SYBR Green I Analysis of the Trinucleotide Repeat Responsible for Huntington's Disease p
The following amplification protocol was used:
Parameter
Value
Cycles
35
Type
Quantification
Segment 1
Segment 2
Target temperature 1°c]
97
68
Incubation time Is]
0
25
Temperature transition rate [OC/s I
20
20
Acquisition mode
None
Single
Display mode
Fl
The following melting peak protocol was used:
Parameter
Value
Cycles
Type
Melting Curves
Segment I
Segment 2
Segment3
Target temperature [0C]
95
60
95
Incubation time [sl
0
0
0
Temperature transition rate [OC/s I
20
0.20
0.05
Acquisition mode
None
None
Cont.
Display mode
Fl
To assign accurate lengths to products the samples were amplified according to
the above protocols with the inclusion of a 5'-fluorescein-Iabeled primer used at
0.50 uM. The capillary tops were removed and each sample was spun down into a
microfuge tube. The samples were sized on a commercial sequencing apparatus
[Genomics Core Facility, University of Utah]. The lengths of the two PCR products in each sample were determined.
Results
A gradient of product T mS was observed due to the polymorphic size of the CAG
repeat region. The smallest normal allele observed was at 56bp (CAGn= 8) while
the largest normal allele observed was 113bp (CAGn = 27). The corresponding Tms
were 79.82°C and 84.77°C, respectively. The smallest expanded (disease-causing)
allele was 155 bp (CAG n = 40) while the largest expanded allele was 179bp (CAGn
= 48). The corresponding Tms were 85.85°C and 86.61°C. We observed a 1.08°C separation between the longest normal allele (27 CAG repeats) and the shortest Huntington disease allele (40 CAG repeats). All 9 HD samples had two widely separated
peaks that indicated heterozygous composition. The T mS of the 18 alleles from the
9 HD samples correlated with the respective lengths (R2=0.99) as shown in Fig. 1.