BACKGROUND: High resolution melting is a post-PCR-based method for detecting DNA sequence variation by measuring changes in the melting of a DNA duplex. Melting of double-stranded DNA molecules is influenced by several factors. We evaluated the influence of the DNA isolation method in the melting curve analysis to detect genetic variations. METHODS: We isolated DNA from whole blood of 547 subjects by two different methods: Maxwell 16 Instrument and DNA FlexiGene Kit. A fragment of 159 bp was amplified and analyzed by high resolution melting. Those samples that showed a different melting curve pattern were sequenced. RESULTS: Of the samples extracted with the Maxwell 16 Instrument, 42% showed variation compared with 0.18% of the samples extracted with DNA FlexiGene Kit. After sequencing, we showed that all samples extracted with the Maxwell 16 Instrument were false positive except one, which coincided with the only sample that showed variation in those extracted with the DNA FlexiGene Kit. CONCLUSION: The method used to extract DNA is an important factor to consider in the analysis of melting curves obtained by high resolution melting, as it may influence the melting behaviour of the samples, giving false positive results in the detection of genetic variants.
BACKGROUND: High resolution melting is a post-PCR-based method for detecting DNA sequence variation by measuring changes in the melting of a DNA duplex. Melting of double-stranded DNA molecules is influenced by several factors. We evaluated the influence of the DNA isolation method in the melting curve analysis to detect genetic variations. METHODS: We isolated DNA from whole blood of 547 subjects by two different methods: Maxwell 16 Instrument and DNA FlexiGene Kit. A fragment of 159 bp was amplified and analyzed by high resolution melting. Those samples that showed a different melting curve pattern were sequenced. RESULTS: Of the samples extracted with the Maxwell 16 Instrument, 42% showed variation compared with 0.18% of the samples extracted with DNA FlexiGene Kit. After sequencing, we showed that all samples extracted with the Maxwell 16 Instrument were false positive except one, which coincided with the only sample that showed variation in those extracted with the DNA FlexiGene Kit. CONCLUSION: The method used to extract DNA is an important factor to consider in the analysis of melting curves obtained by high resolution melting, as it may influence the melting behaviour of the samples, giving false positive results in the detection of genetic variants.
Authors: Katja Zappe; Christine Pirker; Heidi Miedl; Martin Schreiber; Petra Heffeter; Georg Pfeiler; Stefan Hacker; Werner Haslik; Sabine Spiegl-Kreinecker; Margit Cichna-Markl Journal: Int J Mol Sci Date: 2021-11-20 Impact factor: 5.923