VNTRs and Electrophoresis – Vocabulary Flashcards

• Core forensic idea: Non-coding ("junk") DNA makes up ≈ 80 % of the human genome and changes freely → rich source of person-specific markers

“Junk” DNA & Its Forensic Relevance

• Lies between functional genes; exerts little to no selective pressure

• Replication errors are rarely corrected → high mutation rate (especially in repetitive regions)

• Four functional classes in the genome

  • Protein-coding DNA (genes)

  • Repetitive DNA (includes VNTRs, telomeres, satellites)

  • Pseudogenes (non-functional copies of genes)

  • Unique non-coding DNA (regulatory, intronic, etc.)

• Why useful?

  • Extremely polymorphic → likelihood of two unrelated people sharing lengths is very low (except identical twins)

  • Inherited in Mendelian fashion → trace family and paternity relationships

Genes, Alleles & Chromosomal Context

• Genes spread unevenly across the 23 pairs of chromosomes, but reside at the same locus on maternal & paternal copies

• Alternative DNA sequences at a locus = alleles; diploid humans carry max. 2 alleles per locus

• Gene stretches are separated by non-coding DNA; VNTRs often sit near chromosome ends (telomeres)

Polymorphisms

• Definition: inter-individual DNA differences

  • Sequence polymorphism → one base replaced by another (SNP)

  • Size/Length polymorphism → bases inserted or deleted (indels, VNTRs)

• More abundant in non-coding regions than coding ones

Variable Number Tandem Repeats (VNTRs)

• A subtype of size polymorphisms; a.k.a. mini-satellites

• Basic characteristics

  • Repeat unit length: 6 – 100 bp

  • Copy number: a few to many thousands in tandem (head-to-tail)

  • Entire locus can span hundreds→thousands of base pairs

• Mutation mechanism

  • DNA polymerase slippage during replication forms loops → whole repeat units added or deleted (never partial repeats)

  • Change occurs one whole repeat at a time

• Telomeric association

  • VNTRs frequently follow the telomeric repeat \text{TTAGGG} which “caps” chromosomes

VNTR Terminology

• Repeat Unit = number of bases per single repeat (e.g. “6 bp”)

• Repeat Sequence = exact base string (e.g. “CGTCAT”)

• Copy Number = consecutive repetitions of the unit (e.g. 6)

• Example (Page 8):

  • Sequence: ACGTGCT ACGT CAT CGT CAT CGT CAT CGT CAT CGT CAT AACGTGC

  • Repeat unit = 6 bp, repeat sequence = “CGTCAT”, copy number = 6

VNTR Alleles: Diversity, Inheritance & Counting

• Each distinct copy number = one allele (thousands possible)

• Alleles change by ±1 repeat unit per mutational event

• Allele count formula when repetitive block length is known: \text{Number of alleles}=\frac{\text{Total repeat region length}}{\text{Repeat unit length}}

  • Example: 500 bp block / 10 bp repeat = 50 possible length alleles

• Mendelian inheritance: one maternal + one paternal allele per person; different homologs may bear different copy numbers

• Probability of two unrelated people sharing an allele combination is low; identical twins are the only guaranteed match

VNTR Testing – Five-Step Workflow

1. DNA Collection

2. Restriction Enzyme (RE) Digestion

3. Gel Electrophoresis

4. Southern Blot Transfer

5. Probe Hybridisation (plus optional re-probing)

Step 1 – DNA Collection

• Requires relatively large, good-quality samples (blood, semen, tissue)

• Process: extraction → purification → quantification → concentration normalisation for cross-sample comparison

Step 2 – Restriction Enzyme Digestion

• REs = bacterial immune proteins recognising palindromic sequences and cutting DNA

  • Read 5'→3' identically on both strands (e.g. GAATTC / CTTAAG)

• Two cut types

  • Blunt ends – cut exactly in the middle

  • Sticky ends – staggered cleavage, leaving single-stranded overhangs

• Cutting frequency

  • Formula (approx.): \text{Cuts}=\frac{N}{4^{n}}

  • N = genome size, n = recognition site length (bp)

  • Shorter sites → more cuts; e.g. in human genome (3.2 × 10^9 bp):

  • HaeIII (4-bp site “CCGG”) → ≈ 12.5 M cuts

  • EcoRI (6-bp “GAATTC”) → ≈ 781,250 cuts

  • NotI (8-bp “GCGGCCGC”) → ≈ 48,828 cuts

• Post-digestion mixture

  • Contains thousands of fragments; only some hold VNTRs, each fragment’s length reflects flanking RE sites + VNTR copy number

Step 3 – Gel Electrophoresis

• Porous agarose/polyacrylamide gel acts as molecular sieve

• Electric field drives negatively charged DNA toward the positive electrode

• Separation strictly by size: shorter fragments run faster/farther

• Features

  • Multiple wells → load ladder (size standard) + numerous samples side-by-side

  • DNA ladder fragments of known sizes (acts like a ruler)

• VNTR smear: because a digest contains many fragments of similar/overlapping lengths, bands smear instead of discrete lines → requires blotting/probing to visualise specific VNTRs

