SYBR Green I Analysis of the Trinucleotide Repeat Responsible for Huntington's Disease E'I
86
~ M r-----~~~------------~
~
E
R2= 0.96
f- 82 1--- -/'-- - - - - - - - --1
80 ~~-------------------I
60 80 100 120 140 160 180
HD Length (bp)
Fig. 2. Tm-length correlation in control and Huntington's disease samples. In addition to the
Huntington's disease samples in Fig. 1, 16 more alleles (from 8 heterogygous normal samples)
are included. Note that the lower end of the graph includes two unusually short PCR products.
Also, an unusully long but non-disease-causing product is present (113 bp)
~
86
IV
U
.#/'
/
>.
U
.... 84
.I:.
C'I
d.
E 82
I/ '
A=0.9936x + 0.3161
/
R2= 0.99
y ..
82
84
86
T ITI (ABI 5700)
Fig. 3. Correlation of Tms obtained on the LightCycler instrument and ABI 5700. Huntington's
disease samples were first amplified on the LightCycler instrument and then melting curves
obtained on both LightCycler and ABI 5700. Tms were calculated from derivative melting curve
data by non-linear least squares fitting of multiple Gaussian curves using custom software
developed to accept data from both instruments
The LightCycler software is more flexible than the ABI 5700 software. This
flexibility can be important in detecting heterozygotes. With SYBR Green I detection, as the cycle number increases, the higher temperature peak is favored over
the lower temperature peak (Fig. 4). Therefore, the number of cycles before the
melting curve is obtained during amplification is important. If the samples vary
in starting template concentration or the efficiency varies between tubes, optimal
melting results would be expected after different numbers of cycles. One solution
to this problem is to acquire multiple melting curves during amplification, for
example, at 30, 35, and 40 cycles.
This increases the ability of the LightCycler instrument to detect multiple
peaks in a heterozygous sample. The melting protocols within such a run are dif-
86
~ M r-----~~~------------~
~
E
R2= 0.96
f- 82 1--- -/'-- - - - - - - - --1
80 ~~-------------------I
60 80 100 120 140 160 180
HD Length (bp)
Fig. 2. Tm-length correlation in control and Huntington's disease samples. In addition to the
Huntington's disease samples in Fig. 1, 16 more alleles (from 8 heterogygous normal samples)
are included. Note that the lower end of the graph includes two unusually short PCR products.
Also, an unusully long but non-disease-causing product is present (113 bp)
~
86
IV
U
.#/'
/
>.
U
.... 84
.I:.
C'I
d.
E 82
I/ '
A=0.9936x + 0.3161
/
R2= 0.99
y ..
82
84
86
T ITI (ABI 5700)
Fig. 3. Correlation of Tms obtained on the LightCycler instrument and ABI 5700. Huntington's
disease samples were first amplified on the LightCycler instrument and then melting curves
obtained on both LightCycler and ABI 5700. Tms were calculated from derivative melting curve
data by non-linear least squares fitting of multiple Gaussian curves using custom software
developed to accept data from both instruments
The LightCycler software is more flexible than the ABI 5700 software. This
flexibility can be important in detecting heterozygotes. With SYBR Green I detection, as the cycle number increases, the higher temperature peak is favored over
the lower temperature peak (Fig. 4). Therefore, the number of cycles before the
melting curve is obtained during amplification is important. If the samples vary
in starting template concentration or the efficiency varies between tubes, optimal
melting results would be expected after different numbers of cycles. One solution
to this problem is to acquire multiple melting curves during amplification, for
example, at 30, 35, and 40 cycles.
This increases the ability of the LightCycler instrument to detect multiple
peaks in a heterozygous sample. The melting protocols within such a run are dif-