Detection of a ingle Base Substitution in Single Cells by Melting Peak Analysis n
a. WUd typt prOOt mutant cdls
b. WUd typt probt control cdIs
14.0
~
~ 10.0
;
'-' 6.0
..
14.0
R "CI
i4' 10.0
~
.. 6D
~ OD I--~~--1-----t-~~~~==~
a
~ 2.0
! o.o~--~~~--~--~~-===~
~ -2 .0
:I
ro:: .2.0
" ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ w
Tanptraturt \'C)
" ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ w
Tanptraturt ("C)
Co Mutant probt mutant ceUs
4.0
d. Mutant probt control cdls
4.0
~
~
"CI 3.0
;;r
"CI 3.0
;;r
~2D
8
51.0
~2.o
8
5\.0
~
aDD --~--~------~--~==~--------:I
~
aOD ~-----+--~--~~~C:----------ro:: .ID
:s
ro:: _ ID
" ~ ~ ~ ~ ~ ro ~ ~ M ~ w
Tanptraturt ("C)
" ~ ~ ~ U ~ ~ ~ ~ M ~ w
Temperature ("C)
= mutant cells
-noDNA
= (ontto! cells
I Wid type peak
I Motantpeak
Fig.2a-d. Detection of the BRCAI 2841GT mutation in single cells with wild-type probe (a, b)
and mutant probe (c, d). The cells were from cell lines from a heterozygote carrier of the mutation (a, c) and a normal control (b, d). Each graph contains three lines from three separate cells.
The black line at the bottom is the no DNA (no cell) control. Mutant and wild-type probe were
used in a single tube. The heterozygous cells clearly show two distinct melting peaks with both
probes (a, b). The wild-type cells show a single peak at 53°C with the mutant probe (d) and at
59°C with the wild-type probe (b)
Presence of the BRCAl 3519G~T mutation was assessed in DNA from a small
number (10-50) of normal and tumor cells from paraffin-embedded tissue
from a carrier of this mutation. Two tumors were studied. The normal tissue
showed a double peak in the melting curve, in keeping with heterozygosity for
the mutation. The tumor DNA showed a single mutant peak (Fig. 3), indicating
loss of the normal allele. Since BRCAl is considered a tumor suppressor gene,
loss of the normal allele is expected in tumor tissue. With the present technique it is possible to detect loss of the normal allele in very small foci of aberrant tissue.
Mutation Detection
in Miuodissected
Tumor Samples
a. WUd typt prOOt mutant cdls
b. WUd typt probt control cdIs
14.0
~
~ 10.0
;
'-' 6.0
..
14.0
R "CI
i4' 10.0
~
.. 6D
~ OD I--~~--1-----t-~~~~==~
a
~ 2.0
! o.o~--~~~--~--~~-===~
~ -2 .0
:I
ro:: .2.0
" ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ w
Tanptraturt \'C)
" ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ w
Tanptraturt ("C)
Co Mutant probt mutant ceUs
4.0
d. Mutant probt control cdls
4.0
~
~
"CI 3.0
;;r
"CI 3.0
;;r
~2D
8
51.0
~2.o
8
5\.0
~
aDD --~--~------~--~==~--------:I
~
aOD ~-----+--~--~~~C:----------ro:: .ID
:s
ro:: _ ID
" ~ ~ ~ ~ ~ ro ~ ~ M ~ w
Tanptraturt ("C)
" ~ ~ ~ U ~ ~ ~ ~ M ~ w
Temperature ("C)
= mutant cells
-noDNA
= (ontto! cells
I Wid type peak
I Motantpeak
Fig.2a-d. Detection of the BRCAI 2841GT mutation in single cells with wild-type probe (a, b)
and mutant probe (c, d). The cells were from cell lines from a heterozygote carrier of the mutation (a, c) and a normal control (b, d). Each graph contains three lines from three separate cells.
The black line at the bottom is the no DNA (no cell) control. Mutant and wild-type probe were
used in a single tube. The heterozygous cells clearly show two distinct melting peaks with both
probes (a, b). The wild-type cells show a single peak at 53°C with the mutant probe (d) and at
59°C with the wild-type probe (b)
Presence of the BRCAl 3519G~T mutation was assessed in DNA from a small
number (10-50) of normal and tumor cells from paraffin-embedded tissue
from a carrier of this mutation. Two tumors were studied. The normal tissue
showed a double peak in the melting curve, in keeping with heterozygosity for
the mutation. The tumor DNA showed a single mutant peak (Fig. 3), indicating
loss of the normal allele. Since BRCAl is considered a tumor suppressor gene,
loss of the normal allele is expected in tumor tissue. With the present technique it is possible to detect loss of the normal allele in very small foci of aberrant tissue.
Mutation Detection
in Miuodissected
Tumor Samples