High-sensitivity detection of the A3243G mutation of mitochondrial DNA by a combination of allele-specific PCR and peptide nucleic acid-directed PCR clamping.
High-sensitivity detection of the A3243G mutation of mitochondrial DNA by a combination of allele-specific PCR and peptide nucleic acid-directed PCR clamping.
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DOI：
10.1373/clinchem.2004.033761
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发表时间：
2004-11
影响因子：
9.3
通讯作者：
M. Urata;Y. Wada;S. H. Kim;Worawan Chumpia;Y. Kayamori;N. Hamasaki;D. Kang
中科院分区：
文献类型：
--
作者：
M. Urata;Y. Wada;S. H. Kim;Worawan Chumpia;Y. Kayamori;N. Hamasaki;D. Kang
背景线粒体DNA（mtDNA）的A3243G突变与许多常见疾病有关，包括糖尿病和伴有乳酸性酸中毒和中风样发作的线粒体脑肌病（MELAS）。对于这种突变的检测，等位基因特异性PCR灵敏度很高，但需要严格控制PCR条件；因此它不足以用于常规临床测试。我们的目标是开发一种常规可用的 PCR 方法，用于定量检测 A3243G 突变的低水平异质性。方法在肽核酸(PNA)存在下进行A3243G突变的定量等位基因特异性PCR，其中PNA与野生型mtDNA互补，其中一种引物的3'末端与突变体的核苷酸位置3243匹配。结果 通过我们的方法，在多种条件下（DNA，5-100 ng；DNA，5-100 ng），野生型 mtDNA 的扩增与突变型 mtDNA 的扩增相比被抑制了 7000 倍。退火温度，61-66℃；和PNA，1.5-3.5微摩尔/升。因此，A3243G 突变的 0.1% 异质性可以通过该方法可靠地定量。 40 名健康志愿者的血液样本显示异质性 <0.06%，表明 0.1% 具有诊断意义。结论 PNA 在多种条件下保持了等位基因特异性 PCR 的特异性，这对于常规临床检测非常重要。
BACKGROUND The A3243G mutation of mitochondrial DNA (mtDNA) is involved in many common diseases, including diabetes mellitus and mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (MELAS). For detection of this mutation, allele-specific PCR is highly sensitive but requires strict control of PCR conditions; it thus is not adequate for a routine clinical test. We aimed to develop a routinely available PCR method for quantitative detection of low-level heteroplasmy of the A3243G mutation. METHODS Quantitative allele-specific PCR for the A3243G mutation was performed in the presence of peptide nucleic acid (PNA), in which PNA is complementary to the wild-type mtDNA, with one primer having a 3' end matched to nucleotide position 3243 of the mutant. RESULTS With our method, amplification of wild-type mtDNA was suppressed 7000-fold compared with amplification of the mutant mtDNA under a broad range of conditions: DNA, 5-100 ng; annealing temperature, 61-66 degrees C; and PNA, 1.5-3.5 micromol/L. Hence, 0.1% heteroplasmy of the A3243G mutation can be reliably quantified by this method. Blood samples form 40 healthy volunteers showed <0.06% heteroplasmy, suggesting that 0.1% is diagnostically significant. CONCLUSIONS PNA maintains the specificity of allele-specific PCR over a wide range of conditions, which is important for routine clinical testing.