Translational Genomics and Epigenomics Laboratory, Olivia Newton-John Cancer Research Institute, Heidelberg, Victoria, Australia; Department of Surgery, University of Melbourne, Austin Health, Heidelberg, Victoria, Australia;
Translational Genomics and Epigenomics Laboratory, Olivia Newton-John Cancer Research Institute, Heidelberg, Victoria, Australia;
Clin Chem. 2016 Jul;62(7):1012-9. doi: 10.1373/clinchem.2016.256388.
The quantification of genomic chimerism is increasingly recognized for its clinical significance after transplantation. Before the measurement of chimerism, accurate genotyping of genetic polymorphisms for informative alleles that can distinguish donor DNA from recipient DNA is essential. The ease of allelic discrimination of small deletion and insertion polymorphisms (DIPs) makes DIPs attractive markers to track chimerism. Current methodologies for the genotyping of DIPs are largely based on "open-tube" approaches. "Closed-tube" approaches involving no or minimal post-PCR handling are preferred. We compared 3 distinct methodologies to determine an optimal platform for DIP genotyping.
Genomic DNA from 19 normal individuals was genotyped for 6 small biallelic DIPs using high-resolution melting analysis (HRMA), probe-free droplet digital PCR (ddPCR), and microfluidic electrophoresis of PCR products. For HRMA, 3 different platforms were compared.
Our newly developed probe-free ddPCR approach allowed the genotype of each DIP to be determined by fluorescence intensity based on amplicon size. Microfluidic electrophoresis also allowed genotypes to be determined by amplicon size. HRMA assays allowed the genotype of each DIP to be determined by melting profile. Genotyping results were concordant between the 3 methodologies. HRMA was the most readily performed methodology and was robust across 3 separate HRMA-capable platforms.
We demonstrated the effectiveness of probe-free ddPCR to accurately genotype small biallelic DIPs. Nevertheless, HRMA proved to be the optimal approach for genotyping small DIPs because closed-tube approaches are preferred owing to rapid and less laborious workflows and least risk of PCR contamination.
移植后，基因组嵌合体的定量因其临床意义而越来越受到重视。在测量嵌合体之前，必须对能够区分供体 DNA 和受体 DNA 的遗传多态性的信息性等位基因进行准确的基因分型。小缺失和插入多态性（DIP）等位基因易于区分，这使得 DIP 成为追踪嵌合体的有吸引力的标记。目前用于 DIP 基因分型的方法主要基于“开管”方法。“闭管”方法不涉及或仅涉及最小的 PCR 后处理，是首选方法。我们比较了 3 种不同的方法，以确定 DIP 基因分型的最佳平台。
使用高分辨率熔解分析（HRMA）、无探针液滴数字 PCR（ddPCR）和 PCR 产物微流控电泳，对 19 名正常个体的基因组 DNA 进行 6 个小双等位基因 DIP 的基因分型。对于 HRMA，比较了 3 种不同的平台。
我们新开发的无探针 ddPCR 方法允许根据扩增子大小基于荧光强度确定每个 DIP 的基因型。微流控电泳也允许根据扩增子大小确定基因型。HRMA 分析允许根据融解曲线确定每个 DIP 的基因型。3 种方法的基因分型结果一致。HRMA 是最容易进行的方法，并且在 3 个独立的 HRMA 兼容平台上都很稳健。
我们证明了无探针 ddPCR 准确基因分型小双等位基因 DIP 的有效性。然而，HRMA 被证明是基因分型小 DIP 的最佳方法，因为闭管方法由于快速、省力的工作流程和最小的 PCR 污染风险而更受欢迎。
