Profiling Reviewer

DNA Profiling — Exam Reviewer

1. FOUNDATIONS

Why are humans unique if we're 99.9% identical?
Our DNA is 3 billion base pairs long, so that 0.1% difference = ~3 million variations called polymorphisms — the basis of DNA profiling.

Polymorphism vs. Mutation

  • Polymorphism — naturally built-in variation; present in ≥1–2% of the population; cannot be passed down if it occurs in conserved regions

  • Mutation — non-natural change; can be inherited parent-to-child

2. HUMAN GENOME REGIONS

Conservative (Conserved) Region (~40%)

  • Contains protein-coding exons and functional non-coding elements (promoters, tRNA, rRNA)

  • Must stay stable and unchanged — codes for critical proteins

  • Polymorphism here = rare, and if it occurs, it ends at that individual (they cannot reproduce, or die)

  • Exons → code for amino acids; must remain conserved

  • Promoters → "on/off switch" for transcription; work with RNA polymerase

  • rRNA → aligns codons ("Universal Constant" across all species)

  • tRNA → translates nucleic acid to amino acid

Non-Conservative Region (~60%)

  • Contains introns and intergenic DNA

  • NOT essential for life; changes here are harmless (no protein coding)

  • Primary source of genetic markers for forensics/identity testing

  • Introns → spacers between exons; removed during mRNA splicing

  • Intergenic DNA → ~60% of genome; highly variable; mutations accumulate harmlessly

  • Genetic disorders only manifest from changes in the conserved region

3. FOUR TYPES OF DNA POLYMORPHISM

Type

Description

Size

Notes

SNP (Single Nucleotide Polymorphism)

Variation in ONE base

1 bp

Most common type

STR (Short Tandem Repeat)

Short repeated sequences

1–7 bp repeat; 5–100 repeats

Microsatellite DNA; method of choice in forensics

VNTR (Variable Number Tandem Repeat)

Longer repeated sequences

6–100 bp repeat; 10–1,500 repeats

Minisatellite DNA; clustered near telomeres

Interspersed Elements

Mobile elements (SINEs, LINEs)

Variable

Move around chromosomes; unreliable for identity testing

SNP, STR, and VNTR stay at fixed chromosomal locations → reliable genetic markers
Interspersed elements move around → treated as indel polymorphisms → NOT used in identity testing

VNTR vs. STR Quick Comparison

Minisatellite (VNTR)

Microsatellite (STR)

Repeat length

15–50 bp

2–6 bp

Total array size

500 bp – 20 kb

50–500 bp

Location

Subtelomeric

Genome-wide

Use

Some paternity labs

Forensic standard

4. DNA FINGERPRINTING vs. DNA PROFILING

DNA Fingerprinting (Traditional)

DNA Profiling (Modern)

Term coined by

Alec Jeffreys (1985)

Standard modern term

Primary method

RFLP via restriction enzymes

STR via PCR

DNA needed

Large, high-quality, undegraded

Very little; degraded OK

Separation

Gel electrophoresis

Capillary electrophoresis

Visualization

Southern Blotting + autoradiogram (X-ray)

Electropherogram (digital peaks)

Output

Barcode-like bands

Peaks on a graph

Speed

Days to weeks

Hours

Precision

Lower resolution

Single base pair resolution

Key person: Sir Alec Jeffreys, Leicester University, UK — discovered DNA fingerprinting while studying the myoglobin gene in seals vs. humans. Found that all organisms share identical sequences but differ in number of repeats.

5. PROCEDURE (TRADITIONAL — RFLP)

Step 1 — DNA Extraction
Goal: release DNA from the nucleus. Methods: organic, inorganic, or solid-phase.

