DNA Structure and Replication & Gene Expression-Rock Pocket Mouse

Bio quiz

Explain how recombinant plasmids are used to clone genes and make protein products

Recombinant plasmids are small DNA rings used to insert a target gene into a host cell so it can copy the gene or make a protein.

Key Steps

• Isolation: Extract the target gene from source DNA and obtain a plasmid vector (circular bacterial DNA).

• Cutting: Use restriction enzymes to cut both the plasmid and the target gene at specific sites, creating matching "sticky ends."

• Ligation: Use DNA ligase to glue the target gene into the cut plasmid, forming a recombinant plasmid.

• Transformation: Introduce the recombinant plasmid into host bacteria, usually through heat shock or electroporation.

• Selection: Grow bacteria on selective media (like antibiotics) to find cells that took up the plasmid.

• Cloning and Production: Allow bacteria to multiply (cloning the gene) and trigger the cells to read the gene and produce the desired protein.

importance of the Human Genome Project

• Disease Understanding: It helped scientists find genes linked to rare and common health conditions, including cancer and heart disease.

• Personalized Medicine: The data allows doctors to tailor treatments and medical care to a patient's unique genetic makeup.

• Open Science: The project set a standard for sharing scientific data freely and openly with researchers worldwide.

• Evolutionary Insights: Comparing human DNA with other organisms helps trace human migration and understand evolutionary history.

• Biotechnology Growth: It launched the modern genomics and biotechnology industries, making DNA sequencing faster and cheaper.

What’s a genome?

A genome is the complete set of genetic material (all the DNA) in an organism.

What does PCR stand for? Why is it important?

Polymerase Chain Reaction (PCR) is a foundational technology in modern biology and medicine because it allows scientists to amplify a single copy or a few copies of a specific DNA segment into millions or billions of copies within hours.

Without PCR, genetic sequencing, modern medical diagnostics, and forensic science would be virtually impossible.

Why is PCR important in modern biology and medicine?

PCR is a laboratory tool that copies tiny pieces of DNA or RNA so scientists can study them easily.

Medical Diagnostics

• Finds germs like COVID-19, HIV, and tuberculosis.

• Detects infections early, before your body makes antibodies.

Genetic Testing

• Finds gene mutations that cause inherited disorders.

• Screens newborn babies for hidden health issues.

• Helps doctors pick the right treatment for cancer.

Forensic Science

• Takes a tiny sample, like a single hair or drop of blood, from a crime scene.

• Copies the DNA to match a suspect.

Biological Research

• Copies and reads DNA codes.

• Helps scientists study how different living things connect and evolve.

What are the 3 main steps of PCR cycle?

A PCR machine changes temperatures repeatedly across 20 to 40 cycles. Each cycle contains three main, distinct temperature-controlled steps:

[Double-Stranded DNA] 
        |
        v  (94–98°C)
  1. DENATURATION  --> Strands separate
        |
        v  (50–65°C)
    2. ANNEALING   --> Primers bind to single strands
        |
        v  (72°C)
   3. EXTENSION    --> Taq polymerase synthesizes new DNA

What happens during each stage of PCR?

1. Denaturation

• Temperature: ~94–98 °C

• What happens: High heat is applied to the sample to break the hydrogen bonds holding the double-stranded DNA together. This separates the DNA into two single strands, which will act as templates for the new DNA.

2. Annealing

• Temperature: ~50–65 °C

• What happens: The temperature is lowered to allow short DNA sequences called primers to bind to specific, complementary target sequences on the single-stranded DNA templates. These primers mark the starting point for DNA replication.

3. Extension (Elongation)

• Temperature: ~72 °C

• What happens: The temperature is raised slightly to the optimal working condition for Taq polymerase (a heat-stable DNA polymerase enzyme). The enzyme binds to the primers and synthesizes a new complementary strand of DNA by adding free nucleotides to the growing chain.

