Forensic DNA Analysis and CRISPR Systems: Key Concepts
Posted on Oct 27, 2026 in Biotechnology
1) Chelex Extraction Steps
1. Hydration: Submerge the biological stain in sterile water at room temperature for 30 minutes to soften the sample matrix.
2. Debris Removal: Spin down the mixture via centrifugation and discard the substrate carrier (e.g., fabric or swab fragment).
3. Reagent Addition: Introduce the ion-exchange resin (Chelex) along with Dithiothreitol (DTT) and Proteinase K to the remaining cellular pellet.
4. Lysis Incubation: Maintain at 56°C to enzymatically digest cell membranes and release internal cellular contents.
5. Initial Separation: Run a brief centrifugation cycle.
6. Denaturation Boiling: Heat the solution to 100°C for 8 minutes to deactivate cellular proteins and denature double-stranded DNA into single strands.
7. Final Sedimentation: Perform a final spin to precipitate the resin beads and cellular debris.
8. Harvesting: Collect the pure supernatant containing single-stranded genetic material for downstream quantification.
Q2) Core Differences Between STR and SNP
* Abundance: STRs occur every 15 kb, while SNPs are found more frequently, roughly every 1 kb.
* Allele Count: STRs have 5 to 20 alleles per locus, whereas SNPs are limited to just 2 alleles.
* Fragment Size: STR amplicons are large, but SNPs are highly compact.
* Mixtures: STRs easily distinguish multiple contributors, but SNPs make mixture parsing highly problematic.
* Artefacts: STRs are prone to baseline stutter peaks, while SNPs show clean profiles with zero stutter.
3) Types of Samples DNA Can Be Obtained From
DNA can be extracted from blood, semen, epithelial cells, hair shafts, and tissue. It can also be preserved on FTA paper. Although environmental samples may contain inhibitors, they can still yield DNA suitable for forensic testing.
4) Capillary Electrophoresis Advantages and Disadvantages
Advantages: Provides automated sample loading, controlled temperature conditions, and rapid separation, producing high-quality electropherograms. It also reduces contamination risk through automated polymer flushing.
Disadvantages: The instruments are costly for low-volume labs and may suffer from dye cross-talk between capillaries, requiring regular maintenance.
5) Forensic Issues
Key issues encountered in forensic DNA work include degradation of DNA, inhibition of PCR reactions, contamination at various stages, and the presence of mixed DNA profiles. These factors complicate interpretation and may reduce the reliability of results.
6) Samples Run in Capillary Electrophoresis
A typical capillary electrophoresis run includes the positive control, negative control, and the allelic ladder, alongside casework samples. The ladder must fall within an acceptable sizing range to validate the run.
7) Two Advantages of STR Over SNP?
STRs offer stronger discrimination because they have many alleles per locus. They also allow clearer interpretation of mixed samples, whereas SNP mixtures are difficult to resolve due to their two-allele limitation.
8) Solutions for PCR Inhibition
PCR inhibition can be reduced by diluting the DNA extract, increasing the amount of Taq polymerase, or adding BSA/NaOH to neutralize inhibitory substances. Filtration methods such as Microcon-100 devices or low-melt gel plugs can also remove inhibitors.
9) What PCR Inhibition Does
PCR inhibitors interfere with cell lysis, damage DNA, and block polymerase activity. This results in weak amplification, partial profiles, allelic dropout, or complete failure of the reaction.
10) Effects of Degradation
Degraded DNA leads to preferential loss of larger alleles, especially at loci such as D2, D18, and FGA. Smaller STR fragments are more likely to amplify, and heterozygotes may appear homozygous due to dropout. High-molecular-weight markers like VNTRs cannot be analysed when degradation is severe.
11) Technique Used in Sexual Assault Cases?
Sexual assault samples are processed using differential extraction, which separates epithelial cells from sperm cells. When sperm are absent, Y-chromosome markers can be used to confirm the presence of male DNA.
Chapter 1: Forensic Issues
* Forensic Sample Realities: Exposed to harsh environments (damp, sunlight), limited in quantity, degraded, mixed, and prone to contamination or PCR inhibition.
* Degraded DNA: Environmental factors (water, UV, nucleases) destroy high molecular weight markers (VNTRs and RFLPs become impossible).
• STR Advantage: STR allele ranges are small, increasing the chance of survival. Larger alleles (D2, D18, FGA) drop out first.
• MiniSTRs: Re-designed primers positioned closer to the repeat region create a smaller reading frame to rescue degraded samples. Not ideal for large multiplexes due to overlap.
* PCR Inhibition:
• Inhibitors: Soil, sand, wood, plant matter (outdoors); textile dyes/denim (heme/hematin in blood, melanin in hair, urea in urine, leather).
• Mechanisms: Interfere with cell lysis, degrade DNA, or directly inhibit Taq polymerase activity.
