaaagtctgacaa

aaactatttttgtgaagactgttgta

Forward

0.10

70

M224

7

aactgactaaactaggtgccacgtcgtgaaagtctgacaa

aattgatacacttaacaaagatacttc

Forward

0.13

74

SRY

10831

/SRY

1532

10

aactgactaaactaggtgccacgtcgtgaaagtctgacaa

ttgtatctgactttttcacacagt

Forward

0.03

74

M18

17

aactgactaaactaggtgccacgtcgtgaaagtctgacaa

gtttgtggttgctggttgtta

Forward

0.05

78

M157

18

aactgactaaactaggtgccacgtcgtgaaagtctgacaa

caccaaaggtcatttgtggt

Reverse

0.20

78

M81

14

aactgactaaactaggtgccacgtcgtgaaagtctgacaa

cttggtttgtgtgagtatactctatgac

Reverse

0.03

82

M163

25

aactgactaaactaggtgccacgtcgtgaaagtctgacaa

cacaaaggaattttttttgag

Reverse

0.51

86

M212

20

aactgactaaactaggtgccacgtcgtgaaagtctgacaa

gcattctgttaatataaaacacaaaa

Forward

0.20

86

M9

22

aactgactaaactaggtgccacgtcgtgaaagtctgacaa

catgtctaaattaaagaaaaataaagag

Reverse

0.40

90

12f2

29

aactgactaaactaggtgccacgtcgtgaaagtctgacaa

aacatgtaagtctttaatccatctc

Forward

0.02

94

M33

29

aactgactaaactaggtgccacgtcgtgaaagtctgacaa

cagttacaaaagtataatatgtctgagat

Reverse

0.18

98

M175

46

aactgactaaactaggtgccacgtcgtgaaagtctgacaa

cacatgccttctcacttctc

Forward

0.28

106

a

The detection orientation has been probed relative to the YCC information reported in

[32]

.

78

J.J. Sanchez et al. / Forensic Science International 137 (2003) 74–84

2.7. Statistical methods

Gene diversities and standard errors were calculated

according to the methods of Nei

[23]

.

3. Results

3.1. DNA purification methods

DNA purified with Qiagen columns and DNA from FTA

1

paper with bloodstains in all cases gave satisfactory results
(

Fig. 1

). DNA from buccal cells on FTA

1

paper gave

variable intensities of the results of samples.

3.2. Design of primers

When no band or only a very weak band was observed,

suggesting that the affinities of the primers were suboptimal,
the primers were redesigned. In one case, the PCR amplifica-
tion was very weak and four different sets of primers were
tried before an acceptable yield was obtained. It was not
possible to understand the reason since the primer set best
suited from a theoretical point gave the lowest yield. In three
cases with unsatisfactory yields, the primers were redesigned

with ‘GC’ at the 3

0

end with successful results. Twenty-one of

the 25 primer pairs worked satisfactorily at the first design.

3.3. PCR buffer and efficiency of multiplex PCR
amplification

We found that the best results of amplification of all 25

DNA targets were obtained by increasing the concentration
to 8 mM MgCl

2

. Higher concentrations inhibited the ampli-

fication (data not shown).

3.4. Quality of DNA primers for template PCR
amplification

Unpurified primers could be combined into multiplexes

up to seven systems while HPLC purified primers could be
combined to amplify at least 25 templates in one reaction.

3.5. Titration of primer concentrations in PCR
amplification

It was necessary to titrate primer concentrations to obtain

a balanced PCR multimix for minisequencing. The final
concentrations of primers ranged from 0.11 to 0.46 mM.

Fig. 2. Electropherogramme with 35 Y chromosome SNP profiles from a male donor. GeneScan analysis of SNaPshot

TM

minisequencing of

the Y chromosome SNP multiplex.

J.J. Sanchez et al. / Forensic Science International 137 (2003) 74–84

79

3.6. Sensitivity of the target multiplex PCR amplification

In our hands, the lower limit for reproducible results was

approximately 100 pg DNA with a range up to approxi-
mately 10 ng and an optimum at 1–2 ng (

Figs. 2 and 3

).

3.7. Purification of the PCR template amplification
product

Both spin column and enzymatic purified PCR amplifica-

tion products gave satisfactory minisequencing typing reac-
tions. The recovery with the Exo I-SAP was 100% while the
column purification had a recovery of approximately 80%
(data not shown).

3.8. Design of DNA primers for minisequencing

None of the 35 detection primers had to be redesigned.

3.9. Quality of DNA primers for minisequencing

Clear, homogeneous peaks were obtained only if the

purity of the primers was higher than approximately 90%.
If the purity was less, the signal from degenerated primers
(n

1, n 2, etc.) would decrease the discrimination.

