Abstract：
Detecting low-abundance mutations is of particular interest in the fields of biology and medical science. However, most currently available molecular assays have limited sensitivity for the detection of low-abundance mutations. Here, we established a platform for detecting low-level DNA mutations with high sensitivity and accuracy by combining enhanced-ice-COLD-PCR (E-ice-COLD-PCR) and pyrosequencing with di-base addition (PDBA). The PDBA assay was performed by selectively adding one di-base (AG, CT, AC, GT, AT, or GC) instead of one base (A, T, C, or G) into the reaction at a time during sequencing primer extension and thus enabling to increase the sequencing intensity. A specific E-ice-COLD-PCR/PDBA assay was developed for the detection of the most frequent BRAF V600E mutation to verify the feasibility of our method. E-ice-COLD-PCR/PDBA assay permitted the reliable detection of down to 0.007% of mutant alleles in a wild-type background. Furthermore, it required only a small amount of starting material (20 pg) to sensitively detect and identify low-abundance mutations, thus increasing the screening capabilities in limited DNA material. The E-ice-COLD-PCR/PDBA assay was applied in the current study to clinical formalin-fixed paraffin-embedded (FFPE) and plasma samples, and it enabled the detection of BRAF V600E mutations in samples that appeared as a wild type using PCR/conventional pyrosequencing (CP) and E-ice-COLD-PCR/CP. E-ice-COLD-PCR/PDBA assay is a rapid, cost-effective, and highly sensitive method that could improve the detection of low-abundance mutations in routine clinical use.
摘要：
检测低丰度突变在生物学和医学科学领域特别感兴趣。然而,目前大多数可用的分子检测方法对低丰度突变的检测灵敏度有限.这里,我们通过结合增强-ice-COLD-PCR(E-ice-COLD-PCR)和焦磷酸测序和双碱基添加(PDBA),建立了一个高灵敏度和准确性检测低水平DNA突变的平台.通过选择性添加一种二碱(AG，CT,AC,GT,AT,或GC)而不是一个碱基(A，T,C,或G）在测序引物延伸期间的时间进入反应，从而能够增加测序强度。开发了一种特异性的E-ice-COLD-PCR/PDBA测定法，用于检测最常见的BRAFV600E突变，以验证我们方法的可行性。E-ice-COLD-PCR/PDBA测定允许在野生型背景中可靠地检测低至0.007%的突变等位基因。此外，它只需要少量的起始材料（20pg）来灵敏地检测和鉴定低丰度突变，从而提高了对有限DNA材料的筛选能力。本研究将E-ice-COLD-PCR/PDBA测定应用于临床福尔马林固定石蜡包埋（FFPE）和血浆样品，并且它能够使用PCR/常规焦磷酸测序（CP）和E-ice-COLD-PCR/CP检测野生型样品中的BRAFV600E突变。E-ice-COLD-PCR/PDBA检测是一种快速，成本效益高,和高度敏感的方法，可以提高常规临床使用的低丰度突变的检测。
