DNA Profiling Notes

Short Tandem Repeats & DNA Profiling

Outcomes

  • Understand polymorphic DNA.
  • Learn the terminology of DNA Profiling.
  • Understand the structure and forms of STRs.
  • Learn what a DNA profile is and how to interpret an electropherogram.
  • Understand how DNA profiling has changed over the years.
  • Learn the problems encountered in DNA Profiling.

DNA Profiling

  • Our DNA is full of genes that make proteins.
  • The sequence of bases in these locations are important.
  • Changes to bases (letters) alter the instructions.
  • Substantial change can make the instructions nonsensical.
  • The majority of our DNA is non-coding and can be repetitive.
  • Example given: THE CAT AND DOG ARE SAD OUR NAN HAS FLU NOT FUR becomes THE CAT AND OGA RES ADO URN ANH ASF LUN OTF UR

Microsatellites Vs Minisatellites

  • Repeating (polymorphic) DNA is used in DNA Profiling.
  • In Forensic Profiling, SHORT TANDEM REPEATS (STRs), also known as MICROSATELLITES, are used.
  • VARIABLE NUMBER TANDEM REPEATS (VNTRs), also known as MINISATELLITES, are another type of repeating DNA.
  • The difference is size: STRs have repeating units of 2-6bp, VNTRs are above 6bp.
  • These are called di-nucleotides, tri-nucleotides, tetra-nucleotides etc. Example: TTG TTG TTG TTG TTG TTG TTG TTG TTG TTG TTG TTG TTG

Locus and STR Nomenclature

  • Locus: A location.
  • STRs have a code based on their location.
  • Example: D21S11:
    • D means DNA.
    • 21 is the chromosome number.
    • S means it’s an STR.
    • 11 means it was the 11th marker discovered in the chromosome.

Alleles

  • Allele: The DNA sequence that exists at that location; in STRs, this is the length of the tandem repeat.
  • If a repeat sequence of ATGG appears 12 times in tandem, the allele is 12.
  • For each STR, there is a range of possible repeat motifs (e.g., ATGG could appear 6-21 times).
  • Possible alleles: 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21.

Homozygous vs. Heterozygous

  • Homozygous: Two copies of one allele (same size).
    • Example: 2, 2
  • Heterozygous: One copy of two alleles (different sizes).
    • Example: 5, 8
  • Tetra-nucleotide repeat example provided.

Amplifying a DNA profile

  • A DNA profile comprises 16 STR markers consistently found in our DNA (plus Amelogenin - the sex marker).
  • Process involves:
    • Extract DNA to obtain the best quality and quantity of sample.
    • Amplify STR markers by PCR.
    • Use Capillary Electrophoresis to separate fragments by size and obtain the DNA profile.

Profiling Through the Ages

  • July 2014: DNA17 was introduced, containing more markers and mini-STRs to improve amplification in degraded samples.
  • Prior to this, SGM and SGM+ were used.
  • Originally, DNA profiling (fingerprinting) was performed using VNTRs.

Electropherogram Interpretation

  • Distinguishes between sequencing vs. fragment analysis using computer software.
  • Illustrates homozygous and heterozygous alleles.
  • Numerical allele score = number of 4bp repeats.

Allelic Ladder

  • Allelic ladders contain all possible allele options.
  • Examples:
    • D10S1248
    • D16S539
    • D2S133
    • D2S441
    • D8S1179
    • D21S11
    • D18S51
    • D22S1045
    • D19S433
    • TH01
    • FGA
    • D3S1358
    • D1S1656
    • D12S391
    • SE33

DNA Profile

  • A full DNA profile indicates whether a person is homozygous or heterozygous at every location.

Match Probability

  • Even with a perfect sample, it's crucial to determine the probability that someone else's DNA could match the profile.
  • Match probability is calculated using the Hardy-Weinberg equation.

Hardy-Weinberg Equation

  • p2+2pq+q2=1p^2 + 2pq + q^2 = 1
    • $p^2$ : Homozygotes
    • $2pq$ : Heterozygotes
  • Even the most common allele frequencies result in a match probability of 1 in 16 billion, but we use a conservative statement of 1 in a billion.
  • "Of the order of 1 in a billion"

Importance of Maths

  • Maths is extremely important to be able to answer the question in court “what’s the chance that someone else could have left X at the crime scene?”
  • The likelihood changes based on sample quality.

Stutter Peaks

  • Numerous problems can occur when examining a DNA profile. Key issues in Forensic Science are mixture interpretation and degradation, leading to analysis of peak number and height.
  • Stutter is a general issue related to CE.
  • Using 4bp repeating units (tetra-nucleotide repeats) reduces the chance of stutter.

Stutter Mechanism

  • Step 1: DNA polymerase extends through four repeat units.
  • Step 2: Enzyme dissociates, and strands separate.
  • Step 3: Strands re-anneal, but the template strand loops out, causing the extending strand to align out-of-register by one repeat unit.
  • Step 4: Final stutter product is formed, one repeat unit shorter than the template strand.

Problems in DNA Profiling

  • Mixtures analysis
  • Degradation
  • Noise
  • Pull up / Bleed through
  • Locus drop out (LDO)
  • Allelic imbalance (AI)
  • ADI-ADO high stutter

Next Tasks

  • Taste PTC paper to determine your alleles.
  • Consider forensic ethics.