Rapid Genotyping of 2-bp and 9-bp Deletion Mutations Using the LightCycler Instrument m
The following master mix was used for the reaction:
Volume [flll
[final)
LighlCycler-DNA Master Hybridization Probes
MgCl2 stock solution
Primers (10 f1M)
Hybridization probes (10 f1M)
Mutation detection probe
Anchor probe
H20 (PCR grade)
Total volume
2.0
2.4
0.4+0.4
0.4
0.8
11.6
18.0
Ix
4mM
0.2 f1M
0.2 f1M
0.4 f1M
To complete the amplification mixtures, 18 fJl of master mix and 2 fJl of the
genomic DNA template (30 ng) were added to each capillary.
The thermal cycling was carried out as follows:
• Denaturation at 95°C for 30 s
• Amplification
Parameter
Value
Cycles
Type
Target temperature 1°C)
Incubation time [s)
Temperature transition rate [O C/s]
Acquisition mode
Gains
• Melting Curve Analysis
40
Quantification
Segment I
Segment 2
95
o
20
56
5
20
None
Single
Pl = 1;P2= 15;F3=30
Parameter
Value
Cycles
Type
Target temperature rOC]
Incubation time [s]
Temperature transition rate [OC/s 1
Acquisition mode
Gains
• Cooling at 40°C for 2 min
Melting curves
Segment 1
Segment 2
95
45
30
20
20
20
None
None
PI = I; P2= 15; F3=30
Segment 3
72
4
20
None
Segment 3
85
o
0.2
Continuous
For detection of 9-bp deletion by SYBR Green I -based method, the following procedure was followed.
LightCycier PCR