At the end of one cycle, the target DNA sequence has doubled. The process then repeats, exponentially growing the amount of DNA.

u Describe/label a diagram of DNA structure

• base pairs are help together with hydrogen bonds (3 C-G, 2 A-T)

• Phosphate Group: Be able to draw

• Deoxyribose Sugar: Know which end is 5’/3’

• Nirtrogenous Base: Know matching pairs (purine vs pyramidines)

• Anti-parallel

• complmentary

Chargaff’s Rules (1950)

1.The base composition varies between species.

2.Within a species, the amount of A is about equal to the amount of T, and the amount of C is about equal to the amount of G

• This was a clue to the structure of DNA because it helped Watson & Crick figure out how that structure and nitreogenous bases pairings work. Also, Chargaff stated that the composition is within a species.

• It was the basis to predict unknown percentages of Bases.

How was Chargaff’s Rules a clue to the structure of DNA?

James Watson and Francis Crick used these numerical ratios as a vital puzzle piece when building their physical model of DNA:

• Complementary Base Pairing: The 1:1 ratios suggested that bases bind together in specific pairs A with T and G with C.

• Uniform Width of the Double Helix: Because a two-ringed purine always pairs with a one-ringed pyrimidine A with T, G with C, the total width of the ladder remains constant down the entire molecule.

• The Double Helix Blueprint: This specific locking mechanism explained how genetic information could copy accurately

Rosalind Franklin (1950s)

• Franklin’s x-ray diffraction photograph was taken by Wilkins. Wilkins showed this to Watson who was familiar with this kind of helix pattern.

• x ray diffraction: Spots and smudges in images produced by x-ray that were diffracted as they passed through aligned fibers of DNA

• Photo 51 (1952): She captured Photograph 51, a remarkably clear X-ray diffraction image that revealed the helical shape of DNA.

• Data Sharing Without Consent: Her colleague Maurice Wilkins shared Photo 51 and her unpublished data with James Watson without her knowledge.

• The DNA Model (1953): Watson and Crick used her precise mathematical data and imagery to build their famous DNA model, publishing their findings in Nature without crediting her adequately

Watson and Crick (1953)

DNA is a double helix

• Began to build models of a double helix to prove the x-ray

• Franklin confirmed sugar-phosphate backbone was on the outside.

• This arrangement was appealing because it took the phosphate groups in the aqueous surrounding with the hydrophobic bases in the middle

• Nobel Prize

u Explain how Chargaff’s rules and Franklin’s x-ray crystallography images led to our understanding of the structure of DNA

Chargaff's Rules

• Erwin Chargaff discovered that the amount of adenine (A) equals the amount of thymine (T) in DNA.

• He also found that the amount of cytosine (C) equals the amount of guanine (G).

• This showed scientists that bases pair up in a specific way: A pairs with T, and C pairs with G.

Franklin's X-Ray Crystallography

• Rosalind Franklin took clear x-ray pictures of DNA, known as Photo 51.

• Her images revealed an X-shaped pattern, which proved that DNA has a twisted spiral shape, or a double helix.

• The pictures also showed that the sugar-phosphate backbone sits on the outside of the molecule.

Putting It Together

• James Watson and Francis Crick used these two pieces of evidence to build the first accurate 3D model of DNA.

• Franklin's helix shape gave the physical structure.

• Chargaff's base ratios showed how the rungs of the ladder fit evenly inside that structure.

NITROGENOUS BASES

u How does DNA’s structure suit its function as the molecule of inheritance?

• Is semi-conservative → 1 parent + 1 new

• Nucleic acids dictate their own replication from monomers

• The 2 strands are complementary, each stores the information needed to make another

• When a cell copies a DNA molecule, each strand serves as a templete for ordering neuclotides into a new complementary strand

• 2 each an exact replica of the parent

DNA replication is …………………

Origins of Prokaryotes vs Eukaryotes!

