Amplicon DNA melting analysis for mutation scanning and genotyping: Cross-platform comparison of instruments and dyes
Amplicon DNA melting analysis for mutation scanning and genotyping: Cross-platform comparison of instruments and dyes
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DOI：
10.1373/clinchem.2005.063438
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发表时间：
2006-03-01
影响因子：
9.3
通讯作者：
Voelkerding, KV
中科院分区：
文献类型：
--
作者：
Herrmann, MG;Durtschi, JD;Voelkerding, KV
背景资料：最近报道了使用具有特殊“饱和”染料的高分辨率仪器进行PCR产物的基因分型和突变扫描的DNA解链分析。其他仪器和染料的比较性能尚未evaluated.Methods：在饱和DNA染料LC绿色Plus或SYBR绿色I存在下，通过PCR扩增β-珠蛋白基因的110 bp片段，包括镰状细胞性贫血位点（A17 T）。将3种不同基因型（野生型、杂合型和纯合型突变体）的扩增子在9种不同仪器（ABI 7000和7900 HT、Bio-Rad iCycler、Cepheid SmartCycler、Corbett Rotor-Gene 3000、爱达荷州Technology HR-1和LightScanner以及Roche LightCycler 1.2和LightCycler 2.0）上以a.速率为0.1 ℃/s或按照制造商的建议。每个仪器/染料组合的能力，基因型的熔解温度（T-M）和扫描杂合子的曲线shape进行了evaluated.Results：分辨率之间的差异很大，仪器之间的T-M SD（0.018至0.274摄氏度）和19倍（LC绿色Plus）或33倍（SYBR绿色1）的信噪比差异的15倍。这些因素限制了大多数仪器通过扩增子熔解准确地对单核苷酸多态性进行基因分型的能力。平板仪器（96孔）显示出最大的差异，平板间存在空间差异。SYBR绿色I和LC绿色Plus均可用于T-m基因分型，但LC绿色Plus可用于杂合子扫描。但是，由于光谱不匹配，LCGreen Plus不能用于带有氩激光器的仪器。所有与LCGreen Plus兼容的仪器都是。能够通过改变熔解曲线形状来检测杂合子。然而，专门设计用于高分辨率熔解的仪器显示最少的变化，这表明更好的扫描灵敏度和specificity.Conclusion：不同的仪器和染料的基因型纯合子变体和扫描杂合子的能力相差很大的全扩增子熔解分析。(C)2006年美国临床化学协会。
Background: DNA melting analysis for genotyping and mutation scanning of PCR products by use of high-resolution instruments with special "saturation" dyes has recently been reported. The comparative performance of other instruments and dyes has not been evaluated.Methods: A 110-bp fragment of the beta-globin gene including the sickle cell anemia locus (A17T) was amplified by PCR in the presence of either the saturating DNA dye, LCGreen Plus, or SYBR Green I. Amplicons of 3 different genotypes (wild-type, heterozygous, and homozygous mutants) were melted on 9 different instruments (ABI 7000 and 7900HT, Bio-Rad iCycler, Cepheid SmartCycler, Corbett Rotor-Gene 3000, Idaho Technology HR-1 and LightScanner, and the Roche LightCycler 1.2 and LightCycler 2.0) at a. rate of 0.1 degrees C/s or as recommended by the manufacturer. The ability of each instrument/dye combination to genotype by melting temperature (T-m) and to scan for heterozygotes by curve shape was evaluated.Results: Resolution varied greatly among instruments with a 15-fold difference in T-m SD (0.018 to 0.274 degrees C) and a 19-fold (LCGreen Plus) or 33-fold (SYBR Green 1) difference in the signal-to-noise ratio. These factors limit the ability of most instruments to accurately genotype single-nucleotide polymorphisms by amplicon melting. Plate instruments (96-well) showed the greatest variance with spatial differences across the plates. Either SYBR Green I or LCGreen Plus could be used for genotyping by T-m, but only LCGreen Plus was useful for heterozygote scanning. However, LCGreen Plus could not be used on instruments with an argon laser because of spectral mismatch. All instruments compatible with LCGreen Plus were. able to detect heterozygotes by altered melting curve shape. However, instruments specifically designed for high-resolution melting displayed the least variation, suggesting better scanning sensitivity and specificity.Conclusion: Different instruments and dyes vary widely in their ability to genotype homozygous variants and scan for heterozygotes by whole-amplicon melting analysis. (C) 2006 American Association for Clinical Chemistry.