Molecular Pathology Research and Development Laboratory, Department of Pathology, Peter MacCallum Cancer Centre, Locked Bag 1, A'Beckett St, Melbourne, Victoria, 8006, Australia.
BMC Cancer. 2011 Jun 24;11:265. doi: 10.1186/1471-2407-11-265.
Detection of mutations by DNA sequencing can be facilitated by scanning methods to identify amplicons which may have mutations. Current scanning methods used for the detection of germline sequence variants are laborious as they require post-PCR manipulation. High resolution melting (HRM) is a cost-effective rapid screening strategy, which readily detects heterozygous variants by melting curve analysis of PCR products. It is well suited to screening genes such as BRCA1 and BRCA2 as germline pathogenic mutations in these genes are always heterozygous.
Assays for the analysis of all coding regions and intron-exon boundaries of BRCA1 and BRCA2 were designed, and optimised. A final set of 94 assays which ran under identical amplification conditions were chosen for BRCA1 (36) and BRCA2 (58). Significant attention was placed on primer design to enable reproducible detection of mutations within the amplicon while minimising unnecessary detection of polymorphisms. Deoxyinosine residues were incorporated into primers that overlay intronic polymorphisms. Multiple 384 well plates were used to facilitate high throughput.
169 BRCA1 and 239 BRCA2 known sequence variants were used to test the amplicons. We also performed an extensive blinded validation of the protocol with 384 separate patient DNAs. All heterozygous variants were detected with the optimised assays.
This is the first HRM approach to screen the entire coding region of the BRCA1 and BRCA2 genes using one set of reaction conditions in a multi plate 384 well format using specifically designed primers. The parallel screening of a relatively large number of samples enables better detection of sequence variants. HRM has the advantages of decreasing the necessary sequencing by more than 90%. This markedly reduced cost of sequencing will result in BRCA1 and BRCA2 mutation testing becoming accessible to individuals who currently do not undergo mutation testing because of the significant costs involved.
通过 DNA 测序进行突变检测，可以通过扫描方法来识别可能存在突变的扩增子。目前用于检测种系序列变异的扫描方法很繁琐，因为它们需要 PCR 后处理。高分辨率熔解（HRM）是一种具有成本效益的快速筛选策略，通过对 PCR 产物的熔解曲线分析，很容易检测到杂合变体。它非常适合筛选 BRCA1 和 BRCA2 等基因，因为这些基因中的种系致病性突变总是杂合的。
设计并优化了用于分析 BRCA1 和 BRCA2 所有编码区和内含子-外显子边界的分析方法。选择了一组最终的 94 个分析方法，用于 BRCA1（36 个）和 BRCA2（58 个），这些方法在相同的扩增条件下运行。在引物设计上投入了大量的精力，以确保在扩增子中能够重复检测到突变，同时最大限度地减少对多态性的不必要检测。在覆盖内含子多态性的引物中加入了脱氧肌苷残基。使用多个 384 孔板来促进高通量。
使用优化的分析方法检测到了 169 个 BRCA1 和 239 个 BRCA2 已知序列变异。我们还使用 384 个单独的患者 DNA 对该方案进行了广泛的盲法验证。所有杂合变体均通过优化的分析方法检测到。
这是首次使用一组反应条件，以多板 384 孔格式，使用专门设计的引物，对 BRCA1 和 BRCA2 基因的整个编码区进行 HRM 筛选。相对大量样本的平行筛选提高了对序列变异的检测能力。HRM 的优点是将测序所需的工作量减少了 90%以上。这将显著降低测序成本，使 BRCA1 和 BRCA2 突变测试能够为目前由于成本高昂而无法进行突变测试的个人所接受。
