Rapid genotyping of paraoxonase 55 and 192 mutations by melting point analysis using real time PCR technology.
BACKGROUND
Paraoxonase (PON1) enzyme was identified as one of the components of HDL responsible for prevention of lipid peroxides accumulation in low-density lipoprotein (LDL). A triphasic phenotypic frequency distribution of PON1 activity was shown in the human population resulted by two nucleotide interchanges at residues 55 and 192. The paraoxonase isoforms have different effectiveness in hydrolysing lipid peroxides.


METHODS
To date, genotyping for PON1 is mainly performed by PCR RFLP technique, that is time consuming and sensitive to contamination. We developed highly reliable single-step methods for genotyping both PON1 55 and 192 polymorphisms using LightCycler real time PCR technology based on fluorescence resonance energy transfer. After the ultrafast PCR, melting point analysis was performed and fluorescence intensity was monitored simultaneously with slow heating.


CONCLUSIONS
The observed melting temperatures in the PON1 55 and 192 melting point analyses characteristic to the oligonucleotides hybridised to the mutant and wild-type DNA were 57 degrees C, 61 degrees C and 51.5 degrees C, 57.5 degrees C, respectively. The temperature differences in melting points (4 degrees C and 6 degrees C, respectively) offer a powerful tool for rapid, reliable mutation detection for 55 and 192 polymorphisms even in routine diagnostic laboratories or large epidemiological studies.
Pocsai
Zsuzsa
Z
Department of Preventive Medicine, School of Public Health, Medical and Health Science Centre, University of Debrecen, P.O. Box 9, Debrecen H-4012, Hungary.
Tóth
Zsuzsa
Z
Paragh
György
G
Széles
György
G
Adány
Róza
R
D043303:Aryldialkylphosphatase / Q000235:genetics / Q000378:metabolism*; D004252:DNA Mutational Analysis / Q000379:methods*; D017931:DNA Primers; D005453:Fluorescence; D005838:Genotype; D006801:Humans; D009154:Mutation; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes; D016133:Polymerase Chain Reaction / Q000379:methods*; D011110:Polymorphism, Genetic; D015203:Reproducibility of Results; D044366:Transition Temperature
D017931:DNA Primers; D015345:Oligonucleotide Probes; D043303:Aryldialkylphosphatase