• Solutions: Dilute the sample, increase Taq concentration, add BSA (Bovine Serum Albumin) or NaOH to neutralize, or use Microcon-100 filters.
* Contamination Sources: Crime scene (environmental), between samples during prep, or via post-PCR amplified DNA. Monitored using substrate controls, negative controls, and staff elimination databases.
* DNA Mixtures: Two or more profiles present. Evaluated via peak area/height ratios. Requires highly polymorphic markers (e.g., D18S51 with 51 alleles vs TPOX with 15).
* Low Copy Number (LCN) DNA: Defined as <100 pg of template DNA. Enhanced by increasing PCR cycles to 28–34 cycles.
Chapter 2: Single Nucleotide Polymorphisms (SNPs)
Definition: A single base change at a precise genomic position. Must occur in at least 1% of the population (otherwise classified as a rare mutation).
* Frequency: Occurs roughly every 1,000 base pairs (3–5 million differences per genome). Requires 50–80 SNPs to match the statistical discriminating power of standard STR kits.
* Detection Methods:
• RFLP: Outdated; requires too much sample.
• Sequencing: Uses ddNTPs (dideoxyribonucleotides lacking a 3′ hydroxyl group) to randomly terminate chain extension, resolved via capillary electrophoresis.
• Single Base Extension (SBE): Preferred method (mini-sequencing). Internal primers bind adjacent to the SNP; Taq extends the sequence by exactly 1 base pair using tagged ddNTPs. Allows high-level multiplexing.
* Forensic Applications: Used for phenotyping (predicting pigmentation, hair, skin, and eye color).
* Ethical Constraints: National DNA Databases (NDNAD) strictly block SNPs linked to medical disorders, race, or intelligence data.
Chapter 3: DNA Processing & Instrumentation
* The 5 Core Steps: 1. Extraction / 2. Quantification / 3. Amplification / 4. Detection / 5. Interpretation.
* 1. Extraction Techniques:
Organic: Reagents include SDS and Proteinase K (lysis), DTT (disrupts disulfide bonds), EDTA (binds Mg2+ to halt nucleases), and Phenol/Chloroform to isolate DNA into the upper aqueous layer. Excellent for high molecular weight DNA.
• Qiagen: Solid-phase commercial extraction where DNA binds to a silica gel membrane in an ethanol buffer, washed, and eluted in water.
• FTA Paper: Absorbent cellulose paper embedded with protective chemicals. Bypasses quantification step; a direct punch goes straight to PCR. Prone to static jumping.
• Differential Extraction: Used in sexual assault cases to isolate epithelial cells (female fraction) from sperm cells (male fraction). Epithelial cells are lysed first via SDS/Proteinase K; sperm nuclei are subsequently lysed by adding DTT.
• Magnetic Bead Extraction: Uses superparamagnetic iron oxide beads to capture nucleic acids. Eliminates centrifugation, is highly automated, and limits cross-contamination.
* 2. Quantification Assays:
• Slot Blot: Historical baseline. Primate-specific colorimetric probe. Detects down to 150 pg.
• PicoGreen: Fluorescent intercalating dye for dsDNA. Non-human specific and highly carcinogenic.
• Real-Time PCR (qPCR): Measures fluorescence mid-cycle. SYBR Green is cheap but non-specific (binds any dsDNA/primer-dimer). TaqMan Probes use a 5′ reporter dye and 3′ quencher; Taq polymerase’s 5′ exonuclease activity cleaves the probe during extension to emit a target-specific signal. Includes an Internal PCR Control (IPC) to verify reaction integrity.
* 3. Amplification & 4. Detection: Multiplex PCR utilizes fluorescent dyes (5-FAM, JOE, NED, ROX) to label loci. Loci sharing colors are strategically separated by molecular size ranges (e.g., SGMplus kit).
Capillary Electrophoresis uses injection formamide, POP4 polymer (STRs) or POP6 (sequencing), silica capillaries, and an argon laser paired with a 2D CCD chip.
Chapter 4: Data Interpretation & Artifacts
* Run Controls: Every run requires a Positive Control, a Negative Control (monitors contamination), and at least two Allelic Ladders (alleles must fall within ±0.5 bp of ladder peaks to be designated).
* Preferential Amplification (Pref. Amp): Occurs when one allele amplifies significantly more than its paired heterozygote. Rule: Peak height ratio >60% = Designate; <60% = Do Not Designate.
* Allelic Drop-out / Drop-in: Caused by low template DNA or baseline imbalance. Mitigated by running samples in duplicate.
* Dye Blobs: Broad, loose peaks caused by fluorescent dyes detached from primers migrating independently. Prevented by filtering the PCR product post-amplification.
* Voltage Spikes: Sharp, pointed peaks occurring at the identical location across all color channels due to electronic surges. Spikes disappear upon sample re-injection.
