Zainuddin et al
SUPPLEMENTARY INFORMATION
Methods
TP53 mutation detection
PCR amplification of exons 4 to 8 of the TP53 gene was performed in the following
conditions; 0.2-0.5µg of genomic DNA, 1X PCR buffer, 1.5mM MgCl2, 200µM dNTP,
0.125µM of each primer and 0.7U HotStarTaq DNA polymerase (Qiagen). Final reaction
volumes were 50μl each. The cycling program started with an initial cycle of 2-minute
denaturation step at 95°C, 1 minute annealing step at 61°C and 1 minute extension step at
72°C, followed by 40 cycles of 30 sec at 95°C, 30 seconds at 61°C and 45 seconds at 72°C.
The final extension step was 5 min at 72°C. 7μl of each PCR products was analyzed on a 2%
agarose gel in TBE.
PCR conditions for p53CA and D17S1678 were similar to the conditions described
above. The p53CA PCR reaction mixture was denatured for 3 minutes at 94°C and cycled 35
times (94°C for 1 min, 60°C for 2 min and 72°C for 5 min), followed by a 10-minute
extension at 72°C. The D17S1678 PCR reaction mixture cycling program was similar to the
p53CA program, except that 56°C annealing temperature was applied.
PCR amplification using an alternative set of TP53 primers for each of exon 4 to 8
was performed in the following conditions; 0.2-0.5µg of genomic DNA, 1X PCR buffer,
2mM MgCl2, 200µM dNTP, 0.2µM of each primer and 1U AmpliTaq Gold DNA polymerase
(Applied Biosystems). Final reaction volumes were 25μl each. The cycling program started
with 40°C for 10 minutes and a 10-minute denaturation step at 95°C, followed by 40 cycles of
30 sec at 94°C, 30 seconds at 56°C and 45 seconds at 72°C. The final extension step was 7
min at 72°C. 7μl of each PCR products was analyzed on a 2% agarose gel in TBE.
MDM2 SNP309 and TP53 codon 72 genotyping
PCR amplification was made in the following conditions; 0.2-0.5µg of genomic DNA, 1X
PCR buffer, 1.5mM MgCl2, 800µM dNTP, 2µM of each primer and 1U HotStarTaq DNA
polymerase (Qiagen). Final reaction volumes were 50μl each. The cycling program started
with an initial cycle of 10-minute denaturation step at 95°C, followed by 35 cycles of 30 sec
at 95°C, 30 seconds at 57°C and 1 minute at 72°C. The final extension step was 7 min at
72°C.
To differ between MDM2 genotypes, the resulting 154bp PCR products were digested
with 5 units of MspA1 I (New England Biolabs, Beverly, MA) at 37°C for 16 hours. The
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Zainuddin et al
digested products were then electrophoresed on a polyacrylamide gel using Genegel™ Excel
12.5/24 kit (GE Healthcare Bio-Sciences AB) at 14°C for 1 hour and 40 minutes and silverstained. The G/G homozygote product is cleaved by MspA1 I and yields 59- and 95bp bands,
the T/T homozygote is not cleaved by the enzyme and yields a single 154bp band, whereas
the T/G heterozygote contains all three bands.
The 312bp PCR products of TP53 exon 4 were digested with 1 unit of BstUI (New
England Biolabs, Beverly, MA) at 60°C for 5 hours. The codon 72 WT Arg/Arg homozygote
product is cleaved by BstUI and yields 259- and 53bp bands, the Pro/Pro homozygote is not
cleaved by the enzyme and yields a single 312bp band, while the Arg/Pro heterozygote
contains all three bands.
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Zainuddin et al
Supplementary Table 1. Primers for amplification of TP53 mutationa and MDM2 SNP309
analysis.
Primer name
Primer sequence (5’3’)
TP53 exons 4 to 8b (GenBank accession number X54156)
ACTGAAGACCCAGGTCCAGATGAA
p53 – 4 sense
AGGGTGAAGAGGAATCCCAAAGTT
p53 – 4 antisense
TGTTCACTTGTGCCCTGACTTTCA
p53 – 5-6 sense
p53 – 5-6 antisense AGGGCCACTGACAACCACCCTTA
CAAGGCGCACTGGCCTCATCTT
p53 – 7-8 sense
p53 – 7-8 antisense ACCGCTTCTTGTCCTGCTTGCTTA
Alternative set of TP53 exons 4 to 8 primersc
CCTGGTCCTCTGACTGCTCT
p53 – 4 sense
GCCAGGCATTGAAGTCTCAT
p53 – 4 antisense
TGTTCACTTGTGCCCTGACT
p53 – 5 sense
AACCAGCCCTGTCGTCTCT
p53 – 5 antisense
CAGGCCTCTGATTCCTCACT
p53 – 6 sense
CTTAACCCCTCCTCCCAGCAGAG
p53 – 6 antisense
TCATCTTGGGCCTGTGTTATC
p53 – 7 sense
GGGTCAGAGGCAAGCAGA
p53 – 7 antisense
GGGACAGGTAGGACCTGATTT
p53 – 8 sense
AGGAAAGAGGCAAGGAAAGG
p53 – 8 antisense
Fragment
size (bp)
312
487
611
362
271
185
203
287
Genomic
location
TP53 exon 4
TP53 intron 4
TP53 intron 4
TP53 intron 6
TP53 intron 6
TP53 intron 8
including
splice site
TP53 intron 3
TP53 intron 4
TP53 intron 4
TP53 intron 5
TP53 intron 5
TP53 intron 6
TP53 intron 6
TP53 intron 7
TP53 intron 7
TP53 intron 8
17p deletion (p53CA; GenBank accession number X61505 and D17S1678; GenBank accession
number G06085)
AGGGATACTATTCAGCCCGAGGTG
p53CA-GT
135
25kb upstream
from TP53
ACTGCCACTCCTTGCCCCATTC
p53CA-AC
TTTGGGTCTTTGAACCCTTG
WI-9178-1
116
40kb
telomeric to
CCACAACAAAACACCAGTGC
WI-9178-2
TP53
MDM SNP309d (GenBank accession number AF527840)
CTGCCCACTGAACCGGC
MDM2 sense
GAGGTCTCCGCGGGAGTTC
MDM2 antisense
154
First intron of
the MDM2
promoter
a
TP53 codon 72 polymorphism was assessed using the PCR products of TP53 exon 4.
Montesinos-Rongen M, Roers A, Kuppers R, Rajewsky K, Hansmann ML. Mutation of the
p53 gene is not a typical feature of Hodgkin and Reed-Sternberg cells in Hodgkins disease.
Blood 1999; 94: 1755-60. cCases which showed no PCR product using the standard PCR
primer set were evaluated using an alternative set of TP53 primers for each of exon 4 to 8.
d
Bougeard G, Baert-Desurmont S, Tournier I, Vasseur S, Martin C, Brugieres L, et al. Impact
of the MDM2 SNP309 and p53 Arg72Pro polymorphism on age of tumour onset in LiFraumeni syndrome. J Med Genet 2006;43 (6):531-3.
b
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