Abstract
Unbalanced and degraded mixtures (UDM) are very common in forensic DNA analysis. For example, DNA signals from criminal suspects are masked by a large amount of DNA from victims, or cell-free fetal DNA (cffDNA) in maternal plasma is masked by a high background of maternal DNA. Currently, detecting minor DNA in these mixtures is complex and challenging. We developed a new set of SNP-SNP microhaplotypes with short amplicons, and we successfully genotyped them using the new method of amplification-refractory mutation system PCR (ARMS-PCR) combined with SNaPshot technology based on a capillary electrophoresis (CE) platform. This panel reflects a high polymorphism in the Southwest Chinese Han population and thus has excellent potential for mixture studies. We evaluated the feasibility of this panel for UDM detection and noninvasive prenatal paternity testing (NIPPT). Fifteen SNP-SNPs detected minor DNA of homemade DNA mixtures, with a sensitivity of 0.025–0.05 ng and a specificity of 1:1,000. In addition, the panel successfully genotyped degraded DNA from single and mixed samples. Finally, 15 SNP-SNPs were applied to 26 trios. All samples displayed positive results with at least one marker to detect cffDNA. Besides, all fetal alleles in maternal plasma were confirmed by genotyping fetal genomic DNA from amniocentesis and paternal genomic DNA from peripheral blood. The results indicated that the SNP-SNP strategy based on the CE platform was useful for UDM detection and NIPPT.
Highlights
Unbalanced two-person DNA mixtures are frequently encountered in forensic cases (Tan et al, 2018)
We previously developed a set of 15 new single nucleotide polymorphism (SNP)-SNPs and used amplification-refractory mutation system (ARMS)-PCR combined with SNaPshot technology to construct a panel based on the capillary electrophoresis (CE) platform (Zhang et al, 2020)
This study demonstrates that SNP-SNPs markers can be applied to detect unbalanced degraded mixture (UDM) and cellfree fetal DNA (cffDNA) through CE-based ARMS-PCR and SNaPshot technology
Summary
Unbalanced two-person DNA mixtures are frequently encountered in forensic cases (Tan et al, 2018). Effective isolation and genotyping of minor DNA is required and essential for forensic practice. Degradation of DNA and unbalanced DNA mixtures make genotyping more difficult and challenging. The DNA fragments of degraded samples are usually 100–200 bp, including naturally degraded stains and cellfree fetal DNA (cffDNA) found in maternal plasma and other locations (Freire-Aradas et al, 2012). When an unbalanced mixed sample undergoes degradation, an unbalanced degraded mixture (UDM) is formed. Many problems occurred while detecting the UDM, such as poor sample recovery, stutter artifacts, unbalanced peak heights, allele dropouts, and complicated interpretation of the results
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