A novel method of detecting mitochondrial C1494T and A1555G mutations by using the base-quenched probe technique in a single PCR assay / 中华耳鼻咽喉头颈外科杂志

ABSTRACT

<p><b>OBJECTIVE</b>In the present study we describe a new method to detect the mitochondrial DNA (mtDNA) mutations A1555G and C1494T by using the base-quenched probe technique in a single PCR reaction.</p><p><b>METHODS</b>6-carboxyfluorescein (FAM) was directly conjugated to the 3' end of the probe. Four vectors, representing the four possible genotype combinations, were constructed as the amplification template for the methodology established. In present study A1555G and C1494T mutations in 117 individuals with hearing loss were detected by the base-quenched probe method and were further validated by the direct DNA sequencing analyses.</p><p><b>RESULTS</b>From the melting curve we could distinguish the four haplotypes accurately. And there were complete concordance between the base-quenched probe method and direct DNA sequencing.</p><p><b>CONCLUSION</b>This method is suitable for clinical test of mtDNA mutations A1555G and C1494T in individuals with hearing loss.</p>