g Applications in Genetics
Parallel LightCycier
PCR Analysis
For the parallel LightCycler PCR mutation analysis, the following protocol was
used:
• Denaturation at 95°C for 150 s
• Amplification
Parameter
Value
Cycles
Type
Target temperature [0C]
Incubation time [sJ
Temperature transition rate [OC/s]
Acquisition mode
Gains
• Melting curve analysis
45
Quantification
Segment 1
95
0
20
None
FI=l
Parameter
Value
Cycles
Type
Target temperature [DC]
Incubation time [s]
Temperature transition rate [OC/s]
Acquisition mode
• Cooling at 40°C for 300 s
Results
Melting curves
Segment 1
95
0
20
None
Segment 2
Segment 3
55
72
20
20
20
20
Single
None
F2=15
F3=30
Segment 2
Segment 3
45
85
60
0
20
0.1
None
Continuous
Figure 1 displays the online fluorescence curves of parallel LightCycler-assisted
PCR amplification of HFE, AAT, factor v, APOBlOO and APOE gene fragments
from a sample with negative and positive controls.
Genotypes were determined by subsequent melting curve analysis (Fig. 2A-G),
which revealed single-characteristic melting temperatures (Tm) associated with
the wild-type and mutant alleles. As shown in Fig. 2A-F, the following Tm profile
was found measuring the LCRed640 fluorescence in channel 2 (F2): the HFE
H63D mutant and wild-type alleles were associated with a Tm of 73°C and 67SC,
respectively (Fig. 2A); the HFE C282Y mutation and the wild-type alleles were
associated with a T m of 61 SC and 56.0°C, respectively (Fig. 2B); in the case of the
AAT gene, the PiM and the PiZ alleles were associated with aT m of 69°C and 64°C,
respectively (Fig. 2C); the mutated factor V allele showed a T m of 57°C, whereas
the wild-type allele was associated with a Tm of 65°C (Fig. 2D); the APOBlOO
C9774T and G9775A mutant alleles and the wild-type allele were associated with