3.10. Annealing temperature of minisequencing
primers

Annealing temperatures from 50 to 60 8C gave almost the

same overall results in the 35 SNP multiplex when judged by
inspection of the peak areas.

3.11. Y chromosome SNP typing results

Fig. 2

shows a representative electropherogramme of

typing of 35 Y chromosome SNPs in an individual. In
one of the 194 males, typing could no reaction was obtained
in M81. The same lack of reaction in M81 was found in the
son of the investigated man. The remaining 34 Y chromo-
some SNPs were detected in the man and his child. All other
male samples gave a full 35-Y-SNP profile.

3.12. Reproducibility of Y chromosome SNP typing
with minisequencing

SNP typing was performed twice in all 194 male Danes

and the duplicate types were consistent. In each minisequen-
cing experiments, at least one sample with known types for
all 35 SNPs was included, and concordant assignments of
SNP types were obtained in all cases.

Four samples were typed for the 11 SNPs SRY2627,

M213, M35, M153, SRY

4064

, M17, M18, M9, SRY

10831

,

92R7, and P25 as part of an interlaboratory exercise of the
European DNA Profiling Group, and correct results were
obtained.

50

100

250

500

1000 2000 4000 8000

0

5

10

15

20

25

DNA (pg)

Relative Fluorescence Units

(%)

Fig. 3. Sensitivity of the 35 Y chromosome SNP typing assay.
For each DNA concentration, the relative fluorescence units
(RFUs from GeneScan) of investigations of four SNPs detected
with each of the four dyes: blue, green, yellow and red were
collated from typing of two individuals. For each DNA
concentration, the median RFU value of the two individuals
was calculated for each dye, and for each concentration the
median RFUs were normalized as a percentage of the total RFUs
of all the RFUs for the dye in question. Finally, for each DNA
concentration, the median of the normalized RFU values for all
four dyes was calculated as a percentage of the sum of all
normalized median RFU values of all concentrations. Thus, the
sum of RFUs in the figure sum up to 100%. The error bars
indicate the standard error of the mean (S.E.M.).

Fig. 4. Effect of excess DNA from females on the 35 Y
chromosome SNP typing assay. The relative fluorescence units
(RFUs from GeneScan) of mixtures of male DNA and female
DNA in great excess. The RFUs were calculated as indicated in

Fig. 3

. In general, there was a dose response relation between the

concentration of male DNA and the RFU signal strength, while
female DNA had practically no influence on the RFU signal in
the concentration range investigated.

80

J.J. Sanchez et al. / Forensic Science International 137 (2003) 74–84

3.13. Male–female mixtures of DNA

Female DNA did not influence the results of Y chromo-

some SNP typing when added in concentrations more than
300 times the concentrations of male DNA (

Fig. 4

).

3.14. Y chromosome SNP population data in Danes

Table 3

shows the frequency distribution of the 35 SNPs

investigated in 194 male Danes. No SNP signal was obtained
in 15 female Danes. A total of 19 SNPs showed variation
while 16 SNPs were monomorphic in the male Danes
studied.

Two signals were obtained in P25 and 92R7 in some

individuals (cf discussion). DNA from individuals with two

signals in theses systems was investigated with STR-tech-
nique. Only one STR-profile was obtained in each individual
demonstrating that contamination of DNA was not the
reason for the two signals in P25 and 92R7.

4. Discussion

We have developed a PCR multiplex-based system for

typing of a large number of SNPs using Y chromosome
SNPs as an example. An important part of the work was to
explore the various aspects of the multiplex PCR methods.
The 35 Y chromosome SNPs presented here are not our final
set of Y chromosome SNPs for population studies or forensic
genetic applications.

Table 3
Frequencies of 35 Y chromosome SNP markers in male Danes

Locus

Fragment number

a

Polymorphism

b

Frequency (number)

Frequency (%)

M2/sY81

1

A/G

194/0

100.0/0.0

M9

2

C/G

85/109

43.8/56.2

M17

3

4G/3G

162/32

83.5/16.5

M18

3

No ins./2 bp ins.

