DliJI Applications in Genetics
We describe here a homogenous assay for the simultaneous genotyping of
both mutations in the SRD5A2 gene that combines RapidCycle PCR with allelespecific fluorescent probe melting profiles on the LightCycler. In order to allow
the simultaneous analysis of the two polymorphic codons in a single reaction,
two reporter dyes with different excitation and emission spectra, LightCycler
Red 640 (LCRed640) and LightCycler Red 705 (LCRed7s0), and the LightCycler
color compensation software that corrects for the temperature-dependent
crosstalk between the emission spectra of these dyes were utilized. The method
is fast and robust and thus is ideally applicable to SRD5A2 genotyping in both
clinical and research settings.
Materials
Equipment LightCycler instrument
Reagent amplification primer (MWG Biotech, Eberswalde, Germany)
Hybridization probes (TIB MOLBIOL, Berlin, Germany)
Reagents The following reagents were purchased from Roche Diagnostics, Mannheim:
Sample Preparation
Design of Primers
and Fluorogenic
Probes
High-Pure PCR Template Preparation Kit
LightCycler-DNA Master Hybridization Probes
LightCycler glass capillarycuvettes
Procedure
Genomic DNA was isolated from whole blood or buffy coats using the High-Pure
PCR Template Preparation Kit. The DNA was resuspended in 10 mmolll Tris (pH
7.4), containing 0.1 mmoUI EDTA at a concentration of 20 ngl~.
The amplification primers were designed using the primed input program from the
Whitehead Institute for Biomedical Research (website: www-genome.wi.mit.edul
cgi-binlprimer/primer3.cgi). The amplified fragment harboring both polymorphic
sites in exon 1 of the SRD5A2 gene is 198 bp in size (GenBank locus L03843)
(Table 1). The primers were synthesized by standard phosphoramidite chemistry
(MWG-Biotech, Eberswalde, Germany). Primers and probes for the detection of the
alanine-threonine substitution at codon 49 and of the valine-leucine substitution
at codon 89 in exon 1 of the human SRD5A2 gene are given in Table 1.
Two pairs of hybridization probes labeled with different fluorophores were used
in one reaction in order to genotype both polymorphic codons simultaneously. For
codon 49 the LCRed640-labeled detection probe was designed to be complementary to the sense strand of the 49A allele with the polymorphic nucleotide seven
bases from the 3' end (underlined nt.), as shown in Table 1 and Fig. 1. The resulting A-C mismatch between detection probe and the 49T allele is the most unstable mismatch possible with this polymorphism, ensuring a maximum difference in
the melting temperatures (T m) between both alleles. Due to an extremely high GC
We describe here a homogenous assay for the simultaneous genotyping of
both mutations in the SRD5A2 gene that combines RapidCycle PCR with allelespecific fluorescent probe melting profiles on the LightCycler. In order to allow
the simultaneous analysis of the two polymorphic codons in a single reaction,
two reporter dyes with different excitation and emission spectra, LightCycler
Red 640 (LCRed640) and LightCycler Red 705 (LCRed7s0), and the LightCycler
color compensation software that corrects for the temperature-dependent
crosstalk between the emission spectra of these dyes were utilized. The method
is fast and robust and thus is ideally applicable to SRD5A2 genotyping in both
clinical and research settings.
Materials
Equipment LightCycler instrument
Reagent amplification primer (MWG Biotech, Eberswalde, Germany)
Hybridization probes (TIB MOLBIOL, Berlin, Germany)
Reagents The following reagents were purchased from Roche Diagnostics, Mannheim:
Sample Preparation
Design of Primers
and Fluorogenic
Probes
High-Pure PCR Template Preparation Kit
LightCycler-DNA Master Hybridization Probes
LightCycler glass capillarycuvettes
Procedure
Genomic DNA was isolated from whole blood or buffy coats using the High-Pure
PCR Template Preparation Kit. The DNA was resuspended in 10 mmolll Tris (pH
7.4), containing 0.1 mmoUI EDTA at a concentration of 20 ngl~.
The amplification primers were designed using the primed input program from the
Whitehead Institute for Biomedical Research (website: www-genome.wi.mit.edul
cgi-binlprimer/primer3.cgi). The amplified fragment harboring both polymorphic
sites in exon 1 of the SRD5A2 gene is 198 bp in size (GenBank locus L03843)
(Table 1). The primers were synthesized by standard phosphoramidite chemistry
(MWG-Biotech, Eberswalde, Germany). Primers and probes for the detection of the
alanine-threonine substitution at codon 49 and of the valine-leucine substitution
at codon 89 in exon 1 of the human SRD5A2 gene are given in Table 1.
Two pairs of hybridization probes labeled with different fluorophores were used
in one reaction in order to genotype both polymorphic codons simultaneously. For
codon 49 the LCRed640-labeled detection probe was designed to be complementary to the sense strand of the 49A allele with the polymorphic nucleotide seven
bases from the 3' end (underlined nt.), as shown in Table 1 and Fig. 1. The resulting A-C mismatch between detection probe and the 49T allele is the most unstable mismatch possible with this polymorphism, ensuring a maximum difference in
the melting temperatures (T m) between both alleles. Due to an extremely high GC