Typing Highly Degraded DNA Using Target Enrichment

Forensic genetic profiling is the process of targeting unique positions within the human genome for identity testing of biological DNA evidence. Forensic profiling of highly degraded DNA samples is one of the primary challenges faced by forensic analysts. These compromised biological samples are difficult to genetically profile, due to the highly fragmented nature of the target molecules, using traditional methods which centers around the detection of short tandem repeats (STRs). For STR typing to be successful, DNA must be relatively intact in order to amplify by PCR. Molecular biology approaches have been developed that may be applied to severely degraded samples to increase the capability of DNA profiling. Targeting single nucleotide polymorphisms (SNPs) holds potential as their amplicons can be designed to be substantially smaller than those for STRs, making these markers a viable alternative for typing degraded (fragmented) DNA. Additionally, rolling circle amplification (RCA) can be exploited as a tool as it has the capacity to amplify all genomic DNA in a circular template present in a sample. A circular molecule essentially creates an infinitely long template for amplification. RCA generates linear tandem copies of the circular template sequence. However, nuclear DNA is not circular and thus RCA cannot be used to its full potential. CircLigase II is an enzyme that circularizes single-stranded DNA. Thus, it may be possible to generate circular DNA from the highly degraded fragments of challenged samples. Molecular inversion probes (MIPs) are an alternative circle-based enrichment approach. A MIP is a single-stranded oligonucleotide that contains two target-specific arms flanking a SNP of interest (capture) and internal PCR primer binding sites for controlled amplification. The two target-specific arms hybridize to the target DNA, the gap is filled resulting in the complementary state of the SNP of interest, the MIP dissociates from the target, and the target site is amplified employing the internally incorporated primer binding sites. Coupled to massively parallel sequencing (MPS), both circle-based approaches were attempted with limited to no success. Reverse Complement PCR (RC-PCR) was pursued to address the same problem of analyzing degraded DNA. RC-PCR is an innovative, one-step PCR target enrichment technology adapted for the amplification of highly degraded (fragmented) DNA. It provides simultaneous amplification and tagging of a targeted sequence construct in a single, closed-tube assay. A human identification (HID) RC-PCR panel was designed targeting 27 identity SNPs generating targets only 50 base pairs in length. In a single reaction, the complete sequencing construct is produced which is essential for MPS library preparation. The RC-PCR approach produced reliable and concordant genotyping results as well as demonstrated a sensitivity of detection of a majority of alleles down to 60 pg of input DNA. In addition, RC-PCR showed robustness tolerating known PCR inhibitors, especially calcium and collagen. The RC-PCR system may be an effective alternative to current forensic genetic methods in the analysis of highly degraded DNA.