Module 8 Learning Objectives

Experiments Identifying DNA as Genetic Material and DNA Structure

Griffith’s Experiment (1928)

• Worked with Streptococcus pneumoniae (a bacterium).
• Two strains:
  • S strain: smooth & deadly (has capsule).
  • R strain: rough & harmless (no capsule).
• Four Test Groups:
  1. Live S → mouse dies
  2. Live R → mouse lives
  3. Heat-killed S → mouse lives
  4. Heat-killed S + live R → mouse dies
• Conclusion: Something from dead S cells transformed the harmless R cells into killers.
  • He called it a “transforming principle” but didn’t know it was DNA yet.

Avery, MacLeod, McCarty (1944)

• Repeated Griffith’s experiment but treated the bacteria with enzymes.
• Killed off proteins = transformation still happened.
• Killed off RNA = still happened.
• Killed off DNA = transformation STOPPED.
• Conclusion: DNA is the transforming principle and carries genetic info!

Hershey & Chase (1952) – “Blender Experiment”

• Worked with bacteriophages (viruses that infect bacteria).
• They labeled:
  • DNA with radioactive phosphorus (P-32).
  • Protein with radioactive sulfur (S-35).
• Then they:
  1. Let the viruses infect bacteria.
  2. Used a blender to shake off the virus coats.
  3. Looked to see what entered the bacteria.
• Conclusion: Only the radioactive DNA entered the cell and not protein, so DNA = the genetic material!

Structure of DNA: Discoveries

Chargaff (1950s)

• Analyzed DNA and found:
  • A = T
  • C = G
• This became known as the base-pairing rule (AT/GC rule).

Rosalind Franklin (1951)

• Used X-ray diffraction.
• Took Photo 51, which showed a spiral shape.

Watson & Crick (1953)

• Used Franklin’s image + Chargaff’s rules.
• Built the double helix model:
  • Sugar-phosphate backbone on the outside.
  • Bases paired in the middle: A-T, C-G.
• They figured out:
  • DNA is made of two strands.
  • It's antiparallel (they run opposite directions).
  • It twists into a helix.

Structural Features of DNA

DNA Composition

• DNA is made of nucleotides.
• Each nucleotide has 3 parts:
  1. Phosphate group (PO₄³⁻)
  2. 5-carbon sugar (deoxyribose)
  3. Nitrogenous base:
     • Purines: A (adenine), G (guanine) – 2 rings
     • Pyrimidines: C (cytosine), T (thymine) – 1 ring

DNA Strand

• DNA Strand = Nucleotides linked in a chain.
• Connected by phosphodiester bonds between sugar and phosphate.
• Has directionality: 5′ → 3′.
• Backbone = sugar + phosphate.
• Bases stick out like rungs on a ladder.

Double Helix Structure (Watson & Crick's Model)

• Two strands, twisted like a spiral staircase.
• Antiparallel: one strand runs 5′→3′, the other 3′→5′.
• Base-pairing rules (Chargaff’s Rule):
  • A pairs with T (via 2 hydrogen bonds).
  • C pairs with G (via 3 hydrogen bonds).

Double-Stranded Structure of DNA and Semiconservative Replication

Double-Stranded DNA Structure (review + new insight)

• DNA is two strands twisted into a double helix.
• Strands are antiparallel: one is 5′→3′, the other is 3′→5′.
• Held together by hydrogen bonds between base pairs:
  • A–T (2 hydrogen bonds).
  • C–G (3 hydrogen bonds).
• This is the AT/GC Rule (aka Chargaff’s Rule): A always pairs with T, and C always pairs with G.

Semiconservative Replication

• This is how DNA makes copies of itself before a cell divides.
• Each new DNA molecule has:
  • 1 old (parent) strand.
  • 1 new (daughter) strand.
• Proven by Meselson & Stahl (1958):
  • Used nitrogen isotopes (N-15 & N-14) and centrifugation.
  • Showed DNA is copied in a semiconservative way: One old strand, one new strand in each helix.

Steps of DNA Replication:

1. Unwind the helix (by helicase).
2. Separate the strands (breaking H bonds).
3. Add complementary bases using the AT/GC rule.
4. Result: 2 DNA molecules, each half-old + half-new.

DNA Replication

What is DNA Replication?

• It’s how a cell copies its DNA before division.
• It happens during the S phase of the cell cycle.

Key Players (Enzymes)

Leading vs Lagging Strand

• Leading Strand: built continuously toward the fork.
• Lagging Strand: built in short Okazaki fragments, away from the fork, then glued by ligase.
• DNA polymerase can only add nucleotides in the 5′ → 3′ direction, so replication is asymmetrical.

Replication Fork

• The Y-shaped area where DNA is being split + copied.
• One fork on each side of the origin of replication.

Central Dogma and Gene Expression

What is the Central Dogma?

• It's the flow of genetic information in cells: DNA → RNA → Protein

1. Transcription = DNA → RNA (in the nucleus)
2. Translation = RNA → Protein (at the ribosome)

• DNA is your recipe book.
• RNA is the handwritten copy.
• Protein is the baked cake.
• Your DNA holds all the instructions, but it can’t leave the nucleus, so you make a messenger (mRNA) that goes out and gets turned into something functional — a protein.
• Proteins = enzymes, hormones, muscle fibers — basically everything your body does or builds.

Transcription

What is Transcription?

• It’s when the cell copies a gene from DNA into mRNA.
• Happens in the nucleus.

3 Stages of Transcription:

1. Initiation
   • RNA polymerase binds to a region of DNA called the promoter.
   • It unwinds the DNA so it can be read.
2. Elongation
   • RNA polymerase moves along the DNA and builds mRNA by adding complementary RNA nucleotides.
   • Follows base-pairing rules, but with U instead of T:
     • A → U
     • T → A
     • C → G
     • G → C
3. Termination
   • RNA polymerase hits a terminator sequence and stops.
   • The mRNA strand is released.

