Clinical chemistry · 2007
Expanded instrument comparison of amplicon DNA melting analysis for mutation scanning and genotyping.
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Herrmann Mark G, Durtschi Jacob D, Wittwer Carl T, Voelkerding Karl V
Аннотация
BACKGROUND: Additional instruments have become available since instruments for DNA melting analysis of PCR products for genotyping and mutation scanning were compared. We assessed the performance of these new instruments for genotyping and scanning for mutations.
METHODS: A 110-bp fragment of the beta-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 degrees C/s or when this was not possible, at 0.50 degrees 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 degrees 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: A 110-bp fragment of the beta-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 degrees C/s or when this was not possible, at 0.50 degrees 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 degrees 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.