High-Speed Melting Analysis: The Effect of Melting Rate on Small Amplicon Microfluidic Genotyping.
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 sin...
| Publicado en: | Clinical Chemistry Vol. 63; no. 10; pp. 1624 - 1633 |
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| Autores principales: | , , , , , , |
| Formato: | Journal Article |
| Publicado: |
Oxford University Press / USA
2017
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| Acceso en línea: | Ver este registro en EBSCOhost |
| fields | @attributes: recordID: 1 pdfLink: plink: https://search.ebscohost.com/login.aspx?direct=true&db=ccm&AN=126388742&site=ehost-live header: @attributes: shortDbName: ccm uiTerm: 126388742 longDbName: CINAHL Complete uiTag: AN controlInfo: bkinfo: dissinfo: jinfo: jid: 00099147 10CS jtl: Clinical Chemistry issn: 00099147 maglogo: N pubinfo: dt: 2017 vid: 63 iid: 10 pid: 622 pub: Oxford University Press / USA artinfo: ui: 126388742 10.1373/clinchem.2017.276147 126388742 ppf: 1624 ppct: 9 formats: fmt: @attributes: type: P tig: atl: High-Speed Melting Analysis: The Effect of Melting Rate on Small Amplicon Microfluidic Genotyping. aug: au: Pryor, Robert J. Myrick, Joseph T. Palais, Robert A. Sundberg, Scott O. Paek, Jeanette Y. Wittwer, Carl T. Knight, Ivor T. affil: Department of Pathology, University of Utah Health Sciences Center, Salt Lake City, UT sug: ab: 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. pubtype: Academic Journal doctype: Journal Article ougenre: Article language: English refInfo: holdings: @attributes: islocal: N |
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