Post-Transcription Modifications (Eukaryotes Only):

1. 5′ Cap: added to front; helps ribosome attach later.
2. 3′ Poly-A Tail: added to end; protects mRNA.
3. Splicing: removes introns (non-coding junk), keeps exons (expressed sequences).

• This makes the mRNA mature and ready to leave the nucleus.

Genetic Code

Genetic Code

• The “language” of mRNA that tells the ribosome which amino acid to add when building a protein.

Codon

• A codon = a group of 3 RNA bases (nucleotides).
  • Example: AUG, GCU, UAA.
• Each codon codes for 1 amino acid.
• mRNA is read 3 bases at a time during translation.

Start & Stop Codons:

• Start codon = AUG (also codes for methionine).
• Stop codons = UAA, UAG, UGA

Key Features of the Genetic Code:

1. Redundant: multiple codons code for the same amino acid (Ex: GGU, GGC, GGA, GGG all = glycine).
2. Unambiguous: each codon only means ONE amino acid.
3. Universal: nearly all living things use the same code.

From Code to Protein:

• mRNA codons are read by the ribosome.
• tRNA brings amino acids that match each codon.
• The amino acids are linked together to form a polypeptide chain (aka a protein).

Translation

What is Translation?

• It’s when the ribosome reads the mRNA and builds a polypeptide (protein) using amino acids.
• Happens in the cytoplasm, either floating freely or on the rough ER.

3 Stages of Translation:

1. Initiation
   • mRNA binds to the small ribosomal subunit.
   • The start codon (AUG) is recognized.
   • A tRNA with methionine pairs with AUG.
   • Large subunit joins the party.
2. Elongation
   • Ribosome reads one codon at a time.
   • Matching tRNA brings amino acid.
   • Amino acids are linked by peptide bonds.
   • Ribosome shifts over to the next codon.
3. Termination
   • Ribosome hits a stop codon (UAA, UAG, UGA).
   • A release factor binds instead of tRNA.
   • Ribosome disassembles, and the finished polypeptide is released.

Gene Expression: Prokaryotes vs. Eukaryotes

Big Picture

• Gene expression = transcription + translation
• Both cell types do it — but the how and where are different.

Prokaryotes (like bacteria):

• No nucleus → transcription & translation happen at the same time.
• No RNA processing — mRNA is ready to go right after it’s made.
• One circular chromosome.
• Genes often grouped into operons (multiple genes under one promoter).

Eukaryotes (like humans):

• Transcription happens in the nucleus.
• mRNA must be processed before leaving the nucleus:
  • Add 5′ cap
  • Add 3′ poly-A tail
  • Splice out introns
• Translation happens in the cytoplasm.
• Genes are individual (one gene per promoter).

Quick Comparison Table:

Gene Cloning

What is Gene Cloning?

• It’s the process of making many copies of a specific gene (or DNA segment).
• Used in research, medicine (like insulin), and biotech (GMOs, etc.).

Basic Steps of Gene Cloning:

1. Isolate the Gene of Interest
   • Cut it out of DNA using a restriction enzyme (molecular scissors).
   • These enzymes cut at specific sequences and leave "sticky ends."
2. Insert Gene into a Plasmid
   • A plasmid is a small, circular DNA found in bacteria.
   • Cut the plasmid with the same restriction enzyme.
   • Use DNA ligase to "glue" the gene into the plasmid.
   • This creates a recombinant plasmid.
3. Transform Bacteria
   • Insert the recombinant plasmid into a bacterial cell.
   • This is called transformation.
   • Now the bacteria carry and copy the gene as they divide!
4. Select Transformed Bacteria
   • Use antibiotic resistance to identify which bacteria picked up the plasmid.
   • Only those with the plasmid will grow on an antibiotic plate.
5. Let the Bacteria Multiply
   • Bacteria divide rapidly → making millions of copies of your gene.
   • You can now isolate the gene, or have the bacteria make the protein it encodes!

Gel Electrophoresis

What is Gel Electrophoresis?

• It’s a lab technique that separates DNA, RNA, or proteins based on size.
• Basically, running a current through a gel and letting the molecules wiggle through it like a race — smaller fragments move faster

How It Works (Step-by-Step):

1. Prepare the Gel
   • Use a gel (usually agarose) that acts like a spongey net.
   • Load your DNA samples into tiny wells at one end.
2. Apply Electrical Current
   • DNA is negatively charged (because of phosphate groups).
   • Current pulls DNA toward the positive end (red = +).
3. Separation Happens
   • Small DNA fragments zip through the gel faster.
   • Large fragments move slower and stay near the top
4. Visualize the Bands
   • Use a stain (like ethidium bromide) and UV light to see the DNA bands.
   • Each band = DNA of a specific size.

PCR (Polymerase Chain Reaction)

What is PCR?

• PCR = Polymerase Chain Reaction
• It’s a lab technique used to make millions of copies of a specific DNA segment FAST.

3 Main Steps (repeated for ~30 cycles):

1. Denaturation (95°C)
   • Heat is used to separate the DNA strands.
2. Annealing (50–65°C)
   • Cool it down a bit so primers can attach to the target sequence.
   • Primers = short bits of DNA that tell the enzyme where to start copying
3. Extension (72°C)
   • Taq DNA polymerase adds nucleotides to build new DNA strands.

• One cycle = DNA doubled
• 30 cycles = over a billion copies

What You Need for PCR:

• Template DNA (the piece you want to copy)
• Primers (to target the region)
• DNA polymerase (Taq)
• Nucleotides (dNTPs)
• Thermocycler (machine that controls the temps)