Browsing by Subject "Polymerase Chain Reaction / methods"
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Item Developmental Validation of a MPS Workflow with a PCR-Based Short Amplicon Whole Mitochondrial Genome Panel(MDPI, 2020-11-13) Cihlar, Jennifer Churchill; Amory, Christina; Lagace, Robert; Roth, Chantal; Parson, Walther; Budowle, BruceFor the adoption of massively parallel sequencing (MPS) systems by forensic laboratories, validation studies on specific workflows are needed to support the feasibility of implementation and the reliability of the data they produce. As such, the whole mitochondrial genome sequencing methodology-Precision ID mtDNA Whole Genome Panel, Ion Chef, Ion S5, and Converge-has been subjected to a variety of developmental validation studies. These validation studies were completed in accordance with the Scientific Working Group on DNA Analysis Methods (SWGDAM) validation guidelines and assessed reproducibility, repeatability, accuracy, sensitivity, specificity to human DNA, and ability to analyze challenging (e.g., mixed, degraded, or low quantity) samples. Intra- and inter-run replicates produced an average maximum pairwise difference in variant frequency of 1.2%. Concordance with data generated with traditional Sanger sequencing and an orthogonal MPS platform methodology was used to assess accuracy, and generation of complete and concordant haplotypes at DNA input levels as low as 37.5 pg of nuclear DNA or 187.5 mitochondrial genome copies illustrated the sensitivity of the system. Overall, data presented herein demonstrate that highly accurate and reproducible results were generated for a variety of sample qualities and quantities, supporting the reliability of this specific whole genome mitochondrial DNA MPS system for analysis of forensic biological evidence.Item Optimization and Evaluation of qPCR Duplex Assay for mtDNA Copy Number Quantification(2020-05) Johnson, Gretchen A.; Planz, John V.; Phillips, Nicole R.; Zascavage, Roxanne R.Purpose: The mitochondrial genome (mtDNA) encodes thirteen essential proteins in oxidative phosphorylation, the cell's primary energy-generating process. Depending on the cell type and stage of development, each cell contains an average of 103 to 104 copies of mtDNA. Current methods of quantification of mtDNA copy number can be imprecise due to low efficiencies of assays and inherent imbalance of mtDNA copy number with nuclear DNA (nDNA) copy number. Accurate quantification of both mtDNA and nDNA is important when calculating the ratio of mtDNA to nDNA. The goal of this project is to optimize a duplex assay that will give precise and accurate estimates in human samples. Methods: Here we employ synthetic oligomer standards for an absolute real-time qPCR assay. The significance of using absolute qPCR is that the standard curve allows for the direct comparison of unknowns to obtain a copy number. The mitochondrial target is a site in the minor arc (MinArc), and the nuclear target is a single copy locus ([beta]2M). The accuracy of this assay was evaluated using a standard reference material (SRM2372a) and the precision was evaluated via replications. Results: This design resulted in high R2 values for the standards as well as sufficiently high efficiencies. The precision of the assay was analyzed over 6 replicated runs and was deemed effectively reproducible. The accuracy was assessed with the use of a standard reference material (SRM 2372a) and was found to be problematic [Romsos et al., 2018]. This could be from a possible dilution bias of the SRM, effectively changing the copy number ratios in a difficult to predict way [Malik et al., 2011]. An attempt to mathematically correct the data was made but did not provide any solution. Conclusion: The optimization of this assay is ongoing due to the error in accuracy. The assay has proven to be precise and reproducible with sufficient efficiency. Possible future directions include sonication of samples and SRMs to examine if dilution bias could be the cause of inaccurate SRM quantification. Other methods of possibly reducing dilution bias mentioned in Malik et al. [2011] include manual shearing and the use of DNA carriers such as tRNA. Another avenue of future research could include a different method of mathematically correcting the data post run to improve accuracy. This assay has the potential to provide data which can be used to indicate overall mitochondrial health and can be utilized in various research areas such as aging, cancer, forensics and neurodevelopment.Item Typing Highly Degraded DNA Using Target Enrichment(2020-05) Kieser, Rachel E.; Budowle, Bruce; Phillips, Nicole R.; Coble, Michael D.; Berg, Rance E.; Salvatore, MichaelForensic 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.