Abstract:
BACKGROUND:High-resolution DNA melting analysis of small amplicons is a simple and inexpensive technique for genotyping. Microfluidics allows precise and rapid control of temperature during melting. METHODS:Using a microfluidic platform for serial PCR and melting analysis, 4 targets containing single nucleotide variants were amplified and then melted at different rates over a 250-fold range from 0.13 to 32 °C/s. Genotypes (n = 1728) were determined manually by visual inspection after background removal, normalization, and conversion to negative derivative plots. Differences between genotypes were quantified by a genotype discrimination ratio on the basis of inter- and intragenotype differences using the absolute value of the maximum vertical difference between curves as a metric. RESULTS:Different homozygous curves were genotyped by melting temperature and heterozygous curves were identified by shape. Technical artifacts preventing analysis (0.3%), incorrect (0.06%), and indeterminate (0.4%) results were minimal, occurring mostly at slow melting rates (0.13-0.5 °C/s). Genotype discrimination was maximal at around 8 °C/s (2-8 °C/s for homozygotes and 8-16 °C/s for heterozygotes), and no genotyping errors were made at rates >0.5 °C/s. PCR was completed in 10-12.2 min, followed by melting curve acquisition in 4 min down to <1 s. CONCLUSIONS:Microfluidics enables genotyping by melting analysis at rates up to 32 °C/s, requiring <1 s to acquire an entire melting curve. High-speed melting reduces the time for melting analysis, decreases errors, and improves genotype discrimination of small amplicons. Combined with extreme PCR, high-speed melting promises nucleic acid amplification and genotyping in < 1 min.
journal_name
Clin Chemjournal_title
Clinical chemistryauthors
Pryor RJ,Myrick JT,Palais RA,Sundberg SO,Paek JY,Wittwer CT,Knight ITdoi
10.1373/clinchem.2017.276147subject
Has Abstractpub_date
2017-10-01 00:00:00pages
1624-1632issue
10eissn
0009-9147issn
1530-8561pii
clinchem.2017.276147journal_volume
63pub_type
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