Rapid Detection of Missense Mutations in the Prostatic Steroid Sa-Reductase Gene 1m
Results
Forty-five cycles of amplification were performed with genomic DNA with different SRD5A2 genotypes and a template-free control using the fluorescence resonance transfer detection system outlined in Fig. 1. The fluorescence signal was
measured in channel F2 at the end of each annealing phase and increased as
product accumulated. Under our conditions, the fluorescence signal appeared
above background levels after 30 cycles. No increase in fluorescence signal was
observed in the absence of template. When analyzed on an agarose gel, we found
a pure amplification product of the expected size.
The process of hybridization and melting of the detection probes to the target
was monitored by melting curve analysis. By plotting the negative derivative of
the fluorescence signal with temperature versus temperature (-(dP/dTJ vs T),
peaks are obtained at the respective melting temperatures (Tm).
The detection probes match perfectly with the alleles coding for alanine
(sequence GCC) and leucine (sequence CTA) at co dons 49 and 89, respectively.
Accordingly, when examining codon 49, we observed a Tm at 62.5°C with a DNA
homozygous for the sequence GCC, whereas a DNA coding for threonine
(sequence ACC) led to a markedly lower Tm of 51.0°C. Heterozygous samples contain both types of targets and, thus, generated both peaks (Fig. 2a). The fluorescent signal acquired in channel 3 was used to genotype codon 89. However, due to
the interference of fluorescence signals caused by LCRed640, which are also
recorded in channel 3, the emission signal at 705 nm had to be corrected for the
contribution of LCRed640 by utilizing the crosstalk compensation module of the
LightCycler system. By doing so, the alleles coding for leucine (sequence CTA) and
valine (sequence GTA) at codon 89 were clearly distinguishable, with melting
peaks at 57SC and 49.0°C, respectively (Fig. 2b).
With different samples showing different amplification efficiencies, the derivative melting curves were highly reproducible with melting peaks differing by less
than 0.8°C for the same allele, allowing easy and unambiguous assignment of
genotypes to the respective melting curves (Table 2).
To evaluate the reliability of the fluorescence genotyping, 100 human DNA
samples were genotyped for both SRD5A2 polymorphisms by both the conventional allele-specific restriction fragment analysis and the homogenous fluorescence assay. The genotypes determined with both methods were in 100% concordance. The genotyping of the 100 samples on the LightCycler was completed within 3 h, while the ASRA protocol took 12 h and required several manual sample
processing steps.
Comments
The homogeneous protocol presented here combines a number of advantages:
• The fluorescence assay is robust and reliable as documented by the complete
concordance of 100 genotypes determined with both the conventional allelespecific restriction fragment analysis and the LightCycler protocol.
Results
Forty-five cycles of amplification were performed with genomic DNA with different SRD5A2 genotypes and a template-free control using the fluorescence resonance transfer detection system outlined in Fig. 1. The fluorescence signal was
measured in channel F2 at the end of each annealing phase and increased as
product accumulated. Under our conditions, the fluorescence signal appeared
above background levels after 30 cycles. No increase in fluorescence signal was
observed in the absence of template. When analyzed on an agarose gel, we found
a pure amplification product of the expected size.
The process of hybridization and melting of the detection probes to the target
was monitored by melting curve analysis. By plotting the negative derivative of
the fluorescence signal with temperature versus temperature (-(dP/dTJ vs T),
peaks are obtained at the respective melting temperatures (Tm).
The detection probes match perfectly with the alleles coding for alanine
(sequence GCC) and leucine (sequence CTA) at co dons 49 and 89, respectively.
Accordingly, when examining codon 49, we observed a Tm at 62.5°C with a DNA
homozygous for the sequence GCC, whereas a DNA coding for threonine
(sequence ACC) led to a markedly lower Tm of 51.0°C. Heterozygous samples contain both types of targets and, thus, generated both peaks (Fig. 2a). The fluorescent signal acquired in channel 3 was used to genotype codon 89. However, due to
the interference of fluorescence signals caused by LCRed640, which are also
recorded in channel 3, the emission signal at 705 nm had to be corrected for the
contribution of LCRed640 by utilizing the crosstalk compensation module of the
LightCycler system. By doing so, the alleles coding for leucine (sequence CTA) and
valine (sequence GTA) at codon 89 were clearly distinguishable, with melting
peaks at 57SC and 49.0°C, respectively (Fig. 2b).
With different samples showing different amplification efficiencies, the derivative melting curves were highly reproducible with melting peaks differing by less
than 0.8°C for the same allele, allowing easy and unambiguous assignment of
genotypes to the respective melting curves (Table 2).
To evaluate the reliability of the fluorescence genotyping, 100 human DNA
samples were genotyped for both SRD5A2 polymorphisms by both the conventional allele-specific restriction fragment analysis and the homogenous fluorescence assay. The genotypes determined with both methods were in 100% concordance. The genotyping of the 100 samples on the LightCycler was completed within 3 h, while the ASRA protocol took 12 h and required several manual sample
processing steps.
Comments
The homogeneous protocol presented here combines a number of advantages:
• The fluorescence assay is robust and reliable as documented by the complete
concordance of 100 genotypes determined with both the conventional allelespecific restriction fragment analysis and the LightCycler protocol.