Using long ssDNA polynucleotides to amplify STRs loci in degraded DNA samples

Obtaining informative short tandem repeat (STR) profiles from degraded DNA samples is a challenging task usually undermined by locus or allele dropouts and peak-high imbalances observed in capillary electrophoresis (CE) electropherograms, especially for those markers with large amplicon sizes. We hereby show that the current STR assays may be greatly improved for the detection of genetic markers in degraded DNA samples by using long single stranded DNA polynucleotides (ssDNA polynucleotides) as surrogates for PCR primers. These long primers allow a closer annealing to the repeat sequences, thereby reducing the length of the template required for the amplification in fragmented DNA samples, while at the same time rendering amplicons of larger sizes suitable for multiplex assays. We also demonstrate that the annealing of long ssDNA polynucleotides does not need to be fully complementary in the 5’ region of the primers, thus allowing for the design of practically any long primer sequence for developing new multiplex assays. Furthermore, genotyping of intact DNA samples could also benefit from utilizing long primers since their close annealing to the target STR sequences may overcome wrong profiling generated by insertions/deletions present between the STR region and the annealing site of the primers. Additionally, long ssDNA polynucleotides might be utilized in multiplex PCR assays for other types of degraded or fragmented DNA, e.g. circulating, cell-free DNA (ccfDNA).

从降解DNA样本中获取信息完整的短串联重复序列（short tandem repeat, STR）分型结果，是一项极具挑战性的工作，常因基因座或等位基因丢失、毛细管电泳（capillary electrophoresis, CE）电泳图谱中出现的峰高失衡而受到干扰，尤其针对扩增片段长度较大的遗传标记。本研究表明，以长单链DNA多核苷酸作为聚合酶链式反应（PCR）引物的替代物，可大幅优化现有STR检测技术，用于降解DNA样本中的遗传标记检测。这类长引物能够与重复序列更紧密地退火结合，从而缩短碎片化DNA样本中扩增所需的模板长度，同时可生成适用于多重检测的更长扩增子。我们还证实，长单链DNA多核苷酸的退火过程无需在引物5'端实现完全互补，因此可灵活设计任意长引物序列，用于开发新型多重检测体系。此外，完整DNA样本的基因分型也可借助长引物获益：其与靶标STR序列的紧密结合，能够克服因STR区域与引物退火位点之间存在插入/缺失突变而导致的错误分型结果。除此之外，长单链DNA多核苷酸还可应用于其他类型降解或碎片化DNA的多重PCR检测体系，例如循环游离DNA（circulating cell-free DNA, ccfDNA）。