Rapid Screening for Five Major Cystic Fibrosis Mutations by Melting Peak Analysis n
different dyes, are used for multiplex detection. All detection probes are homologous to the wild-type sequence. When hybridized to the template, the distance
between the two fluorophores attached to the probes is 2 bp for all probe systems.
The following master mix was used to detect CF mutations M508 and R347P with lightCycier PCR
hybridization probes:
Volume [Ill ]
[Final ]
LightCycier FastStart DNA Master Hybridization Probes
MgClz (25 mM)
Primers (10 JiM)
Probes (10 11M)
HIO (PCR grade)
Total volume
2
2.4
1+1+1+1
0.4+0.4+0.4+0.4
5
15
IX
4.0mM
0.5 11M
0.2 JiM
The following master mix was used to detect CF mutations G542X, G551D, and
R553X with hybridization probes:
Volume 1111]
[Finnl ]
LightCycier FastStart DNA Master Hybridization Probes
MgClz (25 rnM)
Primers (10 11M)
Probes (10 11M)
H 2 0 (PCR grade)
Total volume
2
3.4
2.5+2.5
0.4+0.4+0.4+0.4
3
15
IX
5.25 rnM
1.25 11M
0.2 11M
A total of 15 f.Ll master mix and 5 f.Ll of DNA (concentration not determined) were
added to each glass capillary placed in precooled adaptors. Sealed capillaries
were centrifuged with the adaptors (1000 g for 1 min) and placed into the LightCycler rotor.
The following PCR protocol was used for amplification:
• Denaturation of DNA and activation of FastStart polymerase for 10 min at 95°C
• Amplification
Parameter
Value
Cycles
Type
Target temperature roC]
lncubation time lsI
Temperature transition rate [OC/sl
Acquisition Mode
Gains
35
Quantification
Segment I
Segment 2
95
55
0
10
20
20
None
Single
Fl=l; F2=10; F3=80
Segment 3
72
30
20
None