n Methods Useful in Genetics and Oncology
Table 1. Continued
Prothrombin G20210A (GcnBank Accession 1M 17262)
Position
Length
GC(%) Tm (G C)
G20210A primers
ATTGATCAGTTTGGAGAGTAGGGG
26667
24
45.8
63.9
GAGCTGCCCATGAATAGCACT
26820 R
21
52.4
65.8
Product
26667-26820
154
G20210A probe
F-TCTCAGCAAGCCTCAATGCT-P
26777
20
50.0
64.2
• Bertina et al. describe an intronic primer also found in PAC 86Fl4 (GenBank Accession
#Z99572) position 62773. The paired exonic primer described here is located at position
62959 R of the PAC 86F14 sequence. The fluorescein probe is at position 62773.
lightCycier PCR PCR conditions were optimized such that all six loci could be readily amplified
and analyzed under the same protocol. One microliter (50 ng) of each DNA sample to be genotyped was added to 9 ~ of master mix. The mixture was transferred
to LightCyder capillary tubes, sealed, and centrifuged briefly to place the sample
in the tip of the capillary. Tubes were loaded into a LightCyder carousel, transferred to the LightCyder instrument, amplified, and analyzed.
The following master mix was used:
LightCyc1er-DNA Master Hybridization probes
MgClz (25 mM)
Forward primer (5 flM)
Reverse primer (5 flM)
Fluorescein probe (1 101M)
dHlO
Total volume
Volume Illil
IFinal1
1
0.8
4.2
9
Ix
3.0mM
0.51J.M
0.5 flMa
0.1 IlM b
• The final concentration of rever e primer used in the assays for the cystic fibrosis -related
mutation FS08deI and the hemochromatosis mutation C282Y was 0.06 flM.
b The fmal concentration of fluorescein probe used in detecting the MTHFR C677T mutation was 0.2 flM .
To allow uniformity between the six genotyping assays, a standard master mix was
investigated. However, three assays required alteration of the primer or probe concentrations to allow optimal amplification and genotyping by fluorescein quenching. The assays designed to genotype the cystic fibrosis-related mutation F508del
and the hemochromatosis mutation C282Y (G845A) were amplified using 0.5 flM
forward primer and 0.06 flM reverse primer (8:1 asymmetry). This primer asymmetry proved to increase the magnitude of the fluorescence transitions during
melting which, in turn, provided stronger melting peaks for genotyping. Another
Table 1. Continued
Prothrombin G20210A (GcnBank Accession 1M 17262)
Position
Length
GC(%) Tm (G C)
G20210A primers
ATTGATCAGTTTGGAGAGTAGGGG
26667
24
45.8
63.9
GAGCTGCCCATGAATAGCACT
26820 R
21
52.4
65.8
Product
26667-26820
154
G20210A probe
F-TCTCAGCAAGCCTCAATGCT-P
26777
20
50.0
64.2
• Bertina et al. describe an intronic primer also found in PAC 86Fl4 (GenBank Accession
#Z99572) position 62773. The paired exonic primer described here is located at position
62959 R of the PAC 86F14 sequence. The fluorescein probe is at position 62773.
lightCycier PCR PCR conditions were optimized such that all six loci could be readily amplified
and analyzed under the same protocol. One microliter (50 ng) of each DNA sample to be genotyped was added to 9 ~ of master mix. The mixture was transferred
to LightCyder capillary tubes, sealed, and centrifuged briefly to place the sample
in the tip of the capillary. Tubes were loaded into a LightCyder carousel, transferred to the LightCyder instrument, amplified, and analyzed.
The following master mix was used:
LightCyc1er-DNA Master Hybridization probes
MgClz (25 mM)
Forward primer (5 flM)
Reverse primer (5 flM)
Fluorescein probe (1 101M)
dHlO
Total volume
Volume Illil
IFinal1
1
0.8
4.2
9
Ix
3.0mM
0.51J.M
0.5 flMa
0.1 IlM b
• The final concentration of rever e primer used in the assays for the cystic fibrosis -related
mutation FS08deI and the hemochromatosis mutation C282Y was 0.06 flM.
b The fmal concentration of fluorescein probe used in detecting the MTHFR C677T mutation was 0.2 flM .
To allow uniformity between the six genotyping assays, a standard master mix was
investigated. However, three assays required alteration of the primer or probe concentrations to allow optimal amplification and genotyping by fluorescein quenching. The assays designed to genotype the cystic fibrosis-related mutation F508del
and the hemochromatosis mutation C282Y (G845A) were amplified using 0.5 flM
forward primer and 0.06 flM reverse primer (8:1 asymmetry). This primer asymmetry proved to increase the magnitude of the fluorescence transitions during
melting which, in turn, provided stronger melting peaks for genotyping. Another