(a) DNA Replication in Prokaryotes (E. coli)

• Single Origin: Replication begins at a single, specific site called the origin of replication on the circular chromosome.

• Bidirectional Growth: The DNA unwinds, forming one replication bubble with two replication forks that move in opposite directions (indicated by the pink arrows).

• Final Product: The forks move all the way around the circular molecule until they meet, producing two identical circular daughter DNA molecules.

• Micrograph: The bottom-left image shows an electron micrograph of a bacterial chromosome actively replicating (often called a theta structure due to its shape).

(b) DNA Replication in Eukaryotes

• Multiple Origins: Eukaryotic chromosomes are long and linear, so replication begins at multiple origins of replication simultaneously to speed up the process.

• Bubble Fusion: Multiple replication bubbles form and expand outward bidirectionally. Eventually, these neighboring bubbles fuse together.

• Final Product: Once all bubbles fuse and the ends are reached, it results in two identical linear daughter DNA molecules.

• Micrograph: The bottom-right image shows an electron micrograph of eukaryotic DNA with several clearly visible replication bubbles along a single strand.

Getting Started?

• DNA replication starts at sites called origins of replication, short streches of DNA that have a specific sequence.

• Proceeds in both directions until done.

What happens at the replication fork?

Topoisomerase: Breaks, swivels, and rejoins the parental DNA ahead of the replication

Helicase: unwinds and seperates the parental strands of DNA

Primase: Synthesizes RNA primers, using parental DNA as a templete

SSBP: Stablized the unwond parental

Describe/ label a diagram of the process of DNA replication

• Parent DNA Strand: The original double-stranded DNA molecule that splits apart to serve as a template.

• Helicase (The Unzipper): An enzyme that breaks the hydrogen bonds between DNA bases, "unzipping" the double helix into two single strands.

• Replication Fork: The Y-shaped region where the two parent DNA strands split apart.

• Single-Strand Binding Proteins (SSBs): Proteins that attach to the separated DNA strands to keep them from sticking back together.

• Primase (The Primer Maker): An enzyme that creates a small piece of RNA called a primer. This gives DNA polymerase a starting place.

• DNA Polymerase (The Builder): The main enzyme that adds new, matching DNA nucleotides to the growing strand. It moves only in the 5' to 3' direction.

• Leading Strand: The new DNA strand built smoothly and continuously toward the replication fork (5' to 3').

• Lagging Strand: The new DNA strand built in short, backward chunks away from the replication fork because of the 5' to 3' restriction.

• Okazaki Fragments: The short pieces of DNA made on the lagging strand.

• DNA Ligase (The Glue): An enzyme that seals the gaps between the Okazaki fragments to make one solid, continuous DNA strand.

What is a recombinant plasmid?

A small, circular piece of DNA (usually from bacteria) that scientists combine with a target gene from another organism.

How do you prepare the DNA and plasmid?

• Use restriction enzymes (molecular scissors) to cut both the plasmid and the target gene at specific DNA sequences.

• This creates matching "sticky ends" that fit together.

What is ligation?

• Use an enzyme called DNA ligase to glue the target gene permanently into the cut plasmid.

• The result is a complete recombinant plasmid.

What is transformation?

• Introduce the recombinant plasmid into a host cell (usually bacteria like E. coli).

• Heat shock or electrical currents create tiny holes in the cell membrane so the plasmid can enter.

How do we find successful cells?

• Grow the bacteria on an antibiotic plate.

• Plasmids carry antibiotic resistance genes, so only bacteria that took up the plasmid survive.

How do we get clones and proteins?

• Gene Cloning: The bacteria divide rapidly, making millions of identical copies of the plasmid and the inserted gene.

• Protein Production: The bacteria read the gene's instructions, transcribe it into mRNA, and translate it to produce large amounts of the desired protein (such as human insulin).

