Discipline: Biological Sciences
Subcategory: Cell and Molecular Biology
Deidra Jordan - Florida International University
Co-Author(s): DeEtta Mills, Florida International University, Miami, FL
Current technology in forensic science uses capillary electrophoresis (CE) instruments to separate DNA amplicons for identification. These profiles are a series of peaks of varying heights that represent alleles from loci that were amplified by PCR. In a single source sample, there should only be two alleles at each locus. But often there are more, indicating there were multiple contributors to the sample–a mixture. Current methodologies only address allelic variation by sequence length and not by the actual nucleotide sequence within that amplicon. Shared alleles in mixtures cannot be easily resolved by length, but should be able to be separated by sequence as the DNA sequence can vary within the amplicon’s length. With traditional CE, DNA amplicons are separated using a denaturing polymer (e.g., POP-7), an entanglement matrix that has a linear relationship between fragment size and migration time. However, Pluronic F-108 is a non-denaturing polymer making it ideal for the detection of single nucleotide polymorphisms (SNP) in the DNA sequence–a process known as single stranded conformation polymorphisms (SSCP). SSCP is based on the single-stranded DNA having a defined conformation and a change in this conformation during separation because of a SNP causes the single stranded DNA to partially reanneal and migrate differently even if only one nucleotide has changed out of several hundred; thus, supporting the hypothesis that CE-SSCP and the F-108 polymer can aid in resolving forensic mixtures. DNA was extracted from buccal swabs containing saliva and cheek cells from different contributors and processed for downstream analysis. The loci D16S539, VWA, and D7S820 were amplified, Sanger sequenced and were separated on the POP-7 polymer. Several SNPs were subsequently identified in each sample. Additional DNA was extracted from those samples and amplified where each sample produced a single peak for the same allele on POP-7. Subsequently, mixtures were created by combining samples and then separating them using the F-108 polymer for SNP analyses. This technique was able to distinguish the presence of SNPs in the amplicon’s sequence even though the length of the amplicon was the same. Results from this study showed that the CE-SSCP technique can aid in resolving forensic mixtures. This method uses the same STR product used for forensics, the same CE instrument but with the non-denaturing F-108 polymer to identify the presence of SNPs. Thus, making the process of screening samples quicker and more efficient for subsequent Next Generation Sequencing analyses. Future research involves creating mixtures with more loci and different mixture ratios to determine a limit of detection with this technique.
Funder Acknowledgement(s): This work is supported by the NSF FGLSAMP FIU Bridge to the Doctorate award HRD #1301998.
Faculty Advisor: DeEtta Mills, firstname.lastname@example.org
Role: I extracted DNA from several different buccal swabs containing saliva and cheek cells from different contributors, and processed them for downstream analysis. I amplified and prepared the DNA samples for Sanger sequencing using the POP-7 polymer and identified SNPs and sequences of each sample. I also identified the alleles of each sequence and sequenced them with the F-108 polymer. Additionally, I created mixture samples using the extracted DNA and analyzed the resulting data.