* n/n+1 Split Peaks: An n peak represents the true STR allele length; an n+1 peak contains an extra base added by Taq polymerase. Split peaks happen when Taq function is sub-optimal or the template is overloaded. Fixed by executing a 60-minute final extension cycle (65–72°C).
* Stutter Peaks: Minor product peaks appearing 4 bp shorter than the true allele peak due to polymerase slippage. Standard threshold is <15%; if >15%, it suggests a mutation or sample mixture.
* Mutations: Somatic mutations display 3 peaks at a locus (some body cells carry the mutated allele, others carry the unmutated version). Trisomy/Translocation also shows 3 distinct peaks at a single locus.
* Primer-Dimer: Low molecular weight artifacts (60–90 bp) where primers bind to each other, consuming reagents and stalling amplification. Resolved using Hot Start PCR.
* Over-Amplification: Too much DNA input causing flat-topping (saturated off-scale peaks) and pull-up (spectral bleeding where intense peaks in one color lane trigger artifact peaks in another, typically blue bleeding into green).
* Sample Contaminants: Extraneous substances that fluoresce independently, producing broad, irregular peaks. Organic extraction successfully eliminates them, whereas Chelex fails to strip out fluorescent contaminants.
Chapter 1: Components of the CRISPR System
Core Equation: Cas9 + gRNA + Target DNA + PAM → DNA Cleavage
Key Components & Roles:
• Cas9 Protein: The enzymatic scissors that binds guide RNA, scans for the PAM site, binds target DNA, and cuts both strands.
• gRNA (Guide RNA): The targeting guide. Combines two elements into an engineered single guide RNA (sgRNA):
• crRNA: Provides the target-specific spacer sequence.
• tracrRNA: Provides the structural scaffold needed for Cas9 association.
• PAM (Protospacer Adjacent Motif): Short DNA sequence directly downstream (3′) of the target site required for Cas9 binding. It is not part of the gRNA sequence.
Mechanics & Outcomes:
• SpCas9 PAM: Typically 5′-NGG-3′.
• Cleavage Site: Double-strand break (DSB) occurs roughly 3–4 nucleotides upstream of the PAM sequence.
Orientation Frameworks
• Guide-centric: PAM is located on the strand that matches the guide RNA sequence.
• Target-centric: PAM is located on the strand that base-pairs with the guide RNA.
Chapter 2: Structure and Function of Cas Proteins
Three Major Functional Duties:
1. Guide RNA Recognition: Associates with gRNA to form a functional surveillance complex.
2. Target Recognition: Interrogates nucleic acids, identifies PAM, and drives guide-target base pairing.
3. Catalysis: Executes double-strand breaks, single-strand nicks, or locks onto targets if catalytically inactive.
Cas9 Domain Anatomy:
• REC Lobe (Recognition Lobe): Interacts with guide RNA and target DNA to form the RNA-DNA heteroduplex.
• NUC Lobe (Nuclease Lobe): Contains catalytic domains responsible for cleavage.
• HNH Domain: Cleaves the DNA strand complementary to the guide RNA.
• RuvC Domain: Cleaves the non-complementary strand.
• PAM-Interacting (PI) Region: Selectively reads and recognizes the PAM sequence.
Chapter 3: CRISPR for Genome Activation & Repression (dCas9)
Core Concepts:
• CRISPRa (Activation): dCas9 fused to an activator domain recruits transcription machinery, increasing mRNA synthesis without a permanent transgene copy.
• CRISPRi (Repression): dCas9 fused to a repressor blocks RNA polymerase or remodels chromatin to stably reduce expression without causing DNA breaks.
Where to Aim (TSS Targeting Windows):
• CRISPRa Window: Targeted between −400 to −50 bp relative to the Transcription Start Site (TSS).
• CRISPRi Window: Targeted between −50 to +300 bp around the TSS.
Effector Domain Toolkit:
• Activators: VP16/VP64 (1st gen, modest); VPR/TV (tripartite, strong); CRISPR-Act3.0 (uses an MS2 loop and SunTag peptide array loading multiple TAD copies to achieve 4–6× the strength of dCas9-TV).
• Repressors: KRAB (most common mammalian repressor); SRDX / TOPLESS N300 (primary plant repressors); dCas9 alone (steric interference).
Modern Horizons (2024–2026):
• Epigenetic Editors (CRISPRoff/CRISPRon): Delivering transient doses of dCas9 fused to writers (like DNMT3A/3L) establishes durable, long-term silencing across cell divisions without sequence modifications.
• Single-AAV Packaging Constraints: AAV payload capacity is strictly limited to ~4.7–5.0 kb. While SpCas9 (1,368 aa) is too bulky, miniaturized tools like CasMINI / dCas12f (529–554 aa) cleanly fit alongside complex transcriptional modifiers inside a single vector.
