Sensitive Detection and Quantification of the JAK2V617F Allele by Real-Time PCR Blocking Wild-Type Amplification by Using a Peptide Nucleic Acid Oligonucleotide
Sensitive Detection and Quantification of the JAK2V617F Allele by Real-Time PCR Blocking Wild-Type Amplification by Using a Peptide Nucleic Acid Oligonucleotide
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DOI：
10.1016/j.jmoldx.2011.04.002
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发表时间：
2011-09-01
影响因子：
4.1
通讯作者：
Hermans, Mirjam H. A.
中科院分区：
文献类型：
--
作者：
Huijsmans, Cornelis J. J.;Poodt, Jeroen;Hermans, Mirjam H. A.
JAK2酪氨酸激酶基因中单个G-to-T错义突变导致V617F氨基酸替代，在几种骨髓增殖性肿瘤中很常见。这种突变等位基因的可靠定量对预测和诊断这组肿瘤具有越来越重要的临床和治疗意义。由于JAK2V617F是体细胞获得的，并且可能随后失去杂合性，因此血液中突变型DNA与野生型DNA的百分比可以在0%到几乎100%之间变化。因此，我们开发了一种实时PCR检测和定量JAK2V617F等位基因负荷的低至高范围。为了使实验符合这些标准，用一种肽核酸寡核苷酸阻断野生型JAK2的扩增。JAK2V617F患者DNA在JAK2野生型DNA中稀释后可线性扩增，扩增范围从0.05%到100%，定量重现性可接受。该方法的灵敏度为0.05% （n = 3 / 3）。在100名健康献血者中的9名中，从血液中分离的DNA中观察到微弱的阳性/背景信号，对应于约0.01%的JAK2V617F等位基因。在一个健康个体中，我们观察到这个信号是重复的。这一发现的临床意义尚不清楚。通过抑制野生型等位基因的扩增，我们开发了一种灵敏的线性实时PCR检测和定量JAK2V617F。[J] .医学诊断杂志，2011,13:558-564；10.1016 / j.jmoldx.2011.04.002)
A single G-to-T missense mutation in the gene for the JAK2 tyrosine kinase, leading to a V617F amino acid substitution, is commonly found in several myeloproliferative neoplasms. Reliable quantification of this mutant allele is of increasing clinical and therapeutic interest in predicting and diagnosing this group of neoplasms. Because JAK2V617F is somatically acquired and may be followed by loss of heterozygosity, the percentage of mutant versus wild-type DNA in blood can vary between 0% and almost 100%. Therefore, we developed a real-time PCR assay for detection and quantification of the low-to-high range of the JAK2V617F allele burden. To allow the assay to meet these criteria, amplification of the wild-type JAK2 was blocked with a peptide nucleic acid oligonucleotide. JAK2V617F patient DNA diluted in JAK2 wild-type DNA could be amplified linearly from 0.05% to 100%, with acceptable reproducibility of quantification. The sensitivity of the assay was 0.05% (n = 3 of 3). In 9 of 100 healthy blood donors, a weak positive/background signal was observed in DNA isolated from blood, corresponding to approximately 0.01% JAK2V617F allele. In one healthy individual, we observed this signal in duplicate. The clinical relevance of this finding is not clear. By inhibiting amplification of the wild-type allele, we developed a sensitive and linear real-time PCR assay to detect and quantify JAK2V617F. (J Mol Diagn 2011, 13:558-564; DOI. 10.1016/j.jmoldx.2011.04.002)