LightCycler PCR for the Polymorphisms -308 and -238 in the TNF Alpha Gene n
Procedure
DNA was isolated by a standard rapid lysis technique using the Qiagen QIAmp
Blood kit.
For the TNF-a mutation analysis, we used complementary designed sensor
probes, fitting to the wild type, covering the point mutations at position -308 and
-238 (GenBank Accession #L1l698). The anchor probe was designed complementary to an adjacent invariant gene segment. Thus, the wild-type allele (GG) is
expected to yield a higher melting temperature than the -308 mutation (AA),
measured on channel two. The -238 mutant shows the same behaviour, except the
measurement is on channel three. The anchor probe was modelled between the
two reporter probes, capable of detecting both mutations in the same run. peR
was performed with the nucleotides mentioned in Table 1.
For the TNF-13 assay (GenBank Accession #M55913), the probes were designed
in a similar manner. Because of contradictory statements in literature concerning
the designation of the wild-type and the mutated allele, the neutral nucleotide
Table 1. Oligonucleotides
TNF-a -308 and -238 (GenBa nk Accession #L11698)
Position Length
GC(%) Tm(°C)
Primers
5'-TTC CTG CAT CCT GTC TGG AA
658
20
50.0
56.8
5' -CAG CGG AAA ACT TCC TTG GT
966-985 20
50.0
58.7
Product
658-985 328 bp
Probes
5'-AAT AGG TTT TGA GGG GCA TGG GGAC
746
25
48.0
65.9
T-LCRed640-P
5'-LCRed705-CCT CGG AAT eGG AGC AGG
816
23
65.2
68.6
GAG GA-P
5'-P-TTC AGC CTC CAG GGT CCT ACA CAC
755
47
53.2
81.0
AAA TCA GTC AGT GGC CCA GAA GA-F
TNF-Il A329G (GcnBank Accession #M55913)
Primers
5' -CCT GCA CCT GCT GCC TGG AT
193
20
65.0
64.8
5' -CAG TCA GAG AAA CCC CAA GGT GA
369
23
52.2
60.9
Product
193-392 200 bp
Probes
5' -LCRed640-TCT CTG TTT eTG CCA TGG
312
28
50.0
66.0
TIC CTC TCT G P
5' -CTC CAT crG TCA GTC TCA TTG TCT
277
34
47.1
66.6
eTG TCA CAC A-F
Preparation
of Template DNA
Primer and Probe
Design
Procedure
DNA was isolated by a standard rapid lysis technique using the Qiagen QIAmp
Blood kit.
For the TNF-a mutation analysis, we used complementary designed sensor
probes, fitting to the wild type, covering the point mutations at position -308 and
-238 (GenBank Accession #L1l698). The anchor probe was designed complementary to an adjacent invariant gene segment. Thus, the wild-type allele (GG) is
expected to yield a higher melting temperature than the -308 mutation (AA),
measured on channel two. The -238 mutant shows the same behaviour, except the
measurement is on channel three. The anchor probe was modelled between the
two reporter probes, capable of detecting both mutations in the same run. peR
was performed with the nucleotides mentioned in Table 1.
For the TNF-13 assay (GenBank Accession #M55913), the probes were designed
in a similar manner. Because of contradictory statements in literature concerning
the designation of the wild-type and the mutated allele, the neutral nucleotide
Table 1. Oligonucleotides
TNF-a -308 and -238 (GenBa nk Accession #L11698)
Position Length
GC(%) Tm(°C)
Primers
5'-TTC CTG CAT CCT GTC TGG AA
658
20
50.0
56.8
5' -CAG CGG AAA ACT TCC TTG GT
966-985 20
50.0
58.7
Product
658-985 328 bp
Probes
5'-AAT AGG TTT TGA GGG GCA TGG GGAC
746
25
48.0
65.9
T-LCRed640-P
5'-LCRed705-CCT CGG AAT eGG AGC AGG
816
23
65.2
68.6
GAG GA-P
5'-P-TTC AGC CTC CAG GGT CCT ACA CAC
755
47
53.2
81.0
AAA TCA GTC AGT GGC CCA GAA GA-F
TNF-Il A329G (GcnBank Accession #M55913)
Primers
5' -CCT GCA CCT GCT GCC TGG AT
193
20
65.0
64.8
5' -CAG TCA GAG AAA CCC CAA GGT GA
369
23
52.2
60.9
Product
193-392 200 bp
Probes
5' -LCRed640-TCT CTG TTT eTG CCA TGG
312
28
50.0
66.0
TIC CTC TCT G P
5' -CTC CAT crG TCA GTC TCA TTG TCT
277
34
47.1
66.6
eTG TCA CAC A-F
Preparation
of Template DNA
Primer and Probe
Design