Laboratory of Pharmaceutical Biotechnology, Ghent University, Ottergemsesteenweg 460, Gent, 9000, Belgium; Imec, Kapeldreef 75, Leuven, 3001, Belgium.
Imec, Kapeldreef 75, Leuven, 3001, Belgium.
Biosens Bioelectron. 2021 May 15;180:113135. doi: 10.1016/j.bios.2021.113135. Epub 2021 Mar 3.
The demand for forensic DNA profiling at the crime scene or at police stations is increasing. DNA profiling is currently performed in specialized laboratories by PCR amplification of Short Tandem Repeats (STR) followed by amplicon sizing using capillary electrophoresis. The need for bulky equipment to identify alleles after PCR presents a challenge for shifting to a decentralized workflow. We devised a novel hybridization-based STR-genotyping method, using Short Tandem Repeat Identification (STRide) probes, which could help tackle this issue. STRide probes are fluorescently labeled oligonucleotides that rely on the quenching properties of guanine on fluorescein derivatives. Mismatches between STRide probes and amplicons can be detected by melting curve analysis after asymmetric PCR. The functionality of the STRide probes was demonstrated by analyzing synthetic DNA samples for the D16S539 locus. Next, STRide probes were developed for five different CODIS core loci (D16S539, TH01, TPOX, FGA, and D7S820). These probes were validated by analyzing 13 human DNA samples. Successful genotyping was obtained using inputs as low as 31 pg of DNA, demonstrating high sensitivity. The STRide probes are ideally suited to be implemented in a microarray and present an important step towards a portable device for fast on-site forensic DNA fingerprinting.
现场或警察局对法医 DNA 分析的需求正在增加。目前，通过聚合酶链反应（PCR）扩增短串联重复序列（STR），并使用毛细管电泳对扩增子进行大小分析，在专门的实验室中进行 DNA 分析。在 PCR 后识别等位基因需要体积庞大的设备，这给向分散式工作流程转移带来了挑战。我们设计了一种新的基于杂交的 STR 基因分型方法，使用短串联重复识别（STRide）探针，这可能有助于解决这个问题。STRide 探针是荧光标记的寡核苷酸，依赖于荧光素衍生物上鸟嘌呤的猝灭特性。不对称 PCR 后，可以通过熔解曲线分析检测 STRide 探针与扩增子之间的错配。通过分析合成 DNA 样本中的 D16S539 基因座，证明了 STRide 探针的功能。接下来，为五个不同的 CODIS 核心基因座（D16S539、TH01、TPOX、FGA 和 D7S820）开发了 STRide 探针。通过分析 13 个人类 DNA 样本验证了这些探针的有效性。使用低至 31 皮克的 DNA 即可成功进行基因分型，证明了其高灵敏度。STRide 探针非常适合于微阵列中使用，并朝着用于快速现场法医 DNA 指纹分析的便携式设备迈出了重要一步。
