Detection of a Single Base Substitution in Single Cells by Melting Peak Analysis n
PCR reaction mix for 2841 mutation:
Volume If1Il
[Final l
FastStart DNA Master Hybridization mix
2
Ix
MgCI2 (20 mM)
2.4
4mM
BRCAI primers (5 j.1M)
1+1
0.251lM
Anchor probe (2 j.1M)
1.5
0.15 J.LM
Wild -type probe (2 j.1M)
0.1 j.1M
Mutant probe (2 j.lM)
0.1 j.1M
Template (I cell)
- 2
HPLC grade water
6.1
The final volume was brought to 18 III per capillary with nuclease-free water.
The final reaction volume was around 20 Ill, but varied depending on the volume
of culture media transferred with the cell. The DNA from the cell becomes available for PCR after the preheat cycle of 8 min at 95°C, which is also needed to activate the hot-start polymerase.
Results and Discussion
PCR and mutation detection were optimized for two different mutations in the
BRCA1 gene: 2841GT (recurrent in the Dutch population [2]) and 3519G-+T
(found in several populations [3]; numbers according to cDNA sequence).
For the 2841GT mutation in BRCA1, the homozygous wild-type PCR product
showed a single peak at 59.2°C, whereas the heterozygous products (mutant)
showed an additional peak at 54°C (Fig. la). The mutant-specific probe showed a
mutant melting peak at 58.3°C and a wild-type peak at 52.4°C (Fig.lb).
Comparable results were obtained with the BRCA1 exon 11 PCR fragment containing the 3519G-+T mutation. In this case the homozygous wild type had a single peak at 64.1°C with the wild-type probe, whereas the heterozygous
mutant/WT showed an additional peak at 60.2°C (Fig. lc). The mutant probe
showed a single peak at 57.2°C for the homozygous wild type and an additional
peak at 63.7°C for the heterozygous sample (Fig. Id). Both wild type probes show
a shoulder in the wildtype sample. This may be due to misincorporations during
PCR.
A single-base substitution was reproducibly detected in single cells from a cell
line containing the BRCA1 2841 GT mutation (Fig. 2). All cells in four series (three
mutant cells per series) showed a heterozygous pattern with the wild type as well
as mutant probe. In none of the experiments loss of an allele due to unequal PCR
amplification was observed. The wild-type cells all showed a single wild-type
peak with both probes. With the wild-type probe, the peak shows a shoulder
towards lower temperature. This probably reflects PCR mutations during the early cycles of the reaction. The shoulder did not result in misinterpretation, but it
Mutation Detection
in DNA from Blood
Mutation Detection
in Single Cells
PCR reaction mix for 2841 mutation:
Volume If1Il
[Final l
FastStart DNA Master Hybridization mix
2
Ix
MgCI2 (20 mM)
2.4
4mM
BRCAI primers (5 j.1M)
1+1
0.251lM
Anchor probe (2 j.1M)
1.5
0.15 J.LM
Wild -type probe (2 j.1M)
0.1 j.1M
Mutant probe (2 j.lM)
0.1 j.1M
Template (I cell)
- 2
HPLC grade water
6.1
The final volume was brought to 18 III per capillary with nuclease-free water.
The final reaction volume was around 20 Ill, but varied depending on the volume
of culture media transferred with the cell. The DNA from the cell becomes available for PCR after the preheat cycle of 8 min at 95°C, which is also needed to activate the hot-start polymerase.
Results and Discussion
PCR and mutation detection were optimized for two different mutations in the
BRCA1 gene: 2841GT (recurrent in the Dutch population [2]) and 3519G-+T
(found in several populations [3]; numbers according to cDNA sequence).
For the 2841GT mutation in BRCA1, the homozygous wild-type PCR product
showed a single peak at 59.2°C, whereas the heterozygous products (mutant)
showed an additional peak at 54°C (Fig. la). The mutant-specific probe showed a
mutant melting peak at 58.3°C and a wild-type peak at 52.4°C (Fig.lb).
Comparable results were obtained with the BRCA1 exon 11 PCR fragment containing the 3519G-+T mutation. In this case the homozygous wild type had a single peak at 64.1°C with the wild-type probe, whereas the heterozygous
mutant/WT showed an additional peak at 60.2°C (Fig. lc). The mutant probe
showed a single peak at 57.2°C for the homozygous wild type and an additional
peak at 63.7°C for the heterozygous sample (Fig. Id). Both wild type probes show
a shoulder in the wildtype sample. This may be due to misincorporations during
PCR.
A single-base substitution was reproducibly detected in single cells from a cell
line containing the BRCA1 2841 GT mutation (Fig. 2). All cells in four series (three
mutant cells per series) showed a heterozygous pattern with the wild type as well
as mutant probe. In none of the experiments loss of an allele due to unequal PCR
amplification was observed. The wild-type cells all showed a single wild-type
peak with both probes. With the wild-type probe, the peak shows a shoulder
towards lower temperature. This probably reflects PCR mutations during the early cycles of the reaction. The shoulder did not result in misinterpretation, but it
Mutation Detection
in DNA from Blood
Mutation Detection
in Single Cells