Step 2 — Restriction Enzyme Digestion

  • RE enzymes cut at palindromic recognition sites (e.g., EcoRI cuts GAATTC)

  • Cuts are staggered/asymmetric (zigzag), creating sticky ends (overhangs)

  • Sticky ends allow rejoining via hydrogen bonds between complementary bases

  • More repeats → longer fragments; fewer repeats → shorter fragments

  • Resulting variable fragments = RFLPs (Restriction Fragment Length Polymorphisms)

  • Problem: Only works on fresh, high-quality DNA — useless on degraded/old samples

Step 3 — Gel Electrophoresis (AGE)

  • Separates fragments by size

  • Stained with EtBr (carcinogenic) or newer stains

  • DNA remains in the gel (fragile)

Step 4 — Southern Blotting

  • Transfers bands from gel onto a membrane

  • Radioactive probes applied to detect specific loci

  • Captured by X-ray film (autoradiogram)

  • Result: barcode pattern = DNA fingerprint

  • Lower/thicker bands = fewer repeats (shorter); Higher bands = more repeats (longer)

6. PROCEDURE (MODERN — STR/PCR)

Step 1 — DNA Extraction (same as traditional)

Step 2 — PCR Amplification

  • Primers bind to conserved flanking sequences (same in all individuals)

  • The STR region in between varies per person → this captures the polymorphism

  • Primers are tagged with fluorescent dyes

  • Fragments differ in length based on number of repeats

Step 3 — Capillary Electrophoresis

  • Same size-separation principle as gel electrophoresis

  • Shorter fragments → migrate faster → arrive at detector earlier

  • Longer fragments → migrate slower → arrive later

  • Detector/laser reads color and time of arrival of fluorescent-tagged fragments

Step 4 — Electropherogram

  • X-axis = fragment size in base pairs

  • Y-axis = fluorescent intensity (concentration of STRs)

  • Expect two peaks per locus (diploid organism — one allele from each parent)

  • Heterozygous peaks = two distinct peaks (different number of repeats from each parent)

  • Homozygous peaks = one peak (same number of repeats from both parents)

Repeat Calculation Formula:

Number of Repeats = (Total Fragment Size − Size of Flanking Region) ÷ Size of One Repeat Unit

Example: Flanking region = 60 bp, repeat unit = 4 bp, fragment = 100 bp → (100−60)/4 = 10 repeats

7. APPLICATIONS

Application

Method Used

Notes

Paternity Testing

Southern blotting

Child with most matching bands = biological child; sparked by a UK immigration case (Alec Jeffreys)

Forensics

STR/PCR

DNA from crime scene matched to suspect

Genealogy & Archaeology

DNA profiling

Trace ancestry, identify historical remains

Organ Transplant

DNA profiling

Match donor and recipient compatibility

DNA Database

NDIS (national) → SDIS (state) → LDIS (local)

Contains convicted offenders and forensic profiles

8. REGION TARGETED BY FIELD

Field

Region Targeted

Purpose

Molecular Genetics

Protein-coding (exons)

Find mutations causing inherited diseases

Forensics / HLA / Paternity

Intergenic (non-coding)

Use polymorphisms for identifying individuals

Molecular Oncology

Both

Find mutations in genes OR regulatory regions leading to cancer

9. QUICK HISTORY TIMELINE

Period

Event

1900s

ABO blood group used for identity testing

1950s–70s

Serum proteins, RBC enzymes, and HLA system used (HLA = gold standard)

1980s

Sir Alec Jeffreys pioneers DNA-based identity testing

1985

First article on DNA fingerprinting published; applied to forensics and paternity

10. KEY TERMS CHEAT SHEET

  • Palindromic sequence — reads the same 5'→3' on both strands (e.g., GAATTC)

  • Flanking region — constant sequences surrounding STRs; same in all individuals; used to design primers

  • RFLP — Restriction Fragment Length Polymorphism; basis of traditional fingerprinting

  • Autoradiogram — X-ray film capture of radioactive probe signals on Southern blot

  • Electropherogram — digital graph output of capillary electrophoresis

  • Genetic markers — genes/sequences used to identify individuals; must be stable and locatable

  • Introns — non-coding spacers between exons; removed during RNA splicing

  • Exons — protein-coding sequences; must stay conserved