19  Experimental Proofs and Methods

19.1 How Scientists Prove Things in Genomics

19.1.1 The Scientific Method in Action

Throughout this book, we’ve learned about amazing discoveries. But how did scientists actually PROVE their ideas?

The scientific method:

  1. Observe something interesting

  2. Ask a question

  3. Form a hypothesis (educated guess)

  4. Design an experiment to test it

  5. Collect and analyze data

  6. Draw conclusions

Let’s explore clever experimental methods that proved key concepts!

19.2 Classic Labeling Experiments

19.2.1 Using Isotopes to Track Molecules

Isotopes = Different forms of the same element

  • Same element, different weight

  • Some are radioactive (can be detected!)

  • Used to “label” and track molecules

Think of isotopes like:

  • Putting a GPS tracker on something

  • Using glow-in-the-dark paint to follow it

  • Tagging an animal to track its migration

19.2.2 The Meselson-Stahl Experiment (1958)

The Question: How does DNA replicate? Three possibilities:

  1. Conservative: Original DNA stays together, new copy is separate

  2. Semi-conservative: Each new DNA has one old strand, one new strand

  3. Dispersive: Old and new DNA mixed together

The Clever Experiment:

Setup:

  • Grew bacteria in medium with heavy nitrogen (¹⁵N) for many generations

  • All DNA became “heavy”

  • Then switched to normal nitrogen (¹⁴N)

  • Let bacteria replicate once, twice, etc.

Detection Method:

  • Centrifuge in special solution

  • Heavy DNA sinks more

  • Light DNA floats more

  • Can see distinct bands!

Results:

  • After 1 replication: ALL DNA was medium weight (half-heavy, half-light)

  • After 2 replications: Half medium, half light

  • This matched semi-conservative replication!

Conclusion: DNA replication is semi-conservative!

  • Each new DNA has one original strand and one new strand

  • Like unzipping the ladder and building a new side for each half

Why this experiment was brilliant:

  • Simple, elegant design

  • Clear, unambiguous results

  • Definitively answered the question

19.2.3 Pulse-Chase Experiments

What they are: Give cells a “pulse” of labeled material, then “chase” with unlabeled material

How it works:

  1. Pulse: Give cells radioactive amino acids for short time

  2. Chase: Switch to normal amino acids

  3. Track: See where the radioactive proteins go over time

What it tells us:

  • Path of proteins through the cell

  • How long processes take

  • Where proteins end up

Example: Tracking protein secretion

  • Pulse: Radioactive amino acids → proteins made in ER

  • Chase: Follow labeled proteins through Golgi → secretion

  • Result: Map the secretory pathway!

19.3 Functional Genomics Approaches

19.3.1 Understanding What Genes Do

Functional genomics = Determining the functions of genes and their products

19.3.2 Gene Knockout Experiments

What it is: Deliberately “break” a gene to see what happens

Logic:

  • If you break gene X and the organism can’t do Y anymore

  • Then gene X must be needed for Y!

Methods:

1. Classical Gene Knockout (in mice):

  • Remove or disrupt a gene

  • Create mice with that gene missing

  • See what goes wrong

  • Identify gene function!

Example:

  • Knock out gene for leptin

  • Mice become obese

  • Conclusion: Leptin controls appetite/weight!

2. RNA Interference (RNAi):

  • Add small RNAs that destroy specific mRNAs

  • Temporarily “turn off” genes

  • See effects

  • Faster than classical knockout

3. CRISPR-Cas9 (modern method):

  • Precisely edit genes

  • Can knockout, knockin, or modify

  • Fast, cheap, accurate

  • Revolutionary!

19.3.3 Gene Overexpression

What it is: Make cells produce too much of a protein

Methods:

  • Insert extra copies of gene

  • Use strong promoters

  • Force high expression

Why do it:

  • See effects of too much protein

  • Study protein function

  • Produce proteins for research or medicine

Example:

  • Overexpress growth factor

  • Cells grow faster

  • Conclusion: That growth factor promotes growth!

19.3.4 Reporter Genes

What they are: Genes that make easily detectable proteins

Common reporters:

  • GFP (Green Fluorescent Protein): Glows green!

  • Luciferase: Makes light (like fireflies)

  • LacZ: Turns blue with specific chemical

How to use them:

  1. Attach reporter gene to gene of interest

  2. Reporter expression shows where/when gene is active

  3. Can see it with microscope or other detection!

Example:

  • Attach GFP to muscle gene promoter

  • See which cells become muscle (they glow green!)

  • Watch development in real-time!

Think of reporters like:

  • Putting a light bulb on a light switch

  • You can see when the switch (gene) is ON!

19.4 ChIP: Chromatin Immunoprecipitation

19.4.1 Finding Where Proteins Bind to DNA

ChIP = Method to find where specific proteins bind on DNA

How it works:

  1. Crosslink: Proteins bound to DNA are “glued” together

  2. Break up: Shear DNA into small pieces

  3. Immunoprecipitate: Use antibody to pull out specific protein

  4. Unglue: Reverse crosslinking

  5. Sequence: Sequence the DNA that was bound

  6. Map: Find where in genome that DNA came from!

Result: Know exactly where in the genome your protein was bound!

