n Applications in Genetics
a Tm of 57°C, 53°C and 62SC, respectively (Fig. 2E); the APOE genotyping
revealed Tms of 56°C and 62SC for the C3932T mutant (APOE*E4) and the wildtype (APOE*E3) alleles, respectively (Fig. 2F). For the detection of the APOE
C4070 T (APOE2*E2) mutation, LCR 705 fluorescence was measured in channel 3
(F3). Two Tms of S7.5°C and 66°C were identified, corresponding to the mutant
and the wild-type alleles, respectively (Fig. 2G). The patient's DNA was found to
contain no mutation except for the heterogenous APOE*E2/E3 genotype.
Comments
With the introduction of real-time PCR technology in combination with melting
curve analysis, a rapid and convenient method for the detection of singlenucleotide polymorphism became available. High throughput of sample analysis
and no requirement of post -PCR sample processing combined with easy handling
are key advantages. This methodical approach has already been applied for the
detection of several known point mutations in genes such as the factor V and prothrombin genes [13, 14], the HFE gene [15] and the AAT gene [16]. In addition,
commercially available mutation detection kits have been developed.
We describe here a LightCycler-assisted real-time PCR, which allows the simple, rapid and accurate evaluation of patients for the presence of the HFE C282Y,
HFE H63D, factor V Leiden, AAT PiZ, APOBlOO C9774T and G977SA and APOE
C3932T and C4070T mutations in a parallel analysis within 60 min. This improved
method offers the possibility of substantially increasing the throughput of sample analysis, of analyzing different samples for the presence of any of these gene
mutations and/or of determining the individual gene mutation pattern. In terms
of the rapidity, flexibility and the subsequent economic advantages, this approach
is suitable for use in clinical and routine laboratory applications.
References
1. Feder IN, Gnirke A, Thomas W, Tsuchihashi Z, Ruddy DA, Basava A, Dormishian F, Domingo
R Jr, Ellis MC, Fullan A, Hinton LM, Jones NL, Kimmel BE, Kronmal GS, Lauer P, Lee VK, Loeb
DB, Mapa FA, McClelland E, Meyer NC, Mintier GA, Moeller N, Moore T, Morikang E, Wolff
RK et al (1996) A novel MHC class-I-like gene is mutated in patients with hereditary
haemochromatosis. Nat Genet l3:399-408
2. Jouanolle AM, Fergelot P, Gandon G, Yaouanq J, Le Gall JY, David V (1997) A candidate gene
for hemochromatosis: frequency of the C282Y and H63D mutations. Hum Genet 100:544-547
3. Crystal RG (1990) Alpha I-antitrypsin deficiency, emphysema, and liver disease Genetic
basis and strategies for therapy. J Clin Invest 85:l343-l352
4. Fabbretti G, Sergi C, Consales G, Faa G, Brisigotti M, Romeo G, Callea F (1992) Genetic variants of alpha-I-antitrypsin (AAT). Liver 12:296-301
5. Bertina RM, Koeleman BP, Koster T, Rosendaal FR, Dirven RJ, de Ronde H, van der Velden
PA, Reitsma PH (1994) Mutation in blood coagulation factor V associated with resistance to
activated protein C. Nature 369:64-67
6. Innerarity TL, Weisgraber KH, Arnold KS, Mahley RW, Krauss RM, Vega GL, Grundy SM
(1987) Familial defective apolipoprotein B-I00: low density lipoproteins with abnormal
receptor binding. Proc Natl Acad Sci USA 84:69l9-6923
a Tm of 57°C, 53°C and 62SC, respectively (Fig. 2E); the APOE genotyping
revealed Tms of 56°C and 62SC for the C3932T mutant (APOE*E4) and the wildtype (APOE*E3) alleles, respectively (Fig. 2F). For the detection of the APOE
C4070 T (APOE2*E2) mutation, LCR 705 fluorescence was measured in channel 3
(F3). Two Tms of S7.5°C and 66°C were identified, corresponding to the mutant
and the wild-type alleles, respectively (Fig. 2G). The patient's DNA was found to
contain no mutation except for the heterogenous APOE*E2/E3 genotype.
Comments
With the introduction of real-time PCR technology in combination with melting
curve analysis, a rapid and convenient method for the detection of singlenucleotide polymorphism became available. High throughput of sample analysis
and no requirement of post -PCR sample processing combined with easy handling
are key advantages. This methodical approach has already been applied for the
detection of several known point mutations in genes such as the factor V and prothrombin genes [13, 14], the HFE gene [15] and the AAT gene [16]. In addition,
commercially available mutation detection kits have been developed.
We describe here a LightCycler-assisted real-time PCR, which allows the simple, rapid and accurate evaluation of patients for the presence of the HFE C282Y,
HFE H63D, factor V Leiden, AAT PiZ, APOBlOO C9774T and G977SA and APOE
C3932T and C4070T mutations in a parallel analysis within 60 min. This improved
method offers the possibility of substantially increasing the throughput of sample analysis, of analyzing different samples for the presence of any of these gene
mutations and/or of determining the individual gene mutation pattern. In terms
of the rapidity, flexibility and the subsequent economic advantages, this approach
is suitable for use in clinical and routine laboratory applications.
References
1. Feder IN, Gnirke A, Thomas W, Tsuchihashi Z, Ruddy DA, Basava A, Dormishian F, Domingo
R Jr, Ellis MC, Fullan A, Hinton LM, Jones NL, Kimmel BE, Kronmal GS, Lauer P, Lee VK, Loeb
DB, Mapa FA, McClelland E, Meyer NC, Mintier GA, Moeller N, Moore T, Morikang E, Wolff
RK et al (1996) A novel MHC class-I-like gene is mutated in patients with hereditary
haemochromatosis. Nat Genet l3:399-408
2. Jouanolle AM, Fergelot P, Gandon G, Yaouanq J, Le Gall JY, David V (1997) A candidate gene
for hemochromatosis: frequency of the C282Y and H63D mutations. Hum Genet 100:544-547
3. Crystal RG (1990) Alpha I-antitrypsin deficiency, emphysema, and liver disease Genetic
basis and strategies for therapy. J Clin Invest 85:l343-l352
4. Fabbretti G, Sergi C, Consales G, Faa G, Brisigotti M, Romeo G, Callea F (1992) Genetic variants of alpha-I-antitrypsin (AAT). Liver 12:296-301
5. Bertina RM, Koeleman BP, Koster T, Rosendaal FR, Dirven RJ, de Ronde H, van der Velden
PA, Reitsma PH (1994) Mutation in blood coagulation factor V associated with resistance to
activated protein C. Nature 369:64-67
6. Innerarity TL, Weisgraber KH, Arnold KS, Mahley RW, Krauss RM, Vega GL, Grundy SM
(1987) Familial defective apolipoprotein B-I00: low density lipoproteins with abnormal
receptor binding. Proc Natl Acad Sci USA 84:69l9-6923