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Forensic Genetics - Week 3 Lecture Notes

Topic = Polymerase Chain Reaction (PCR)

Historical Context & Impact of PCR

  • Invented mid-1980s by Kary Mullis; earned the 1993 Nobel Prize in Chemistry.

  • Conceptual similarity: mimics natural DNA replication but in a test-tube.

  • Transformative for:
    • Forensic science (trace evidence, sex determination, species I.D.)
    • Paternity testing & genealogy
    • Infectious-disease diagnostics
    • Human Genome Project (enabled rapid sub-cloning & mapping)
    • Contamination detection in food, water, pharmaceuticals.

  • Overarching benefit: zooms in on a chosen locus and generates 10^{6}-10^{9} exact copies within hours.

Core Principle

  • PCR relies on repeating three temperature-controlled steps to exponentially amplify a defined DNA segment.

  • Each cycle doubles the quantity of the target; after n cycles the theoretical yield is 2^{n} copies (ignoring reagent limits).

Choosing the Target DNA

  • Typical amplicon lengths 100–1500 bp; forensic labs prefer 100–500 bp because:
    • Shorter fragments amplify efficiently.
    • Degraded/old samples often lack long intact templates.

  • Upper technical limit ≈ 25 kb (long-range PCR).

  • Any genomic feature can be selected: genes, VNTRs, deletions, insertions, SNPs.

Essential Reagents (Master-Mix Components)

  • Template DNA
    • Can be as little as one cell.
    • Quality (integrity, purity) and quantity must be optimized—too little → no product, too much → inhibitors/artefacts.

  • dNTPs (A, C, G, T nucleotides)
    • Raw building blocks for the new strands.

  • Reaction Buffer
    • Maintains pH and ionic strength.
    • Often includes Mg^{2+} because polymerase activity is Mg-dependent.

  • DNA Polymerase
    • Thermostable variant required; usually Taq polymerase from Thermus aquaticus.
    • Functions at >95 degrees withstands denaturation.
    • Modern high-fidelity enzymes offer 3'→5' exonuclease proofreading → lower error rate (critical in forensic typing).

  • Primers (Forward & Reverse)
    • Synthetic oligonucleotides ~20–30 bp that flank the region of interest.
    • Provide free 3'-OH for polymerase initiation.
    • Added in large molar excess so they are not limiting.
    • Orientation: forward primer reads 5'→3' on one strand; reverse primer reads 5'→3' on complementary strand (physically 3'→5' on the template).

Primer Design Key Points

  • Melting temperature estimate: Tm = [2(A+T) + 4(G+C)] - 5 (°C) • Desired Tm range: 50-65 degrees.
    • GC content target: 40–60 % for stable yet specific binding.
    • Forward & reverse primers should match within 1 degree.

  • Databases & software (BLAST, Primer3, NCBI resources) help avoid secondary structures, dimers, and off-target binding.

Thermal Cycling Protocol

  • A PCR run repeats the following three steps 25–35 times:

  1. Denaturation
    • \sim95 degrees for 15–30 s.
    • Disrupts H-bonds, yielding single-stranded templates.

  2. Annealing
    • 50-65 degrees for 20–60 s.
    • Temperature low enough for primers to hybridize, high enough to prevent re-annealing of full template.

  3. Extension / Elongation
    • 72 degrees (optimum for Taq) for ≈1 min per 1 kb.
    • Polymerase incorporates dNTPs, elongating from the primer’s 3' end at ≈1000 bases min⁻¹.

Amplification Dynamics & Strand Types

  • Early cycles: polymerase overruns target boundaries, producing variable-length products.

  • After cycle 3, ‘defined-length’ amplicons accumulate and dominate.

  • Graphical model: parental strands → first-generation intermediates → precise target fragments exponentially prevail.

Post-PCR Analysis

Gel Electrophoresis (Classical)

  • Agarose gel + electric field separates DNA by size (smaller fragments migrate farther).

  • Useful for VNTR analysis: band patterns correspond to allele length.

Capillary Electrophoresis (CE)

  • High-resolution (detects 1 bp differences).

  • Thin capillaries filled with polymer matrix; each acts like a miniature gel.

  • DNA fragments pass a laser detection window; fluorescent signals recorded as peaks (electropherogram).

  • Essential controls:
    • Size ladder in one capillary to calibrate absolute fragment lengths.
    • Alignment marker (two known fragments) in every capillary to normalize inter-capillary variation.

Forensic Workflow Example

  1. Collect biological sample (blood, saliva, etc.).

  2. Extract DNA; quantify and assess quality.

  3. Prepare master-mix (+1 extra volume to avoid pipetting shortage).
    • Ensures identical reagent ratios across cases and controls.

  4. PCR amplify multiple VNTR loci (multiplexing).
    • Each VNTR allele length reflects number of repeat units (e.g.
    \text{520 bp} \rightarrow 35 \text{ repeats}, \text{307 bp} \rightarrow 15 \text{ repeats}).

  5. Separate products by CE; generate electropherogram.

  6. Compare suspect, victim, and crime-scene profiles for match probability.

Ethical / practical considerations:

  • Contamination control (negative/blank controls, separate pre- & post-PCR rooms).

  • Chain of custody in legal contexts; errors or allelic drop-out can mislead.

  • Data privacy: VNTR genotypes are personally identifying; storage & access must follow legislation.

Critical Exam Points

  • List reagents & explain role (Template DNA, dNTPs, Buffer, Taq polymerase, Primers).

  • Describe three thermal steps and associated molecular events.

  • Explain exponential amplification: each new strand becomes template in next cycle → 2^{n} growth.

  • Relate primer design rules to annealing temperature.

  • Connect PCR → VNTR amplification → gel/CE separation → identity determination.

  • Recall key numbers:
    • Typical cycles: 25–35.
    • Taq optimum 72 degree ; denature 95 degree; anneal 50-65 degrees
    • Polymerase speed: ≈1000 bp min⁻¹.
    • Standard primer length: 20–30 bp.