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Expanded Instrument Comparison of Amplicon DNA Melting Analysis for Mutation Scanning and Genotyping
Clin Chem, 53, 8, 1544 - 1548
Methods: A 110-bp fragment of the β-globin gene including the sickle cell anemia locus (HBB c. 20A>T) was amplified by PCR in the presence of LCGreen Plus or SYBR Green I. Amplicons of 4 different genotypes [wild-type, homozygous, and heterozygous HBB c. 20A>T and double-heterozygote HBB c. (9C>T; 20A>T)] were melted on 7 different instruments [Applied Biosystems 7300, Corbett Life Sciences Rotor-Gene 6500HRM, Eppendorf Mastercycler RealPlex4S, Idaho Technology LightScanner (384 well), Roche LightCycler 480 (96 and 384 well) and Stratagene Mx3005p] at a rate of 0.61° C/s or when this was not possible, at 0.50° C steps. We evaluated the ability of each instrument to genotype by melting temperature (Tm ) and to scan for heterozygotes by curve shape.
Results: The ability of most instruments to accurately genotype single-base changes by amplicon melting was limited by spatial temperature variation across the plate (SD of Tm = 0.020 to 0.264° C). Other variables such as data density, signal-to-noise ratio, and melting rate also affected heterozygote scanning.
Conclusions: Different instruments vary widely in their ability to genotype homozygous variants and scan for heterozygotes by whole amplicon melting analysis. Instruments specifically designed for high-resolution melting, however, displayed the least variation, suggesting better genotyping accuracy and scanning sensitivity and specificity.
Methods, 50, 4, S19-S22
Use of the Roche LightCycler Strep B Assay for Detection of Group B Streptococcus from Vaginal and Rectal Swabs
J Clin Microbiol, 43, 8, 4046 - 4051
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