f1iII Applications in Genetics
LightCycier
Master Mix
labeled with fluorescein and LCRed640 or fluorescein and LCRed70S were used
to distinguish between the C3932T and C4070T mutations, respectively.
To simultaneously screen genomic DNA samples for the described mutations, six
different PCR reaction mixtures had to be prepared as follows. A negative control
containing no genomic DNA was run with the samples. A positive control for each
mutation analysis was performed as indicated in Fig. 1 with either heterozygous
or heterozygous and homozygous DNA replacing the sample DNA. LightCyclerassisted real-time PCR of the HFE C282Y and H63D and the AAT gene mutations
was performed in a final reaction volume of 20 Ill, consisting of the following
components:
LightCycler DNA Master Hyb ridization Mix
MgClz stock solution (25 mM)
Primers (20 I!M each)
Hybridization probes (3 I!M each)
H20 (PCR-grade)
Genomic DNA
Volume [~l
[Final)
2
2.4
I + I
I + I
6.6
5
Ix
4mM
I I!M each
0.151!M each
LightCycler-assisted real-time PCR of the factor V and APOBlOO genes was performed in a final reaction volume of 20 III consisting of the following components:
LightCycler Mutation Detection Mix
LightCycler Reaction Mix
H 2 0 (PCR.grade)
Genomic DNA
Volume [~l
(Final)
2
2
11
5
Ix
Ix
LightCycler-assisted real-time PCR of the APOE gene was performed in a final
reaction volume of 20 f!l consisting of the following components:
Volume l~)
(Final)
LightCycler Mutation Detection Mix
2
Ix
LightCycler Reaction Mix
2
Ix
H 2 0 (PCR.grade)
11
Genomic DNA
5