Applications:

  • Find where transcription factors bind

  • Map histone modifications

  • Understand gene regulation

ChIP-seq = ChIP + Next-gen sequencing

  • Maps protein-DNA interactions genome-wide

  • Incredibly powerful!

19.5 CRISPR-Cas9: The Gene Editing Revolution

19.5.1 Molecular Scissors for DNA

CRISPR-Cas9 = Precise gene editing tool (Nobel Prize 2020!)

What it does: Cut DNA at exact locations and make changes

How it works:

  1. Guide RNA: Designed to match target DNA sequence

  2. Cas9 enzyme: Molecular scissors that cut DNA

  3. Complex: Guide RNA + Cas9 finds target

  4. Cut: Cas9 cuts both DNA strands

  5. Repair: Cell repairs cut (can insert changes during repair!)

Think of it like:

  • Find: GPS directs you to exact address (guide RNA)

  • Cut: Scissors cut at that location (Cas9)

  • Fix: Cell repairs, possibly with changes

Applications:

Research:

  • Knockout genes easily

  • Edit specific mutations

  • Study gene function

Medicine (experimental):

  • Fix disease-causing mutations

  • Sickle cell disease trials

  • Cancer immunotherapy

  • Potentially cure genetic diseases!

Agriculture:

  • Create disease-resistant crops

  • Improve nutrition

  • Drought tolerance

Why CRISPR is revolutionary:

  • Easy: Much simpler than old methods

  • Fast: Days instead of months/years

  • Cheap: Affordable for most labs

  • Precise: Target exact locations

  • Versatile: Works in many organisms

19.6 Single-Cell Technologies

19.6.1 Studying Individual Cells

The problem: Bulk measurements average across millions of cells The solution: Single-cell technologies!

19.6.2 Single-Cell RNA-seq (scRNA-seq)

What it does: Measure gene expression in individual cells

Why it matters:

  • See cell-to-cell variation

  • Identify rare cell types

  • Watch cells change over time

  • Understand diseases better

Applications:

  • Cancer: Find rare resistant cells

  • Development: Watch cells differentiate

  • Immunology: Identify immune cell types

  • Neuroscience: Map brain cell types

How it works:

  1. Isolate single cells

  2. Capture mRNA from each cell

  3. Add cell-specific “barcode”

  4. Sequence all cells together

  5. Use barcodes to separate data from each cell

  6. Analyze individual cell gene expression!

19.6.3 Single-Cell Genomics

What it does: Sequence genomes of individual cells

Why:

  • See genetic variation between cells (cancer!)

  • Study rare cells

  • Understand mosaicism

Application: Cancer

  • Sequence many tumor cells

  • See which mutations each cell has

  • Understand tumor evolution

  • Identify dangerous cell populations

19.7 Imaging Technologies

19.7.1 Seeing Genes and Proteins in Action

19.7.2 Fluorescence Microscopy

What it is: Using fluorescent molecules to see biological structures

Key techniques:

  • Immunofluorescence: Antibodies with fluorescent tags

  • Live-cell imaging: Watch processes in real-time

  • Super-resolution: See details smaller than normal light microscopy limit

Example - watching protein movement:

  • Tag protein with GFP (green fluorescent protein)

  • Watch it move in living cells

  • See where it goes, how fast, what it does

  • Like having a spy camera in the cell!

19.7.3 FISH (Fluorescence In Situ Hybridization)

What it does: Find specific DNA or RNA sequences in cells/tissues

How it works:

  1. Design fluorescent probe that binds specific sequence

  2. Add probe to cells/tissue

  3. Wash away unbound probe

  4. Look with fluorescence microscope

  5. See glowing spots where sequence is!

Applications:

  • Count chromosome numbers

  • Detect gene deletions/amplifications

  • See which cells express specific genes

  • Prenatal genetic testing

19.8 Key Takeaways

  • Isotope labeling = Track molecules using heavy/radioactive atoms

    • Meselson-Stahl proved semi-conservative DNA replication

    • Pulse-chase experiments track protein pathways

  • Functional genomics = Determining gene functions

    • Gene knockouts: Break gene, see effects

    • Overexpression: Too much protein, see effects

    • Reporter genes: Visual markers for gene activity

  • ChIP = Find where proteins bind DNA genome-wide

  • CRISPR-Cas9 = Precise gene editing (revolutionary!)

    • Easy, fast, cheap, precise

    • Applications in research, medicine, agriculture

  • Single-cell technologies = Study individual cells

    • scRNA-seq: Gene expression in each cell

    • Reveals cell heterogeneity

  • Imaging = See genes and proteins in action

    • Fluorescence microscopy

    • FISH

  • These methods transformed our understanding of genomics and continue to drive discoveries!

Sources: Information adapted from experimental methods literature, CRISPR research, and functional genomics publications.