Profiling Reviewer (HY)

DNA Profiling — High Yield Exam Reviewer

THE BIG PICTURE

DNA profiling works because humans are 99.9% identical but our 3 billion base pair genome means that 0.1% = ~3 million unique variations (polymorphisms) between individuals.

Polymorphism = the basis of ALL DNA profiling

HIGHEST YIELD FACTS TO MEMORIZE

  • SNP = most common type of DNA polymorphism

  • STR = method of choice in forensic DNA typing

  • VNTR = clustered near telomeres

  • Interspersed elements = NOT used in identity testing (moves around)

  • Polymorphism in conserved region = you cannot reproduce / you die

  • Genetic disorders only occur from changes in the conserved region

  • Sir Alec Jeffreys = father of DNA fingerprinting, Leicester University, UK, 1985

  • He discovered it by studying myoglobin gene in seals vs. humans

  • First identity system used = ABO blood group (1900s)

  • HLA = gold standard before DNA-based testing (1950s–70s)

  • A variant present in ≥1–2% of population = polymorphism (Buckingham)

  • rRNA = Universal Constant (nearly identical across all species for billions of years)

GENOME REGIONS — KNOW THE DIFFERENCE

Conservative

Non-Conservative

Contents

Exons, promoters, rRNA, tRNA

Introns, intergenic DNA

% of genome

~40%

~60%

Can mutate?

Rarely — lethal or sterility

Yes — harmless

Polymorphism here?

Stops at that individual

Normal occurrence

Used for?

Disease diagnosis

Identity testing / forensics

Genetic disorders?

YES — mutations here cause disease

NO

Remember:

  • Exons → code amino acids → must stay conserved

  • Promoters → "on/off switch" → works with RNA polymerase → must stay conserved

  • rRNA → aligns codons | tRNA → translates nucleic acid to amino acid → both conserved

  • Introns → spacers between exons, removed during splicing

  • Intergenic DNA → ~60% of genome, highly variable, best source of genetic markers

4 TYPES OF DNA POLYMORPHISM

Single Nucleotide Polymorphism (SNP)

  • Variation in one single base

  • Most common type of DNA polymorphism

  • Example: 99% have ...ATTCCTATCGAA... / 1% have ...ATTCCCATCGAA...

Short Tandem Repeat (STR) — MOST TESTED

  • Also called microsatellite DNA

  • Repeat unit = 1–7 bp | Repeats = 5–100 times

  • No specific area of clustering — distributed genome-wide

  • Method of choice in forensic DNA typing

  • Modern technique uses STR via PCR

Variable Number Tandem Repeat (VNTR)

  • Also called minisatellite DNA

  • Repeat unit = 6–100 bp | Repeats = 10–1,500 times

  • Clustered near telomeres

  • Used in some paternity testing labs

Interspersed Elements (SINEs, LINEs)

  • Move around chromosomes = unreliable

  • Treated as indel polymorphisms

  • NOT used in forensics or identity testing

  • Example: Person A has it on chr1, Person B doesn't — no valid comparison possible

STR vs VNTR — QUICK COMPARISON TABLE

STR (Microsatellite)

VNTR (Minisatellite)

Repeat length

2–6 bp

15–50 bp

Total array

50–500 bp

500 bp–20 kb

Location

Genome-wide

Subtelomeric

Current use

Forensic standard

Some paternity labs

TRADITIONAL vs MODERN — MOST LIKELY TESTED

Traditional (Fingerprinting)

Modern (Profiling)

Term

DNA Fingerprinting

DNA Profiling

Marker used

RFLP

STR

Key step

Restriction enzyme digestion

PCR amplification

DNA needed

Large, fresh, high quality

Very little, degraded OK

Separation

Gel electrophoresis

Capillary electrophoresis

Visualization

Southern blot + autoradiogram

Electropherogram

Output

Barcode bands

Digital peaks

Speed

Days to weeks

Hours

Precision

Lower

Single base pair resolution

PROCEDURE — HIGH YIELD STEPS

Traditional (RFLP-Based)

DNA Extraction → RE Digestion → Gel Electrophoresis → Southern Blotting

Restriction Enzymes — know these facts:

