Parallel Genotyping of Different Genes: A Rapid Real-Time PCR Approach n
Table 1. Oligonucleotides
U1-Anlitrysin gene (GenBank Accession # K02212)
Position
GC(% )
Primers
GTGTCCACGTGAGCCTTGCTC
11840-11860
63.2
GTTTGTTGAACTTGACCTCGG
12001- 12021
47.6
Probes
CTTCAGTCCCTTTCTCGTCGATGGTC-F
11930- 11955
53.8
LCRed640-CACAGCCTTATGCACGGCCTGGAG- P
11904-11927
62.5
Hemochromatosis gene,II63D mutation (GenBank Accession # Z929 10)
Primers
GCCTCAGAGCAGGACCTTGG
4684-4703
65.0
CAGCTGTTTCCTTCAAGATGC
4973-4993
47.6
Probes
CTTGAAATTCTACTGGAAACCCATGGAGTT4779- 4817
51.3
CGGGGCTCC-F
LCRed640-CACGGCGACTCTCATCATCATAG4745-4777
48.5
AACACGAACA-P
Hemochromatosis gene, C262Y mutation (GenBank Accession I; Z92910)
Primers
TGGCAAGGGTAAACAGATCC
CTCAGGCACTCCTCTCAACC
Probes
AGATATACGTACCAGGTGGAG -F
LCRed640-CCCAGGCCTGGATCAGCCCCTCATTGTGATCTGGG-P
6443- 6462
6813- 6832
6712- 6732
6735- 6769
50.0
60.0
47.6
62.9
Tm (0C)
58.7
56.3
64.9
69.5
63.5
57.9
76.7
70.4
57.3
61.4
50.2
80.5
LCRed640 and hybridized to the wild-type sequence with the fluorescein-labeled
probe spanning the mutation site (Table l). For detection of the factor V Leiden
mutation, specific PCR primers and hybridization probes provided with the
LightCycler Factor V Leiden Mutation Detection Kit (Roche Diagnostics) were
used. The PCR primers amplify a 222-bp fragment of the factor V gene. The two
hybridization probes included a fluorescein-labeled probe spanning the mutation site and hybridizing to wild-type and an adjacent LCRed640-labeled probe.
For detection of the APOBlOO C9774T and G9775A mutations, primers and
hybridization probes provided with the LightCycler Apo B Mutation Detection
Kit (codon 3500) were used. The specific primers amplify a 207-bp fragment of
the APOBlOO gene. The LCRed640-labeled hybridization probe hybridizes to the
wild-type sequence, which harbors the mutation sites. For detection of the APOE
C3932T and C4070T mutations on codon 112 and 158, respectively, primers and
hybridization probes provided with the LightCycler Apo E Mutation Detection
Kit (codon 112 and codon 158) were used. The specific primer pair amplifies a
265-bp fragment of the APOE gene. Two different hybridization probe pairs
Table 1. Oligonucleotides
U1-Anlitrysin gene (GenBank Accession # K02212)
Position
GC(% )
Primers
GTGTCCACGTGAGCCTTGCTC
11840-11860
63.2
GTTTGTTGAACTTGACCTCGG
12001- 12021
47.6
Probes
CTTCAGTCCCTTTCTCGTCGATGGTC-F
11930- 11955
53.8
LCRed640-CACAGCCTTATGCACGGCCTGGAG- P
11904-11927
62.5
Hemochromatosis gene,II63D mutation (GenBank Accession # Z929 10)
Primers
GCCTCAGAGCAGGACCTTGG
4684-4703
65.0
CAGCTGTTTCCTTCAAGATGC
4973-4993
47.6
Probes
CTTGAAATTCTACTGGAAACCCATGGAGTT4779- 4817
51.3
CGGGGCTCC-F
LCRed640-CACGGCGACTCTCATCATCATAG4745-4777
48.5
AACACGAACA-P
Hemochromatosis gene, C262Y mutation (GenBank Accession I; Z92910)
Primers
TGGCAAGGGTAAACAGATCC
CTCAGGCACTCCTCTCAACC
Probes
AGATATACGTACCAGGTGGAG -F
LCRed640-CCCAGGCCTGGATCAGCCCCTCATTGTGATCTGGG-P
6443- 6462
6813- 6832
6712- 6732
6735- 6769
50.0
60.0
47.6
62.9
Tm (0C)
58.7
56.3
64.9
69.5
63.5
57.9
76.7
70.4
57.3
61.4
50.2
80.5
LCRed640 and hybridized to the wild-type sequence with the fluorescein-labeled
probe spanning the mutation site (Table l). For detection of the factor V Leiden
mutation, specific PCR primers and hybridization probes provided with the
LightCycler Factor V Leiden Mutation Detection Kit (Roche Diagnostics) were
used. The PCR primers amplify a 222-bp fragment of the factor V gene. The two
hybridization probes included a fluorescein-labeled probe spanning the mutation site and hybridizing to wild-type and an adjacent LCRed640-labeled probe.
For detection of the APOBlOO C9774T and G9775A mutations, primers and
hybridization probes provided with the LightCycler Apo B Mutation Detection
Kit (codon 3500) were used. The specific primers amplify a 207-bp fragment of
the APOBlOO gene. The LCRed640-labeled hybridization probe hybridizes to the
wild-type sequence, which harbors the mutation sites. For detection of the APOE
C3932T and C4070T mutations on codon 112 and 158, respectively, primers and
hybridization probes provided with the LightCycler Apo E Mutation Detection
Kit (codon 112 and codon 158) were used. The specific primer pair amplifies a
265-bp fragment of the APOE gene. Two different hybridization probe pairs