194/0

100.0/0.0

M19

3

T/A

194/0

100.0/0.0

M32

4

T/C

194/0

100.0/0.0

M33

4

A/C

194/0

100.0/0.0

M35

5

G/C

190/4

97.9/2.1

M40/SRY

4064

6

G/A

190/4

97.9/2.1

M45

7

G/A

86/108

44.3/55.7

M46/Tat

8

T/C

193/1

99.5/0.5

M52

9

A/C

194/0

100.0/0.0

M78

10

C/T

192/2

99.0/1.0

M81

c

11

C/T

193/0

100.0/0.0

M89

12

C/T

4/190

2.1/97.9

M96

13

G/C

190/4

97.9/2.1

M123

14

G/A

193/1

99.5/0.5

M139

15

5G/4G

0/194

0.0/100.0

M151

11

G/A

194/0

100.0/0.0

M153

15

T/A

194/0

100.0/0.0

M154

11

T/C

194/0

100.0/0.0

M157

7

A/C

194/0

100.0/0.0

M163

2

A/C

194/0

100.0/0.0

M167/SRY

2627

16

C/T

194/0

100.0/0.0

M170

17

A/C

119/75

61.3/38.7

M172

18

T/G

189/5

97.4/2.6

M173

19

A/C

89/105

45.9/54.1

M175

20

No del./5 bp del.

194/0

100.0/0.0

M212

21

C/A

194/0

100.0/0.0

M213

21

T/C

4/190

2.1/97.9

M224

10

T/C

194/0

100.0/0.0

SRY

10831

/SRY

1532

22

A/G

32/162

16.5/83.5

12f2

23

Present/absent

189/5

97.4/2.6

92R7

24

GA/A

d

86/108

44.3/55.7

P25

25

C/CA

d

124/70

63.9/36.1

a

Some PCR products contain more than one SNP in the same fragment.

b

Following the Y chromosome consortium nomenclature system

[32]

.

c

One male gave no reaction in minisequencing of M81.

d

Two signals were detected in some individuals

[24,33]

.

J.J. Sanchez et al. / Forensic Science International 137 (2003) 74–84

81

Successful PCR multiplexing depends on a number of

factors. Below, we present some of our considerations
concerning the selection of the SNPs and the generation
of the multiplex PCRs for amplification and minisequen-
cing.

At an early stage, it was decided to use the multicolour

fluorescence electrophoresis technique combined with PCR
multiplexing at approximately 60 8C in high concentrations
of MgCl

2

. The spacing between minisequencing primers

was decided to be four nucleotides because we wanted to
obtain reliable separation in the electrophoresis.

We attempted to avoid SNPs situated in regions reported

to be replicated. Two exceptions were the P25 and 92R7
SNPs that are situated in a region that most probably is part
of a duplication

[24]

. Both SNPs seem to discriminate

between European and other populations

[25]

.

Multiplex PCR amplification primers between 19 and 34

bases pairs long were selected because it was anticipated that
such long primers would work well under multiplex condi-
tions

[26]

.

Qiagen purified DNA from blood samples and blood

stains on FTA

1

paper worked equally well in the assay.

Chelex treated blood samples worked as well (data not
shown). Optimal multiplex SNP typing results were obtained
with 1 ng DNA (range 0.1

20 ng DNA). Thus, quantifica-

tion of DNA is not mandatory for the SNP assay. It should,
however, be noticed that the balance of the amounts of
amplification products of the DNA fragments is changed
with increasing amounts of templates. With increasing
concentrations of PCR amplified fragments, small, fluores-
cent adenosinnucleotide peaks with sizes of PCR amplified
fragments plus one nucleotide were seen, most likely do to
non-template addition of a single adenosin molecules to the
3

0

end of some PCR amplified fragments. At low amounts of

template DNA, loss of signal will occur due to stochastic
phenomena

[27]

.

Commonly used PCR buffers include only KCl, Tris and

MgCl

2

. It has been reported that many primer pairs produ-

cing short amplification products (<200 bp) work better at
higher salt concentration (KCl) in multiplex systems

[26]

.

Increasing the concentration of KCl in the PCR buffer 1.6
and 2-fold in our 35-plex did not increase the yield of PCR
product significantly and had no effect on the synthesis of
fragments >150 bp. Increase of MgCl

2

concentration from 2

to 8 mM increased the yield of amplicons; higher MgCl

2

concentration inhibited the amplification (data not shown).

We used AmpliTaq Gold DNA polymerase (Applied

Biosystems) because this enzyme minimizes primer dimer
formation. Even with a 4-base 3

0

overlap between two

primers we obtained homogeneous PCR products (data
not shown). The most efficient enzyme concentration
seemed to be around 2.5 U/50 ml reaction volume.

In our hands, primer concentrations below 0.01 mM were

insufficient and concentrations above 0.5 mM seemed to
inhibit multiplex PCRS probably by inducing dimer–dimer
formation. Primer concentrations were adjusted to be

approximately 10

3

times more than the concentration of

the template.

We stored dNTPs in small aliquots at

20 8C for up to 8

months. However, we observed that dNTPs were sensitive to
repeated freezing and thawing. As a rule of thumb, the
multiplex PCR would fail if the dNTPs have been frozen
and thawed more than four times. The amount of time in
freezer was less important as it has been reported by others
authors

[28]

.