Step 4 – Southern Blotting

• Invented by Edwin Southern

• Steps

  1. Gel placed on wick soaked in high-salt/alkali solution; nitrocellulose/nylon membrane laid on top

  2. Buffer wicks upward, carrying ssDNA from gel → membrane; alkaline conditions break H-bonds so DNA becomes single-stranded

  3. Membrane now holds immobilised, single-stranded fragments (still a smear)

• Advantages: membrane far sturdier, can undergo harsh washing & multiple probings

Step 5 – Probe Hybridisation (Probing)

• Synthetic ssDNA (or RNA) oligonucleotides complementary to VNTR repeat sequence

• Labelled radioactively (e.g. ^{32}\text{P}) or fluorescently

• Membrane sealed with probe solution → probes hybridise to complementary VNTR-containing fragments

• Excess probe washed away → membrane exposed to X-ray film / imager

• Result: only VNTR fragments with the targeted repeat sequence appear as bands (“bar-code” pattern)

• Multi-locus probes: recognise all VNTR loci with that repeat unit → many bands per lane

• Re-probing: strip off first probe (boil/alkali) and repeat with different probe to survey additional VNTRs (time-consuming)

Single-Locus vs Multi-Locus VNTR Analysis

• Multi-locus probes

  • Advantages: one hybridisation reveals many loci simultaneously

  • Disadvantages: need large DNA quantity, slow, can’t detect mixture/contamination, poor for degraded samples

• Single-locus VNTRs

  • Each probe targets one unique genomic VNTR → each person yields ≤ 2 bands (diploid)

  • Enhances speed, simplifies interpretation, flags mixed/contaminated samples (extra bands)

  • 1980-90s standard panel: six markers (D1S7, D2S44, D4S139, D10S28, D14S13, D17S79) – all highly polymorphic (>19 alleles each)

Probability Calculations with VNTR Alleles

• For a locus with n distinct alleles, number of diploid genotype combinations (order irrelevant) = \frac{(n+1)\times n}{2}

  • Example: D1S7 (28 alleles) → \frac{29\times 28}{2}=406 combinations → random match probability 1/406

  • D4S139 (19 alleles) → \frac{20\times 19}{2}=190 combinations → 1/190 chance

• Combined probability across loci (assuming independence) = product of individual probabilities

  • Example (D1S7 & D4S139): \frac{1}{406}\times\frac{1}{190}=\frac{1}{77,140}

  • Six-marker panel example: \frac{1}{406}\times\frac{1}{351}\times\frac{1}{190}\times\frac{1}{300}\times\frac{1}{465}\times\frac{1}{190}=\frac{1}{7.18\times10^{14}} (≈1 in 7 × 10^14)

• Caveats: allele frequencies differ among ethnic groups → real calculations use population-specific databases; nevertheless, adding loci always lowers chance of coincidental match

Applications

• Criminal investigations: compare DNA from evidence (blood, semen, hair roots) to suspects

  • Example slide: Crime-scene semen matched Suspect 2, excluded Suspect 1

• Paternity & familial tests: child must share one VNTR allele with each biological parent

  • Patterns lacking parental allele lead to exclusion; multiple markers increase confidence

• Mass-disaster victim ID, missing-person cases, migration studies of human populations

Advantages & Disadvantages Summary

• Advantages of VNTR approach:

  • Extremely polymorphic; high discriminatory power

  • Mendelian inheritance allows kinship assessment

  • Early technique that proved DNA could identify individuals → foundation for modern STR/NGS methods

• Disadvantages (particularly multi-locus):

  • Requires large, undegraded DNA amounts

  • Time-consuming (week-long)

  • Complex interpretation when samples are degraded or mixed

  • Outdated: largely replaced by PCR-based STR typing today

Exam Preparation Checklist (from Page 51)

• Define VNTR & list its key characteristics (repeat unit size, copy-number variability, telomeric proximity)

• Outline ALL five steps in VNTR testing, detailing techniques & rationale

• Explain restriction enzymes: palindromic recognition, blunt vs sticky ends, cut-frequency formula \frac{N}{4^{n}}

• Describe electrophoresis mechanics (charge, gel matrix, ladders, lane organisation)

• Discuss pros/cons of multi-locus vs single-locus VNTRs

• Justify why testing multiple VNTRs boosts reliability (qualitative probability argument sufficient)

• Provide real-world examples: e.g. paternity, crime-scene matching

Ethical, Practical & Historical Notes

• Ethical: need for population allele-frequency databases; ensure privacy of genetic fingerprints; identical twins as special case

• Practical: poor for old/degraded samples; mixture detection better with single-locus probes; modern forensics shifted to PCR STRs & capillary electrophoresis for speed & sensitivity

• Historical significance: VNTR fingerprinting first brought DNA evidence into court, leading to 1986 Colin Pitchfork conviction (UK)

Take-Home Message

• VNTRs exploit length variability in non-coding tandem repeats; their high polymorphism underpins early DNA fingerprinting

• Testing involves digestion, separation, blotting & hybridisation; interpretation relies on allele length patterns inherited from parents

• More loci = exponentially lower random-match probability; nonetheless, current practice now favours STR PCR due to greater speed & lower DNA requirements