Real-time fluorescence genotyping of factor V Leiden during rapid-cycle PCR.
Real-time fluorescence genotyping of factor V Leiden during rapid-cycle PCR.
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DOI：
10.1093/clinchem/43.12.2262
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发表时间：
1997-12
影响因子：
9.3
通讯作者：
Marla J. Lay;C. Wittwer
中科院分区：
文献类型：
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作者：
Marla J. Lay;C. Wittwer
一个单步方法因子V莱顿基因分型，使用快速循环PCR和共振能量转移探针的同时荧光分析。在覆盖突变位点的3 ′-荧光素标记的探针存在下，通过使用Cy 5标记的引物不对称扩增含有突变的因子V基因片段。当荧光素探针与Cy 5标记的引物的延伸产物退火时，荧光团足够紧密地接触以发生共振能量转移。随着温度的升高，探针从其目标熔化，降低了共振能量转移。当探针与产物链互补时，它在65 ℃解链;如果存在单碱基突变，探针在57 ℃解链。同时扩增和分析基因组DNA需要20-45分钟，并且在荧光热循环仪加载后不需要样品操作。
A single-step method for factor V Leiden genotyping is presented that uses rapid-cycle PCR and simultaneous fluorescence analysis with resonance energy transfer probes. A fragment of the factor V gene containing the mutation is amplified asymmetrically through use of a primer labeled with Cy5 in the presence of a 3'-fluorescein-labeled probe that covers the mutation site. When the fluorescein probe is annealed to the extension product of the Cy5-labeled primer, the fluorophores are brought into close enough contact for resonance energy transfer to occur. As the temperature increases, the probe melts from its target, decreasing the resonance energy transfer. When the probe is complementary to the product strand, it melts at 65 degrees C; if the single-base mutation is present, the probe melts at 57 degrees C. Concurrent amplification and analysis from genomic DNA takes 20-45 min and requires no sample manipulation after the fluorescence thermal cycler is loaded.