Facts about DNA replication that will become important when we learn about PCR

uDNA strands can only grow from 5’ to 3’

uNucleotides can only be added to the 3’ –OH of another nucleotide

DNA polymerase can only add nucleotides to an existing strand of nucleic acid (either DNA or RNA

DNA Polymerase?

Main Functions

• DNA Replication: It reads an existing template strand and matches complementary bases (A with T, and C with G) to copy the genome in the 5' to 3' direction.

• Proofreading: Many types have 3' to 5' exonuclease activity to catch and remove incorrect bases, ensuring high accuracy.

• DNA Repair: It fills in gaps and replaces damaged or primer sequences during various repair pathways.

The action of helicase creates _____.

replication forks and replication bubbles

Why is the new DNA strand complementary to the 3' to 5' strands assembled in short segments?

DNA polymerase can assemble DNA only in the 5' to 3' direction

The synthesis of a new strand begins with the synthesis of a(n) _____.

RNA primer complementary to a preexisting DNA strand

An old DNA strand is used as a _____ for the assembly of a new DNA strand.

template

Which of these is responsible for catalyzing the formation of an RNA primer?

D

What catalyzes DNA synthesis?

DNA polymerase

Which of the following statements about DNA synthesis is true?

• DNA polymerase adds dNTP monomers in the 3' to 5' direction.

• As DNA polymerase moves along the template strand, each new nucleotide provides a 5' hydroxyl group for the next reaction to occur.

• Nucleotides are added in a random fashion to single-stranded DNA.

• Primers are short sequences that allow the initiation of DNA synthesis.

• Primers are short sequences that allow the initiation of DNA synthesis.

Which part of a deoxynucleoside triphosphate (dNTP) molecule provides the energy for DNA synthesis?

A dNTP carries three phosphate groups. When DNA polymerase adds a new nucleotide to a growing strand, it cleaves off two of these phosphates (as pyrophosphate). Breaking these high-energy bonds releases the energy needed to form the new phosphodiester bond.

Which enzyme is important for relieving the tension in a helix as it unwinds during DNA synthesis?

Topoisomerase

What is the basis for the difference in how the leading and lagging strands of DNA molecules are synthesized?

DNA polymerase can join new nucleotides only to the 3 '

end of a preexisting strand, and the strands are antiparallel

The elongation of the leading strand during DNA synthesis __________.

depends on the action of DNA polymerase

Which way does the daughter strand elongate for the leading strand vs the lagging strand?

Leading: toward the replication fork

Lagging: Away from the replcation fork

The diagram shows a bacterial replication fork and its principal proteins.

In order to insert a human gene into a plasmid, both must _____.

be cut by the same restriction enzyme

What enzyme forms covalent bonds between restriction fragments?

ligase

What is the role of DNA polymerase during DNA synthesis?

DNA polymerase is the enzyme that catalyzes the addition of a nucleotide onto the 3' end of a growing DNA strand.

A hydroxyl is present at the 3' end of the growing DNA strand. What is at the 5' end?

a phosphate group

Addition of a nucleotide onto a DNA strand is an endergonic reaction. What provides the energy to drive the reaction?

Release of pyrophosphate from the incoming nucleotide, and then hydrolysis of the pyrophosphate to inorganic phosphate.

What materials does DNA polymerase require in order to synthesize a complete strand of DNA?

3'-OH end of the new DNA strand

Single-stranded DNA template

All four deoxyribonucleotides triphosphates (containing A, C, T, or G)

The unpaired nucleotides produced by the action of restriction enzymes are referred to as _____.

sticky ends

A researcher is preparing to insert a human gene of interest into a bacterial plasmid in order to clone the human gene. She has genetically engineered the plasmid to carry a gene amp R, which confers resistance to the antibiotic ampicillin. She will include ampicillin in the plating medium when she grows the recombinant bacteria. Why has she engineered the plasmid to include an antibiotic resistance gene?

Recombinant bacteria that have taken up the plasmid can be recognized because they are able to survive in the presence of ampicillin.