The enzymatic purification method is obviously easy, has

an almost 100% recovery and a very limited risk of con-
tamination.

We chose to adjust the length of the minisequencing

primers by means of (1) a part of a neutral sequence of
up to 40 nt and for the longer primers (2) an additional poly-
C part. The neutral sequence was selected in order to obtain a
more balanced base composition. We chose poly-C for the
tail because, in theory, poly-G would give a higher molecular
mass, poly-A would have a risk of depurination during
synthesis, and poly-T tails may interfere with the addition
of 3

0

ddA in the minisequencing reaction (SNaPshot

TM

protocol recommendation, Applied Biosystems).

The quality of minisequencing primers is important

because

primer

batches

with

heterogeneous

primer

sequences consisting of the intended DNA sequence of
‘n’ nucleotides plus a spectrum of shorter nucleotides
(n

1, n 2, n 3, etc.) in many cases will destroy the

minisequencing reaction. In addition, we observed amplifi-
cation failure due to a heterogeneous primer batch in the
PCR multiplex with seven systems even though each of the
seven works in singleplex reactions. Therefore, we recom-
mend that each primer batch is tested before the multiplex
PCR and subsequent analyses, e.g. by minisequencing or
mass spectrometry. Purification of the primers with e.g.
HPLC or gel purification techniques can to some extent
solve these problems.

The minisequencing system was rather insensitive to the

annealing temperature. It was necessary to adjust primer
concentrations from 0.01 to 0.50 mM in the minisequencing
multimix.

The longer extension products had electrophoretic mobi-

lities corresponding to those predicted by the number of
bases. The mobility of shorter extension products with the
same number of bases varied to some extent. This is most
probably due to the fact that differences in the masses of the
various fluorochromes used and in the exact composition of
purines and pyrimidines have a relatively high influence on
the mobility of short DNA molecules.

The SNP-typing results were highly reproducible. A total

of 194 males were SNP typed in duplicate and no discre-
pancies were observed. Furthermore, five of the most poly-
morphic SNPs were analysed by a DNA hybridisation assay
using the Nanogen technology

[29]

. Concordant results were

obtained for all 194 individuals (data not shown).

In one father-child combination, no allele of M81 was

detectable. An amplified fragment was present in the first

82

J.J. Sanchez et al. / Forensic Science International 137 (2003) 74–84

PCR because two other SNPs (M151 and M154) on the
fragment were detected, but no reaction of M81 was detected
in the minisequencing reaction. Work is in progress in order
to determine the nature of the variant.

A total of 19 of 29 SNPs reported to be polymorphic in

Europeans in a previous study

[4]

and 9 of 10 SNPs reported

in another study

[21]

turned out to be polymorphic in the

male Danes studied. The gene diversity for the loci showing
polymorphism ranged from 0.01 to 0.5 (

Table 3

). M173,

M45, 92R7 and M9 were the most polymorphic markers in
Danes. The data were described as frequencies of individual
SNPs and not as Y chromosome haplogroups because the
study was a technical study and the Y chromosome multi-
plex is not ideal for typing of Y chromosome haplogroups. A
larger study of Y chromosome haplogroups in Danes and
other populations will be published elsewhere.

P25 and 92R7 were previously reported as SNPs

[30,31]

.

However, the P25 and 92R7 minisequencing primers were
extended with two different dideoxynucleotides during the
minisequencing reaction of numerous samples. This indi-
cates that at least two different, almost identical fragments
were amplified during the PCR reaction. Hurles et al.

[33]

previously observed that SNP typing of 92R7 gave two
results in some individuals. Further studies have confirmed
that P25 and 92R7 are paralogous sequence variants and that
at least one of the sequence variants in each group of loci is
polymorphic

[24]

.

The multiplex PCR SNP typing format presented here

seems to be useful for forensic casework because small
amounts of DNA (100 pg DNA) can be reliably typed.
The multiplex presented is not our final package for Y
chromosome SNPs for forensic purposes. The way forward
would go either through (1) the development of SNP
packages optimised for an initial screening plus further
packages optimised for the major populations or (2) the
development of a large multiplex package that include Y
chromosome SNPs that can discriminate between individual
lineages in all populations.

Acknowledgements

We thank Dr. Rebecca Reynolds, Roche Molecular Sys-

tems, for advice concerning the design of the multiplex PCR
for template generation in the initial phase of the project. We
thank Ms. AnneMette Holbo Birk for technical assistance.
The work was supported by grants to Juan Sanchez from
Ellen and Aage Andersen’s Foundation and Manuel Morales
Foundation.

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