Herrmann Mark G, Durtschi Jacob D, Bromley L Kathryn, Wittwer Carl T, Voelkerding Karl V
Institute for Clinical and Experimental Pathology, ARUP, Salt Lake City, UT, USA.
Clin Chem. 2006 Mar;52(3):494-503. doi: 10.1373/clinchem.2005.063438. Epub 2006 Jan 19.
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.
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 (Tm) and to scan for heterozygotes by curve shape was evaluated.
Resolution varied greatly among instruments with a 15-fold difference in Tm SD (0.018 to 0.274 degrees C) and a 19-fold (LCGreen Plus) or 33-fold (SYBR Green I) 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.
Different instruments and dyes vary widely in their ability to genotype homozygous variants and scan for heterozygotes by whole-amplicon melting analysis.
最近有报道称，利用配备特殊“饱和”染料的高分辨率仪器对聚合酶链反应（PCR）产物进行基因分型和突变扫描的DNA熔解分析。其他仪器和染料的比较性能尚未得到评估。
在饱和DNA染料LCGreen Plus或SYBR Green I存在的情况下，通过PCR扩增包含镰状细胞贫血位点（A17T）的β-珠蛋白基因的110bp片段。3种不同基因型（野生型、杂合子和纯合突变体）的扩增子在9种不同仪器（ABI 7000和7900HT、Bio-Rad iCycler、Cepheid SmartCycler、Corbett Rotor-Gene 3000、Idaho Technology HR-1和LightScanner，以及罗氏LightCycler 1.2和LightCycler 2.0）上以0.1℃/秒的速率或按照制造商的建议进行熔解。评估了每种仪器/染料组合通过熔解温度（Tm）进行基因分型以及通过曲线形状扫描杂合子的能力。
不同仪器之间的分辨率差异很大，Tm标准差相差15倍（0.018至0.274℃），信噪比相差19倍（LCGreen Plus）或33倍（SYBR Green I）。这些因素限制了大多数仪器通过扩增子熔解对单核苷酸多态性进行准确基因分型的能力。平板仪器（96孔）表现出最大的差异，平板上存在空间差异。SYBR Green I或LCGreen Plus均可用于通过Tm进行基因分型，但只有LCGreen Plus可用于杂合子扫描。然而，由于光谱不匹配，LCGreen Plus不能用于配备氩离子激光的仪器。所有与LCGreen Plus兼容的仪器都能够通过改变熔解曲线形状检测杂合子。然而，专门为高分辨率熔解设计的仪器变化最小，表明具有更好的扫描灵敏度和特异性。
不同的仪器和染料在通过全扩增子熔解分析对纯合变异进行基因分型和扫描杂合子的能力方面差异很大。
