Supplementary Material
Genotyping
DAT1 40-bp VNTR
The following primers were used for PCR amplification of the DAT1 40-bp VNTR in the 3’UTR
region:
5’-TGGGTCCTTGTGGTGTAGGA-3’
type
forward
and
5’-
CTCAAGGCCAGGCAGAGTGTG-3’ type reverse [29]. PCR reaction was carried out with
100 ng of genomic DNA using Taq DNA polymerase (Eurobio, Brunschwig, Basel,
Switzerland) in a 25 µl reaction mix containing 1 × buffer, 0.2 mM dNTPs, 0.03 mM MgCl 2,
0.02 mM of each primer, 1U Taq polymerase. Amplification conditions were as follow: 95°C
for 5 min, 30 cycles of 92°C for 30 sec, 62°C for 30 sec, and 72°C for 30 sec. PCR products
were analysed by electrophoresis on a 1.5% agarose gel at 250 V for 150 min and visualised
with GelRed (Biotium, Chemie Brunschwig AG, Basel, Switzerland). A 50-bp and a 100-bp
DNA ladders (Invitrogen, LuBioScience GmbH, Lucerne, Switzerland) were used to identify
repeat alleles: 6-repeats (6R) were 315 bp, 7-repeats (7R) were 355 bp, 9-repeats (9R) were
435 bp, 10-repeats (10R) were 475 bp, 11-repeats (11R) were 515 bp and 12-repeats (12R)
were 555 bp. By migrating PCR products at 250 V for 150 min, a new repeat allele (9.5R)
was discovered at 454 bp of length (Supplementary Figure S1). For sequencing 9.5R and 10R
homozygous alleles (Supplementary Figure S2), PCR conditions and primers were identical
as those described aboved. Then, PCR products were purified with QIAquick PCR
Purification Kit (Qiagen, Germantown, MD) and sequenced with an ABI PRISM 3100
Genetic Analyzer (PE Applied Biosystems, Foster City,CA).
1
DRD4 48-bp VNTR
The following primers were used for PCR amplification of the DRD4 48-bp VNTR in exon 3:
5’-ACTACGTGGTCTACTCGTCCGGT-3’
type
forward
and
5’-
TCAGGACAGGAACCCACCGA-3’ type reverse [29]. PCR reactions were carried out with
100 ng of genomic DNA using the KAPA2G Robust Hot Start Kit (Kappa Biosystem, Cape
Town, South Africa) in a final volume of 25 µl containing 1 x KAPA2G buffer GC
(containing 1.5 mM MgCl2), 1 x KAPA Enhancer 1, 0.2 mM dNTPs, 0.02 mM of each
primer, 1U KAPA2G Robust Hot Start DNA polymerase. Amplification conditions were as
follows: 95°C for 3 min, 30 cycles of 92°C for 30 sec, 56°C for 30 sec, and 72°C for 30 sec.
PCR products were analysed by electrophoresis on a 1.5% agarose gel at 250 V for 150 min
and visualised with GelRed (Biotium, Chemie Brunschwig AG, Basel, Switzerland). A 50-bp
and a 100-bp DNA ladders (Invitrogen, LuBioScience GmbH, Lucerne, Switzerland) were
used to identify repeat alleles: 2-repeats (2R) were 407 bp, 3-repeats (3R) were 455 bp, 4repeats (4R) were 503 bp, 5-repeats (5R) were 551 bp, 6-repeats (6R) were 599 bp and 7repeats (7R) were 647 bp.
DRD2 –141ins/delC polymorphism (rs1799732)
DRD2 –141ins/delC polymorphism (rs1799732) was genotyped by PCR followed by
restriction enzyme digestion. A 94-bp segment was amplified by PCR with the following
primers, designed by using Primer3 online program (ver. 0.4.0) (http://bioinfo.ut.ee/primer30.4.0/primer3/input.htm).: 5’-GTTCCCGCCTCAAAACAAG-3’ type forward and 5’CCACCAAAGGAGCTGTACCT-3’ type reverse. Target sequences were amplified in a 25-μl
reaction mix containing 100 ng of genomic DNA, 1U of HotStarTaq DNA Polymerase
(Qiagen, Germantown, MD, USA), 1 x buffer (Tris·Cl, KCl, (NH4)2SO4, 15 mM MgCl2 ; pH
8.7), 2 x Q-Solution, 0.2 mM dNTPs, and 0.02 mM of each primer. Amplification conditions
were as follows: 95°C for 15 min, 30 cycles of 94°C for 30 sec, 58°C for 30 sec and 72°C for
2
30 sec. PCR products were then digested overnight at 60°C with 1U of BstN1 (New England
Biolabs, Ipswich, MA, USA). Then fragments were separated on a 1.5% agarose gel with a
50-bp DNA ladder (Invitrogen, LuBioScience GmbH, Lucerne, Switzerland) at 250 V for 60
min and visualised with GelRed (Biotium, Chemie Brunschwig AG, Basel, Switzerland). The
undigested deleted-allele product size was 94 bp (del-allele) and the C-allele showed two
fragments of 54 bp and 40 bp.
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