Petiti Jessica, Rosso Valentina, Croce Eleonora, Franceschi Vanessa, Andreani Giacomo, Dragani Matteo, De Gobbi Marco, Lunghi Monia, Saglio Giuseppe, Fava Carmen, Lo Iacono Marco, Cilloni Daniela
Department of Clinical and Biological Sciences of the University of Turin, San Luigi Gonzaga Hospital, Regione Gonzole 10, 10043 Orbassano (Turin), Italy.
Division of Hematology, Department of Translational Medicine, Università del Piemonte Orientale, Corso Giuseppe Mazzini, 18, 28100 Novara, Italy.
J Clin Med. 2020 Jan 19;9(1):271. doi: 10.3390/jcm9010271.
Acute myeloid leukemia is a heterogeneous hematological disease, characterized by karyotypic and molecular alterations. Mutations in have a role in diagnosis and as a minimal residue disease marker. Often the variant allele frequency during follow up is less than 20%, which represents the limit of detection of Sanger sequencing. Therefore, the development of sensitive methodologies to identify mutations might help to monitor patients' response to therapy. We compared three different methods to identify and monitor mutations in patients' specimens.
Performances of PNA-PCR clamping, droplet digital PCR and Sanger for status identification were evaluated and compared in 96 DNA patients' specimens.
In contrast with Sanger sequencing, our results highlighted the concordance between PNA clamping and digital PCR. Furthermore, PNA-PCR clamping was able to detect more mutated DNA with respect to Sanger sequencing that showed several false negatives independently from the allelic frequency.
We found that PNA-PCR clamping and digital PCR identified mutations in DNA samples with comparable results in a percentage significantly higher compared to Sanger sequencing. PNA-PCR clamping can be used even in laboratories not equipped for sophisticated analyses, decreasing cost and time for characterization.
急性髓系白血病是一种异质性血液疾病，其特征为核型和分子改变。[具体基因名称]中的突变在诊断及作为微小残留病标志物方面发挥作用。随访期间变异等位基因频率通常低于20%，这代表了桑格测序的检测极限。因此，开发敏感的方法来识别[具体基因名称]突变可能有助于监测患者对治疗的反应。我们比较了三种不同方法来识别和监测患者样本中的[具体基因名称]突变。
在96份患者DNA样本中评估并比较肽核酸聚合酶链反应（PNA-PCR）钳夹法、液滴数字PCR和桑格测序法对[具体基因状态]的识别性能。
与桑格测序不同，我们的结果突出了PNA钳夹法和数字PCR之间的一致性。此外，相对于桑格测序，PNA-PCR钳夹法能够检测到更多的突变DNA，桑格测序显示出一些与等位基因频率无关的假阴性结果。
我们发现PNA-PCR钳夹法和数字PCR在识别DNA样本中的[具体基因名称]突变方面结果相当，与桑格测序相比，显著更高比例的样本能得到可比结果。即使在没有配备复杂分析设备的实验室中也可使用PNA-PCR钳夹法，从而降低[具体基因名称]特征分析的成本和时间。
