Molecular Mapping and DNA Profiling

Short Tandem Repeats (STRs) (Microsatellites):
- Repeats of $2-10 ext{ bp}$ .
- Highly polymorphic short DNA sequences repeated in tandem.
Variable Number of Tandem Repeats (VNTRs) (Mini-satellites):
- Repeats of $10-100 ext{ bp}$ .
- Loci have variant repeat numbers between individuals.
- Chromosomes often have variable repeats at loci (heterozygotes).

Excellent Markers: Highly variable and easily detectable.
Detection Methods:
- RFLP and probe-based analysis (e.g., Southern hybridization) for VNTRs, using the repeat sequence as a probe.
- PCR amplification for STRs, using flanking sequences as primers.
Co-dominant: Heterozygotes are recognizable.

Purpose: Determine the phase of, and map distances between, molecular loci.
Phase Determination: Identify which alleles are on the same chromosome (parental arrangement).
- Haplotype: The genotype for closely linked genes or markers on a single chromosome or gamete.
Calculating Map Distance:
- Count recombinations between loci in offspring.
- Map distance = (Number of recombinants / Total offspring) * $100$ .
- Example: $1$ recombination out of $8$ offspring = $12.5 ext{ map units}$ .

Advantages over Genes for Mapping: DNA markers are more numerous, easier to score, and highly polymorphic.
Uses:
- Provide high-resolution genetic maps.
- Identify and clone genes based on map position.
- Identify rare disease-causing alleles.
- Tag desired alleles in plant/animal breeding (Marker Assisted Breeding).
- Assist in genetic counseling/risk calculations.
  - Example (Huntington's Disease): If a marker and disease gene are $20 ext{ mu}$ apart, the chance of inheriting the pathogenic allele with the associated marker is 100 - 20 = 80 ext{%}.
- Assist in genome sequence assembly.

Genomic DNA: Stable, consistent across all cells, and unique to each individual (excluding rare mutations).
Relatedness: Related individuals share related DNA sequences.
Method: Molecular methods identify differences in non-coding genomic regions.

Method: Processes genomic DNA to build a unique banding pattern based on mini-satellite loci (VNTRs).
Pros: Historically used to convict/exonerate individuals.
Cons: Requires large amounts of non-degraded DNA; challenging interpretation of similar bands.

Current Method: Utilizes different, unlinked microsatellite loci (STRs).
Process: PCR amplifies one locus at a time.
Allele Definition: Unambiguously defined by fragment size based on repeat numbers.
Multiplexing: Multiple PCRs in a single reaction, separated by size and identified by different fluorescent dyes.
Pros: Extremely sensitive (small DNA amounts), works on degraded DNA, unambiguous alleles.
Cons: Susceptible to contamination.

Limitation: Requires many more loci due to fewer alleles (up to $4$ ) per locus to build a unique profile.
Applications: Useful for highly degraded DNA, lineage, and evolution studies.

Probability-based: Uses population frequency of alleles; each locus is independent, probabilities are multiplied.
- Example: A profile with a 0.00009 ext{%} chance of occurring randomly in the population.

Forensic Applications:
- Identifying/ruling out suspects.
- Familial matching (e.g., Lonnie Franklin's son).
- Using personal genomics databases (e.g., Joseph DeAngelo via GEDmatch).
- Identifying human remains (e.g., 9/11 victims).
Investigation of Relatedness or Paternity:
- Legal disputes (paternity, immigration).
- Agricultural (breeding stocks, GMO tracking).
- Wildlife (poaching, population analysis).

Exclusion: Simple and definitive; different profiles mean different sources.
Proof of Identity: Impossible to completely prove identity/relatedness; a probability is provided that a match occurred by chance.
Errors:
- False Inclusion: More likely among relatives (e.g., monozygotic twins 100 ext{%}, parent/child 50 ext{%}), or within specific populations.
- False Exclusion: Caused by poor quality/low DNA, contamination, or human error.