n Methods Useful in Genetics and Oncology
fragment
differentiation
2.2
2
, 8
' 6
1<;1.4
::..
I
~ 12
I,
J
!
u. 0.8
0.6
~
0 4
0 2
0
.Q.2 I
I
I
65
70
75
\
/: ,
I I
: I
I
, I
~
I
I
I
eo
85
90
T_oturel't:)
I
95
Fig. 4. Detection of the heteroduplex from ~F508/wt heterozygotes in a 90bp fragment (product 1)
on melting curve analysis. Blue line: fluorescence measured; brown dotted line: melting peaks as
detected by the LightCycler analysis software
In addition, we analyzed a hetero- and a homozygote DNA sample from CFpatients who carried the 1 bp-insertion 3905insT in exon 20 of the CFTR-gene. We
tested amplification products of 63bp and 70bp length (products 10 and 11) and
did not detect a significant difference on melting curve analysis.
We also tested the point mutation G542X (G~T) in exon 11 of the CFTR-gene in
a 62bp amplification product (product 12). A DNA sample from a heterozygote
patient was available. One melting peak was detected which showed a decrease of
T m of OAoC as compared to wildtype-homozygotes (Fig. 5). This melting peak was
interpreted as an overlay of the melting peaks from the heteroduplices, homoduplex wt, and homo duplex mutant.
Comments
We found fragments of different length and G+C content could be distinguished by
melting curve analysis. Multiple specific products may be separated from each other due to different T m in most cases, eliminating the need for gel electrophoresis.
fragment
differentiation
2.2
2
, 8
' 6
1<;1.4
::..
I
~ 12
I,
J
!
u. 0.8
0.6
~
0 4
0 2
0
.Q.2 I
I
I
65
70
75
\
/: ,
I I
: I
I
, I
~
I
I
I
eo
85
90
T_oturel't:)
I
95
Fig. 4. Detection of the heteroduplex from ~F508/wt heterozygotes in a 90bp fragment (product 1)
on melting curve analysis. Blue line: fluorescence measured; brown dotted line: melting peaks as
detected by the LightCycler analysis software
In addition, we analyzed a hetero- and a homozygote DNA sample from CFpatients who carried the 1 bp-insertion 3905insT in exon 20 of the CFTR-gene. We
tested amplification products of 63bp and 70bp length (products 10 and 11) and
did not detect a significant difference on melting curve analysis.
We also tested the point mutation G542X (G~T) in exon 11 of the CFTR-gene in
a 62bp amplification product (product 12). A DNA sample from a heterozygote
patient was available. One melting peak was detected which showed a decrease of
T m of OAoC as compared to wildtype-homozygotes (Fig. 5). This melting peak was
interpreted as an overlay of the melting peaks from the heteroduplices, homoduplex wt, and homo duplex mutant.
Comments
We found fragments of different length and G+C content could be distinguished by
melting curve analysis. Multiple specific products may be separated from each other due to different T m in most cases, eliminating the need for gel electrophoresis.