Seipp Michael T, Pattison David, Durtschi Jacob D, Jama Mohamed, Voelkerding Karl V, Wittwer Carl T
ARUP Institute for Clinical and Experimental Pathology, ARUP Laboratories, 500 Chipeta Way, Salt Lake City, UT 84108, USA.
Clin Chem. 2008 Jan;54(1):108-15. doi: 10.1373/clinchem.2007.097121. Epub 2007 Nov 2.
Multiplexed amplicon melting is a closed-tube method for genotyping that does not require probes, real-time analysis, asymmetric PCR, or allele-specific PCR; however, correct differentiation of homozygous mutant and wild-type samples by melting temperature (T(m)) analysis requires high-resolution melting analysis and controlled reaction conditions.
We designed 4 amplicons bracketing the F5 [coagulation factor V (proaccelerin, labile factor)] 1691G>A, MTHFR (NADPH) 1298A>C, MTHFR 677C>T, and F2 [coagulation factor II (thrombin)] 20210G>A gene variants to melt at different temperatures by varying amplicon length and adding GC- or AT-rich 5' tails to selected primers. We used rapid-cycle PCRs with cycles of 19-23 s in the presence of a saturating DNA dye and temperature-correction controls and then conducted a high-resolution melting analysis. Heterozygotes were identified at each locus by curve shape, and homozygous genotypes were assigned by T(m). We blinded samples previously genotyped by other methods before analysis with the multiplex melting assay (n = 110).
All samples were correctly genotyped with the exception of 7 MTHFR 1298 samples with atypical melting profiles that could not be assigned. Sequencing revealed that these 5 heterozygotes and 2 homozygotes contained the unexpected sequence variant MTHFR 1317T>C. The use of temperature-correction controls decreased the T(m) SD within homozygotes by a mean of 38%.
Rapid-cycle PCR with high-resolution melting analysis allows simple and accurate multiplex genotyping to at least a factor of 4.
多重扩增子熔解是一种用于基因分型的闭管方法，不需要探针、实时分析、不对称PCR或等位基因特异性PCR；然而，通过熔解温度（T(m)）分析正确区分纯合突变体和野生型样本需要高分辨率熔解分析和可控的反应条件。
我们设计了4个扩增子，覆盖凝血因子V（促凝血球蛋白，不稳定因子）F5基因1691G>A、亚甲基四氢叶酸还原酶（NADPH）MTHFR基因1298A>C、MTHFR基因677C>T以及凝血因子II（凝血酶）F2基因20210G>A的基因变异位点，通过改变扩增子长度以及在选定引物的5'端添加富含GC或AT的尾巴，使这些扩增子在不同温度下熔解。我们在饱和DNA染料存在及温度校正对照的情况下，使用循环时间为19 - 23秒的快速循环PCR，然后进行高分辨率熔解分析。通过曲线形状在每个位点鉴定杂合子，并通过T(m)确定纯合基因型。在使用多重熔解分析进行分析之前，我们对先前通过其他方法进行基因分型的样本进行了盲法处理（n = 110）。
除了7个MTHFR 1298样本具有无法分型的非典型熔解曲线外，所有样本均被正确基因分型。测序显示，这5个杂合子和2个纯合子包含意外的序列变异MTHFR 1317T>C。使用温度校正对照使纯合子内的T(m)标准差平均降低了38%。
具有高分辨率熔解分析的快速循环PCR能够实现至少4倍的简单而准确的多重基因分型。
