Expanded instrument comparison of amplicon DNA melting analysis for mutation scanning and genotyping
Expanded instrument comparison of amplicon DNA melting analysis for mutation scanning and genotyping
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DOI：
10.1373/clinchem.2007.088120
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发表时间：
2007-08-01
影响因子：
9.3
通讯作者：
Voelkerding, Karl V.
中科院分区：
文献类型：
--
作者：
Herrmann, Mark G.;Durtschi, Jacob D.;Voelkerding, Karl V.
背景资料：自从对用于基因分型和突变扫描的PCR产物的DNA熔解分析仪器进行比较以来，已有更多的仪器可供使用。我们评估了这些新仪器的基因分型和扫描mutation.Methods的性能：一个110 bp片段的β-珠蛋白基因，包括镰状细胞贫血基因座（HBB c。20 A> T）在LC绿色Plus或SYBR绿色I存在下通过PCR扩增。4种不同基因型的扩增子[野生型、纯合子和杂合子HBB c. 20 A> T和双杂合子HBB c. (9C> T，20 A> T）]在7种不同仪器[Applied Bio-systems 7300，Corbett Life Sciences Rotor-Gene 6500 HRM，Eppendorf Mastercycler RealPlex 4S，爱达荷州Technology LightScanner（384孔）、Roche LightCycler 480（96和384孔）和Stratagene Mx3005 p]以0.61 ° C/s的速率或当这不可能时以0.50 ° C步长进行。我们评估了每种仪器的能力，基因型的熔解温度（T-m）和扫描杂合子的曲线shape.Results：大多数仪器的能力，以准确的基因型单碱基的变化扩增子熔解是有限的空间温度变化在整个板（SD的T-m = 0.020至0.264摄氏度）。其他变量，如数据密度，信噪比，熔解率也影响杂合子scanning.Conclusions：不同的仪器有很大的不同，在他们的能力基因型纯合子变异和扫描杂合子的全扩增子熔解分析。然而，专门为高分辨率熔解设计的仪器显示出最小的变化，表明更好的基因分型准确性和扫描灵敏度和特异性。(c)2007年美国临床化学协会。
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 Bio-systems 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 (T-m) 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 T-m = 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. (c) 2007 American Association for Clinical Chemistry.