Crime Scene Evidence Collection

• The goals of evidence collection at a crime scene are documentation, preservation, and integrity.
• The chain of custody is a chronological record of individuals who have had possession of evidence since collection, and is crucial to prevent unauthorized access and tampering, and to maintain authenticity for court.

Collecting Methods

• Swabbing is best suited for wet or dry fluids, achieved by swabbing damp cotton swabs to collect a concentrated sample on the tip.
• Cutting involves collecting a sample from fabric or embedded materials using sterile scissors to cut around the area.
• Scraping is used when the sample is dried out and adhered, and controls should be taken for all methods for comparison.

Preservation Techniques

• Preservation aims to prevent loss, deterioration, and contamination of evidence.
• Use personal protective equipment (PPE) to prevent cross-contamination between the collector and the evidence.
• Use sterile tools and reagents to collect and handle evidence, with reagents also used in testing.
• The sample condition must be dry and packaged in a paper bag, box, or envelope, but never in plastic to avoid condensation, with all samples frozen except those on non-porous surfaces.

Evidence Item: Collection, Preservation, and Sampling

• Ensure the use of PPE, take pictures, and record the location, time, and condition of the evidence.
• Use appropriate sterile tools for collection.
• Preserve the sample by drying and placing it in the correct package, packaging each piece of evidence separately with controls, and labeling with case number, collector's name, date, and time.
• Sample the evidence using the correct method (Microscope, PCR, fingerprints) and maintain the chain of custody.

DNA Structure and Composition

• DNA is composed of sugar (deoxyribose), a phosphate backbone, and nitrogenous bases.
• DNA is negatively charged and a polar molecule.
• Polarity ensures nucleotides add in the 5'-3' direction, attaching to the 3' prime end, with the negative charge making DNA hydrophilic.
• Base pairings are A-T and C-G, with C-G having 3 hydrogen bonds, making it stronger than A-T, which has 2 hydrogen bonds.
• Double-stranded DNA can become single-stranded through denaturation and melting.
• DNA wraps around histones that eventually form chromosomes through tight winding and compaction.

DNA Replication

• The DNA replication process includes unwinding DNA using helicase, adding primers, adding nucleotides, and sealing the strands.
• The parent copy is used as a template, with each template creating two copies of DNA.

DNA Extraction Goals

• Sufficient DNA quantities and quality are required to achieve the goals of DNA extraction.
• The intention to obtain more than 1 ng of total DNA
• Quality is achieved by minimizing degradation, contamination, and inhibitors.

Organic Extraction and Purification

• Organic extraction and purification isolates DNA from cells using chemicals to lyse cells.
• Lyse cells is done using a buffer, typically containing detergents.
• ProK degrades surrounding proteins like histones.
• Phenol-chloroform extraction separates DNA from proteins, lipids, and other matter; after centrifuging, the top aqueous layer contains DNA, the middle or interphase layer contains proteins, and the bottom organic phenol-chloroform layer contains proteins and lipids.
• DNA is washed using high salt or ethanol to remove any contaminants; lastly, it is eluted with water or a buffer.

Solid Phase Extraction

• Add lysis buffer to the tube with material.
• Add beads to magnetically bind DNA and wash away debris.
• Elute magnetic beads away from DNA and retaining DNA.
• Solid phase extraction is more efficient by eliminating centrifuging steps and minimizing pipetting, which decreases cross-contamination and avoids phenol-chloroform use.

Differential Extraction

• Differential extraction separates a sample mixed with different biological material, often used in sexual assault cases to separate sperm cells from epithelial cells.
• This separation is possible due to the disulfide bonds in the caps of sperm cells allows the cells to resist initial lysis.

Forensic DNA Analysis: PCR

• PCR increases the number of DNA copies for detection, amplifies specific target sequences, and attaches a fluorescent label for fragment detection.
• PCR reagents include DNA, a polymerase, primers, bases, and buffer containing salts like magnesium, BSA, and Tris buffer for pH stability.
• Before denaturing, hot start taq polymerase helps prevent unwanted amplification.
• Denature at 94C: break apart the DNA molecule into two strands.
• Anneal at 56-59C: primers bind to single strands.
• Extend at 72C: Taq polymerase adds nucleotides to primers at the 3'-end.
• Fluorescent molecules added to the 5' end of one primer label fragments.
• Extra extension ensures complete adenylation, meaning every fragment has an A at the end to maintain coherency- 10-45 min at 60C

Electrophoresis

• Electrophoresis moves charged particles through fluid or gel under an electric field.
• Capillary electrophoresis is preferred for forensic DNA analysis due to its speed, sensitivity, use of smaller samples and reagents, and ease of operation.
• The basic components of an electrophoresis system include electricity, a buffer, gel, and polymer.
• For single-stranded DNA for better separation, the prepared sample is diluted in formamide for a CE run.
• With voltage applied, charged particles are directed into the capillary without any liquid.
• Separation involves a liquid polymer and detection uses dye labels.
• The CCD camera and virtual filters collecting fluorescent signals do not detect wavelengths directly; instead, they detect light falling on a specific filter position, with the CCD camera capturing light and virtual filters sorting colors via wavelength.
• Wavelengths are only used for the diffraction grating application.