Conditions of High Resolution Melting Analysis on the Cobas z480 Instrument for the Genotyping of VKORC1 in the Clinical Routine Laboratory.
High resolution melting (HRM) of amplicons is a simple method for genotyping of single nucleotide polymorphisms (SNPs). Albeit many applications reported, HRM seems to be rarely used in clinical laboratories. The suitability of HRM-PCR for the clinical laboratory was investigated for genotyping of SNPs of the vitamin K epoxide reductase complex unit 1 gene.



About 100 DNA samples were analyzed by two different HRM-PCRs on the Cobas z480 instrument and compared with a PCR with fluorescently labeled probes (HybProbe-PCR) on the LightCycler 2.0 instrument as reference.



Reliable genotyping with 100% matching results was obtained, when the amplicon size was small (63 bp) and DNA input was limited by e.g., sample dilution with salt-free water.



DNA extracted by differing methods may be used for genotyping by HRM-PCR. Compared with HybProbe-PCR, HRM-PCR on the Cobas z480 instrument allows for higher through-put, however, at the cost of a higher degree of laboratory standardization and a slower turnaround.
Christian
C
Paar
Verena
V
Hammerl
Hermann
H
Blessberger
Herbert
H
Stekel
Clemens
C
Steinwender
Jörg
J
Berg
D004867:Equipment Design; D005820:Genetic Testing / Q000295:instrumentation / Q000379:methods*; D005838:Genotype; D059014:High-Throughput Nucleotide Sequencing / Q000295:instrumentation; D006801:Humans; D009691:Nucleic Acid Denaturation; D016133:Polymerase Chain Reaction / Q000295:instrumentation; D020641:Polymorphism, Single Nucleotide; D011237:Predictive Value of Tests; D015203:Reproducibility of Results; D064417:Vitamin K Epoxide Reductases / Q000235:genetics*
C578523:VKORC1 protein, human; D064417:Vitamin K Epoxide Reductases