In:
JALA: Journal of the Association for Laboratory Automation, SAGE Publications, Vol. 11, No. 4 ( 2006-08), p. 273-277
Kurzfassung:
DNA melting analysis for the identification of sequence is increasingly used in molecular diagnostics. Recent advances in DNA melting analysis, including high-resolution instrumentation and specialized fluorescent DNA-binding dyes, allow genotyping by whole amplicon melting without probes. With the popularity of melting analysis as a diagnostic tool, there is a need to characterize the ability of commercially available real-time PCR instruments to perform high-resolution amplicon melting analyses. Four real-time instruments varying in sample format, throughput, and heat transfer (Cepheid's SmartCycler, Idaho Technology's HR-1, and Roche's LightCycler 1.2 and LightCycler 2.0) were evaluated for their ability to differentiate homozygous genotypes at the human β-globin sickle cell locus. The melting transition was monitored by including the dye LCGreen Plus in the PCR, and the data were uniformly analyzed with custom in-house software. The wild-type and mutant homozygous genotypes differed by a theoretical T m of 0.09°C and were best discriminated by the high-resolution HR-1 instrument. All instruments could identify a double single nucleotide polymorphism heterozygote by the heteroduplexes formed. However, signal-to-noise ratios varied from 260 to 3500, suggesting that melting instrument design (data acquisition, data density, thermal control) determines the accuracy of genotyping by amplicon melting. (JALA 2006;11:273-7)
Materialart:
Online-Ressource
ISSN:
1535-5535
DOI:
10.1016/j.jala.2006.04.003
Sprache:
Englisch
Verlag:
SAGE Publications
Publikationsdatum:
2006
ZDB Id:
2593238-X
ZDB Id:
2900310-6
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