Rapid Detection of Missense Mutations in the Prostatic Steroid 5a...Reductase Gene DB
Table 1. Oligonucleotides
Human SRD5A2 gene (GenBank Accession #L0483)
Position
Primers
AGCACACGGAGAGCCTGAAG
847
CCGCAAGGGAAAAACGCTAC(R)
1044
Product
847-1044
Probes for codon 49
LCRED640-GCGCGGGCTGGCAGG-PH (R)
881
GCAGCTCCTGCAGGAACCAGGC-FL (R)
897
Probes for codon 89
LCRED705-CCTCTTCTGCCTACAITACITC-PH 998
CACCTGGGACGGTACTTCTGG-FL
976
GCC: Ala nine 49
ACe: Throonlno 49
Length
20
20
198
15
22
22
21
GC (%)
Tm (0C)
60.0
64.0
55.0
61.9
67.7
86.7
67.9
68.2
70.3
45.5
58.1
61.9
64.5
~. -I\CCACACGCiA::;AGCC7CAr'\GC~GGCGGCTACCCGCcr'XCAGCC CGCGCCGCCTGGT'TCC'7CCh~GACCTGCC7T-("'rrCCCCGTCCCCGCGGG-:3
3 -7CGTGTGCCTCTCGGACTTCGGCCGCCGATr:GGCGG, V"GGTCGGGCGCGGCGGACCAAGCACGTCC7CGACGGAAGGAAGCGCCACGGGCGCCC-S
GGACGGT<;GGGCCCG CGGACCM GGACG1CC1"CGACG
50 -CTCTTCC:' iCC ;"CC'TGGGACGGT AC77 CTGGGCC7 CT'l'C7GrCT'A' C' ATT ACT'TCC:"C AGGT AGCGT':'TTTCCCTTGCCC .... ,.GC C" "r; rCCAGCGC A- ']
j
- (".AGAAG("O:::CGGTGGAC"("TGC~ ATfjAAGA~(""I:i(i;AGAACA.C"'GA
"'AA TG • .a.AGGTGTCr ATCGCAAAAAGGGAA~G':CCGCGGCTC ACGTC(,,,cGT - 5
Fig.!. Relative orientation of the fluorophore-Iabeled anchor and detection probes. The A49T
polymorphism is a result of a G to A substitution at nucleotide 888 of the human SRD5A2 gene.
This polymorphism creates an A -C mismatch between the sense strand of the 49T allele and the
detection probe. This mismatch destabilizes the hybrid which results in a decrease in the melting temperature of the probe. In contrast, complete matching of detection probe and sense
strand of the 49A allele results in a higher melting temperature of the hybrid. The V89L polymorphism is a result of a G to C substitution at nucleotide 1008. This polymorphism creates a
C-C mismatch between the antisense strand of the 89V allele and the detection probe. In contrast, complete matching of detection probe and antisense strand of the 89L allele results in
higher stability of the hybrid
content of more than 90% the neighboring nucleotides of this polymorphic site
provide a difficult environment for allele-discriminating hybridization probes.
The probe for detection of the V89L polymorphism was labeled with LCRed705
and hybridizes with perfect match to the antisense-strand of the 89L-allele. It is
true that the resulting C-C mismatch between detection probe and the 89V allele
is rather stable, but the low GC content of the detection probe of 45% favors the
allele-specific discrimination in spite of the relative stability of this mismatch.