Extraction of DNA from trace forensic samples with a modified lysis buffer and chitosan coated magnetic beads

Abstract

The trace amounts of human tissue cells or body fluids left at the crime scene are often mixed with inhibitors such as rust, pigments, and humic acid. The extraction of the DNA from the trace cells is crucial for the investigation of cases. Usually, specially designed magnetic nanoparticles were chosen by the case investigators to enrich and elute DNA, which was then used for polymerase chain reaction (PCR) and short tandem repeat (STR) analysis. The traditional approach often had the following problems, such as low DNA enrichment efficiency, possible DNA breakage, and complex operations. Here, the 1%(w/v) of chitosan (75% deacetylation degree) was used to modify the 50 nm magnetic nanoparticles to gain the Chitosan@Beads, which theoretically carried positively charged in the pH = 5 of lysis buffer so as to adsorb negatively charged DNA through electrostatic interactions. The XPS and FT-IR results demonstrated that chitosan was successfully attached to the surface of magnetic nanoparticles. A set of simulated samples, containing 20 mg/μL of humic acid, pigments, iron ions (Fe²⁺, Fe³⁺), and the coexistence of the above three substances, were prepared to simulate the case scene. Human bronchial epithelial cells were mixed with the 200 μL of the above simulated samples for DNA extraction. 400 μL of lysis buffer, 20 μL of proteinase K (10 mg/mL) and 20 μL of Chitosan@Beads solution (20 mg/mL) was used for cell disruption and DNA enrichment. The extraction sensitivity of Chitosan@Beads was confirmed to be 10 cells, superior to commercial reagent kits. The Chitosan@Beads@DNA can directly use for "In-situ PCR" with elution-free operations. The STR loci rate of DNA extracted by Chitosan@Beads was around 97.9%, higher than the commercial kit (66.7%). In short, we foresee here developed novel Chitosan@Beads and modified lysis buffer could provide a new model for the DNA extraction of forensic trace evidence.

Keywords: Chitosan@Beads; Forensic biological evidence; Inhibitors; Polymerase chain reaction (PCR); STR loci; Short tandem repeat (STR); Trace DNA extraction.