Genotyping of the Methionine- Valine Polymorphism at Codon 129 of the Human Prion Protein 1m
Table 1. Primer sequences and hybridization probes for detection of human prion protein genotype at codon position 129
Human prion prolein gene (GenBank Accession IIHSU29185)
Position
Length GC(%) Tm (OC)
Primer set 1
GTGGAACAAGCCGAGT
25743
16
56.2
59.60
GGTTGGGGTAACGGTG
25927R
16
62.5
60.53
Product
25743-25927
185
Probe set 1
CCGAAATGTATGATGGGCCTGCTCAT-F
25874R
26
50.0
69.25
LCRed640 CACTTCCCAGCA.cGTAGCC-P
25846R
19
63.2
64.82
Primer sel2
CACAGTCAGTGGAACAAG
25735
18
50.0
58.75
GTACACTTGGTTGGGGT
25935R
17
52.9
59.13
Prod uct
25735-25935R 20t
Probe set 2
AGCA.cGTAGCCGCCA-F
25838
15
66.7
63.98
LCRed640-GCCCCCCACCACTGCCC-P
25821
17
82.4
70.40
Complement-valine
CCTTGGCGGCTAC~TGCTGGGAAGTGCCAT25821
60
58.3
GAGCAGGCCCATCATACATTTCGGCAGTGA
Complement-methionine
CCTTGGCGGCTACATGCTGGGAAGTGCCAT- 25821
60
56.7
GAGCAGGCCCATCAIACATTTCGGCAGTGA
Variable nucJeotides in the detection probes are underH ned.
Co mplemenl
5 ' --------------------------------------------------~. 3'
~ ' 2~80~ GGGCAGTGGTGGGGGGCCTTGGCGGCTAcATGCTGGGAAGTGCCATGAGCAGGCCCATCATACATTTCGGCAGTGA 25880 3'
3 ' -
C--------- ~--------------------- 5 '
Deteclio n Probe 1
nehur I
3 ' .... ------------- . . . . .
------ c-- 5 '
Dehor 2
Deteclion Probe 2
Fig. 1. Binding sites for hybridization probes and artificial complement used in the study. Numbering refers to the sequence of the human prion protein gene (GenBank accession #HSU29185;
methionine at codon 129). The polymorphic site is shown in boldface. Probes labeled with fluorescein and LCRed640 are shown in green and red, respectively. The detection probes are complementary to the sequence coding for valine at codon 129 of the prion protein and therefore
contain a cytosine at the polymorphic site