Forensic Genetics - Week 3 Lab Notes

Restriction Enzyme Digestion Results

• Context
  • Two-week practical investigating restriction enzyme digestion of \lambda DNA (phage lambda DNA).
  • Enzymes employed: RsaI and EcoRV.
• Expected gel layout (left ➜ right)
  • DNA ladder (size marker)
  • Undigested \lambda DNA (super-coiled + nicked/open-circle forms ► single high-MW band)
  • Alternating lanes of the two digests prepared in duplicate/triplicate to assess reproducibility:
  • RsaI
  • EcoRV
  • …sequence repeats to fill remaining lanes.
• Interpretative reminders
  • RsaI & EcoRV are 6-base cutters that recognise palindromic sites; each creates characteristic fragment patterns.
  • Comparing band number + sizes with the ladder tells us whether digestion was complete or partial.
  • Undigested control confirms DNA integrity; ladder calibrates fragment length.

Polymerase Chain Reaction (PCR) – Core Concepts

• Definition: Targeted, exponential amplification of a defined DNA region.
• Essential reagents
  • DNA template – the sequence to be copied.
  • Buffer system – maintains optimal \text{pH}, ionic strength, \text{Mg}^{2+} for polymerase activity.
  • dNTPs – \text{dATP, dCTP, dGTP, dTTP}; substrates for DNA synthesis.
  • Primer pair – two short single-stranded oligonucleotides (forward & reverse) that flank the target and provide 3’-OH ends.
  • Taq DNA polymerase – thermostable enzyme from Thermus aquaticus that extends primers.
• Three recurring thermal steps per cycle
  1. Denaturation
  • High T (≈ 94^{\circ}\text{C}) separates dsDNA ⇒ ssDNA.
  1. Annealing
  • Lower T (primer-specific; typically 50–65^{\circ}\text{C}) allows primers to hybridise to complementary sites.
  1. Extension / Elongation
  • Intermediate T (≈ 72^{\circ}\text{C} for Taq) enables strand synthesis (≈ 1\,\text{kb min}^{-1}).
• Amplification mathematics
  • Ideal doubling each cycle: N = N_0 \times 2^{n} (where n = cycle number).

Sex Determination PCR – Principle & Targets

Sex identification exploits sequence differences between X and Y chromosomes.

Amelogenin Gene Test (PCR #1)

• Gene: Amelogenin (tooth-enamel matrix protein).
• Genomic location: homologous loci on both X and Y chromosomes.
• Key polymorphism: 6-bp deletion on the X allele.
• Shared primer set amplifies both alleles.
• Product sizes
  • X_{Amel} = 106\,\text{bp}
  • Y_{Amel} = 112\,\text{bp}
• Gel interpretation
  • Female (XX): single band at 106\,\text{bp}.
  • Male (XY): two bands at 106\,\text{bp} and 112\,\text{bp}.
• Added value
  • Detects mixed DNA samples (both male + female patterns concurrently).

Y-Chromosome Repeat Test (PCR #2)

• Target: Y-specific tandem repeat sequence (absent on X).
• Product size
  • Y_{rep} = 154\,\text{bp}
  • X_{rep} = 0 (no amplification because no template sequence).
• Gel interpretation
  • Female: no band (blank lane, aside from primer-dimer / background).
  • Male: single band at 154\,\text{bp}.
• Combined strategy
  • Running both assays cross-validates conclusions, mitigates allele dropout or PCR failure.

Practical Protocol – Day-of-Lab Setup

Tube Labelling Scheme (per group)

• Four reactions for each PCR system:
  • Your DNA (initials)
  • Partner’s DNA (their initials)
  • Positive male control (M)
  • Positive female control (F)

Master-mix Components & Volumes (per 20 µL reaction)

• ddH_2O – 3\,\mu\text{L}
• 2× HotStar PCR mix (contains Taq, buffer, dNTPs, Mg^{2+}) – 10\,\mu\text{L}
• Forward primer – 1\,\mu\text{L}
• Reverse primer – 1\,\mu\text{L}
• Genomic DNA – 5\,\mu\text{L} (added to tube designated for the specific sample)
• Seal tubes, quick-spin if necessary, then place on PCR rack.

Thermocycler Programs

• Specific annealing temperature is dictated by primer T_m: 56^{\circ}\text{C} for Amel primers, 65^{\circ}\text{C} for Y-repeat primers.

Analytical Considerations & Troubleshooting

• Band intensity correlates with template quantity and amplification efficiency.
• Absence of control bands indicates mastermix failure, thermal-cycler error, or enzyme inhibition.
• Primer-dimer (~20–40 bp) may appear if primer complementarity exists; minimise by optimising primer concentration ✔.
• Partial digestion (restriction assay) or allelic dropout (PCR) can mimic sex chromosomal anomalies; verifying with two independent assays reduces misclassification risk.

Broader Applications & Ethical Notes

• Forensic sex identification of crime-scene DNA—critical for suspect elimination.
• Archaeology & anthropology: determining biological sex of fossil remains when skeletal features are ambiguous.
• Wildlife conservation genetics: assessing sex ratios in endangered populations from non-invasive samples (feces, hair, feathers).
• Medical genetics: detecting sex-chromosome aneuploidy (e.g.
  • Klinefelter: XXY may yield both Y repeat band and doubled X band).
• Ethical caution: genetic sex ≠ gender identity; data privacy and informed consent remain paramount.