Ladas Ioannis, Fitarelli-Kiehl Mariana, Song Chen, Adalsteinsson Viktor A, Parsons Heather A, Lin Nancy U, Wagle Nikhil, Makrigiorgos G Mike
Department of Radiation Oncology, Dana-Farber Cancer Institute and Brigham and Women's Hospital, Harvard Medical School, Boston, MA.
The Broad Institute of MIT and Harvard, Cambridge, MA.
Clin Chem. 2017 Oct;63(10):1605-1613. doi: 10.1373/clinchem.2017.272849. Epub 2017 Jul 5.
The use of clinical samples and circulating cell-free DNA (cfDNA) collected from liquid biopsies for diagnostic and prognostic applications in cancer is burgeoning, and improved methods that reduce the influence of excess wild-type (WT) portion of the sample are desirable. Here we present enrichment of mutation-containing sequences using enzymatic degradation of WT DNA. Mutation enrichment is combined with high-resolution melting (HRM) performed in multiplexed closed-tube reactions as a rapid, cost-effective screening tool before targeted resequencing.
We developed a homogeneous, closed-tube approach to use a double-stranded DNA-specific nuclease for degradation of WT DNA at multiple targets simultaneously. The No Denaturation Nuclease-assisted Minor Allele Enrichment with Probe Overlap (ND-NaME-PrO) uses WT oligonucleotides overlapping both strands on putative DNA targets. Under conditions of partial denaturation (DNA breathing), the oligonucleotide probes enhance double-stranded DNA-specific nuclease digestion at the selected targets, with high preference toward WT over mutant DNA. To validate ND-NaME-PrO, we used multiplexed HRM, digital PCR, and MiSeq targeted resequencing of mutated genomic DNA and cfDNA.
Serial dilution of KRAS mutation-containing DNA shows mutation enrichment by 10- to 120-fold and detection of allelic fractions down to 0.01%. Multiplexed ND-NaME-PrO combined with multiplexed PCR-HRM showed mutation scanning of 10-20 DNA amplicons simultaneously. ND-NaME-PrO applied on cfDNA from clinical samples enables mutation enrichment and HRM scanning over 10 DNA targets. cfDNA mutations were enriched up to approximately 100-fold (average approximately 25-fold) and identified via targeted resequencing.
Closed-tube homogeneous ND-NaME-PrO combined with multiplexed HRM is a convenient approach to efficiently enrich for mutations on multiple DNA targets and to enable prescreening before targeted resequencing.
从液体活检中收集临床样本和循环游离DNA（cfDNA）用于癌症的诊断和预后应用正在迅速发展，因此需要改进方法以减少样本中过量野生型（WT）部分的影响。在此，我们介绍了一种通过酶促降解WT DNA来富集含突变序列的方法。突变富集与在多重闭管反应中进行的高分辨率熔解（HRM）相结合，作为靶向重测序前的一种快速、经济高效的筛选工具。
我们开发了一种均相闭管方法，使用双链DNA特异性核酸酶同时在多个靶点降解WT DNA。无变性核酸酶辅助的探针重叠微等位基因富集法（ND-NaME-PrO）使用与假定DNA靶点的两条链都重叠的WT寡核苷酸。在部分变性（DNA呼吸）条件下，寡核苷酸探针增强了所选靶点处双链DNA特异性核酸酶的消化作用，对WT DNA的偏好远高于突变DNA。为验证ND-NaME-PrO，我们对突变的基因组DNA和cfDNA进行了多重HRM、数字PCR和MiSeq靶向重测序。
对含KRAS突变的DNA进行系列稀释显示，突变富集了10至120倍，等位基因分数检测低至0.01%。多重ND-NaME-PrO与多重PCR-HRM相结合，可同时对10至20个DNA扩增子进行突变扫描。将ND-NaME-PrO应用于临床样本的cfDNA，可实现对10个以上DNA靶点的突变富集和HRM扫描。cfDNA突变富集高达约100倍（平均约25倍），并通过靶向重测序进行鉴定。
闭管均相ND-NaME-PrO与多重HRM相结合，是一种高效富集多个DNA靶点上的突变并在靶向重测序前进行预筛选的便捷方法。