  • Cut at palindromic recognition sites (e.g., EcoRI cuts GAATTC)

  • Palindromic = reads same 5'→3' on both strands

  • Create staggered/zigzag cuts = sticky ends (overhangs)

  • Sticky ends allow DNA fragments to join via hydrogen bonds

  • More repeats = longer fragments

  • Fewer repeats = shorter fragments

  • Resulting fragments = RFLPs

  • Problem: ONLY works on fresh DNA — useless on degraded samples

Southern Blotting — know these facts:

  • Transfers bands from gel to a membrane (gel is fragile)

  • Radioactive probes detect specific loci

  • Captured on X-ray film = autoradiogram

  • Lower/thicker bands = fewer repeats

  • Higher bands = more repeats

  • Pattern = DNA fingerprint (unique barcode per person)

Modern (STR/PCR-Based)

DNA Extraction → PCR Amplification → Capillary Electrophoresis → Electropherogram

PCR — know these facts:

  • Primers bind to conserved flanking sequences (same in everyone)

  • STR region in between varies per person = polymorphism captured

  • Primers tagged with fluorescent dyes

  • Shorter fragments → migrate faster → arrive at detector earlier

  • Longer fragments → migrate slower → arrive at detector later

ELECTROPHEROGRAM — KNOW THIS

  • X-axis = fragment size (base pairs)

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

  • Expect 2 peaks per locus (diploid = 1 allele from mom + 1 from dad)

  • Heterozygous = 2 distinct peaks (different repeats from each parent)

  • Homozygous = 1 peak (same repeats from both parents)

FORMULA (Will Likely Appear in Exam):

No. of Repeats = (Total Fragment Size − Flanking Region Size) ÷ Repeat Unit Size

Example: Flanking = 60 bp, Repeat unit = 4 bp, Fragment = 100 bp
→ (100 − 60) ÷ 4 = 10 repeats

APPLICATIONS — ONE-LINER EACH

Application

Key Point

Paternity Testing

Child with most matching DNA bands = biological child

Forensics

DNA from crime scene matched to suspect

Genealogy/Archaeology

Trace ancestry or identify historical remains

Organ Transplant

Match donor-recipient compatibility

DNA Database

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

FIELD vs REGION TARGETED

Field

Region

Why

Molecular Genetics

Exons (coding)

Find disease-causing mutations

Forensics / Paternity / HLA

Intergenic (non-coding)

Use polymorphisms to ID individuals

Molecular Oncology

Both

Mutations in genes AND regulatory regions

HISTORY TIMELINE — KNOW THE ORDER

Era

Key Event

1900s

ABO blood group — first identity testing system

1950s–70s

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

1980s

Alec Jeffreys pioneers DNA-based identity testing

1985

First DNA fingerprinting article published — forensics + paternity

COMMON EXAM TRAPS

Trap

Correct Answer

Which polymorphism is most common?

SNP

Which is the forensic standard?

STR

Which is unreliable for identity testing?

Interspersed elements

What does RFLP require that STR doesn't?

Fresh, high-quality, undegraded DNA

What does Southern blot use that gel electrophoresis doesn't?

Probes + membrane transfer

Old/traditional technique term?

DNA fingerprinting

Modern technique term?

DNA profiling

Where are VNTRs clustered?

Near telomeres

Who discovered DNA fingerprinting?

Sir Alec Jeffreys

What gene did Jeffreys study?

Myoglobin gene in seals vs. humans

What molecule is the Universal Constant?

rRNA

Flanking regions are _____ in all individuals

The same / conserved

STRs have _____ specific clustering area

No specific area

MEMORY TRICKS

  • SNP = Single = Smallest = Supreme (most common)

  • STR = Short = Standard in forensics

  • VNTR = Very Near Telomere Region

  • Conservative = Constant, Critical, Cannot change

  • Non-conservative = No function, No harm, Nice for markers

  • Fewer repeats → Fragments are Faster and smaller

  • More repeats → Migrate More slowly (bigger)

  • Gel electrophoresis = separates | Southern blot = identifies

  • RFLP needs FRESH DNA | STR can use FORENSIC (old/degraded) DNA