n Methods Useful in Genetics and Oncology
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80 100 120 140 160 180
Amplicon Length (bp)
Fig. I. Tm-Iength correlation in heterozygous Huntington's disease samples. Tms of each allele in
heterogygous Huntington's samples are plotted against amplicon length. Note the wide separation in T m between the normal alleles (lower group) and the disease-causing (CAGn > 39) alleles (upper group). The line represents a second degree polynomial fit to the data points. The Tmlength dependence is less pronounced as the product increases in size
In a within-run 10 tube replicate experiment we found a high degree of Tm
reproducibility with a standard deviation of 0.053°C. The average T m separation
between a normal and an expanded allele in the heterozygous HD samples was
4.04°C, the median Tm separation was 4.32°C. The LightCycler software assigned
2 peaks, representing each allele, in all of the Huntington's samples and in 8 of 22
control samples. 5 of the 22 control samples were homozygous by product length
analysis on the sequencing gel. Therefore, the heterozygote detection by T m analysis was 8/17 (47%) in the control samples. These identified heterozygote samples
were substantially different in size (mean = 18bp, median = 16.5bp). In the 9 heterozygous samples where the LightCycler instrument failed to identify 2 peaks,
the difference in PCR product size was small (mean = 4.3bp, median = 4.5bp).
When the 8 heterozygous normal samples that were assigned 2 peaks were included, the correlation between Tm and product length decreased only slightly (Fig. 2).
Comments
A T m difference of 1 ° C was found between the longest normal sample (27 CAG
repeats) and the shortest Huntington's disease sample (40 CAG repeats) in our
data set. It would be rare to find a normal sample with more than 27 CAG repeats.
Only 6 out of 600 control alleles from people without Huntington's disease were
found to have >27 CAG repeats in a large population study (Kremer, 1994). Melting analysis can be used to screen for HD. We recommend that previously sized
controls corresponding to 27,30, and 35 CAG repeats should be included in the
same run as the unknowns. Any sample with a T m above the 27 repeat sample
should be sized on a sequencing gel.
When the Huntington's disease samples were amplified in the LightCycler
intrument and then melted on both the ABI 5700 heat block instrument and the
LightCycler instrument, similar results were obtained (Fig. 3).
86
G
~
84
E
I82
'j
I
I
I
j
80 100 120 140 160 180
Amplicon Length (bp)
Fig. I. Tm-Iength correlation in heterozygous Huntington's disease samples. Tms of each allele in
heterogygous Huntington's samples are plotted against amplicon length. Note the wide separation in T m between the normal alleles (lower group) and the disease-causing (CAGn > 39) alleles (upper group). The line represents a second degree polynomial fit to the data points. The Tmlength dependence is less pronounced as the product increases in size
In a within-run 10 tube replicate experiment we found a high degree of Tm
reproducibility with a standard deviation of 0.053°C. The average T m separation
between a normal and an expanded allele in the heterozygous HD samples was
4.04°C, the median Tm separation was 4.32°C. The LightCycler software assigned
2 peaks, representing each allele, in all of the Huntington's samples and in 8 of 22
control samples. 5 of the 22 control samples were homozygous by product length
analysis on the sequencing gel. Therefore, the heterozygote detection by T m analysis was 8/17 (47%) in the control samples. These identified heterozygote samples
were substantially different in size (mean = 18bp, median = 16.5bp). In the 9 heterozygous samples where the LightCycler instrument failed to identify 2 peaks,
the difference in PCR product size was small (mean = 4.3bp, median = 4.5bp).
When the 8 heterozygous normal samples that were assigned 2 peaks were included, the correlation between Tm and product length decreased only slightly (Fig. 2).
Comments
A T m difference of 1 ° C was found between the longest normal sample (27 CAG
repeats) and the shortest Huntington's disease sample (40 CAG repeats) in our
data set. It would be rare to find a normal sample with more than 27 CAG repeats.
Only 6 out of 600 control alleles from people without Huntington's disease were
found to have >27 CAG repeats in a large population study (Kremer, 1994). Melting analysis can be used to screen for HD. We recommend that previously sized
controls corresponding to 27,30, and 35 CAG repeats should be included in the
same run as the unknowns. Any sample with a T m above the 27 repeat sample
should be sized on a sequencing gel.
When the Huntington's disease samples were amplified in the LightCycler
intrument and then melted on both the ABI 5700 heat block instrument and the
LightCycler instrument, similar results were obtained (Fig. 3).