HL IB Biology: DNA Replication, PCR, and Electrophoresis Study Guide

DNA Replication: Fundamentals and Importance

DNA replication is a semi-conservative process that is fundamentally dependent on complementary base pairing.

Semi-Conservative Replication

The term "semi-conservative" refers to the fact that when a DNA molecule replicates, one strand of the original "parent" DNA is preserved in each of the resulting "daughter" molecules.

• Template Strand: This is the preserved original strand which serves as a guide for the synthesis of a new strand.

• Synthesis Process: The new strand is constructed from free nucleotides present in the nuclear space surrounding the chromosomes. This process occurs within the nucleus.

• Complementary Base Pairing: Nucleotides are added one by one according to specific rules:

  • If an adenine ($A$) is exposed on the template, a thymine ($T$) is added to the new strand, and vice versa.

  • If a cytosine ($C$) is exposed on the template, a guanine ($G$) is added, and vice versa.

  • Hydrogen bonds only form when the correct bases are paired, ensuring the stability and accuracy of the new molecule.

Biological Significance

In multicellular organisms, DNA replication is essential for:

• Growth: Increasing the number of cells.

• Replacement: Substituting old or damaged cells and tissues.

• Reproduction: Passing genetic information to offspring.

Examiner Tip: Do not confuse a "parent cell" with a "parent organism." A parent cell refers to any cell in the body that divides to produce two daughter cells, serving as the source of the original DNA.

Historical Context and Accuracy

• Genetic Continuity: Keeping one original strand ensures high-degree accuracy and genetic continuity between cell generations. It ensures new cells inherit all genes with the correct sequence of DNA bases.

• Crick and Watson: These scientists proposed the semi-conservative model as part of their discovery of the double-helix structure, though they initially lacked evidence.

• Meselson and Stahl: Later scientists whose research provided the necessary experimental support for the semi-conservative hypothesis.

General Mechanism of Replication

DNA replication serves as preparation for mitosis, doubling the DNA so that two genetically identical daughter cells can be produced.

Key Enzymes and Their Functions

1. Helicase:

   • Unwinding: It flattens the helical structure (analogous to untwisting a rope ladder).

   • Separating: It breaks the hydrogen bonds between base pairs, exposing the bases on both strands (analogous to unzipping a zipper).

2. DNA Polymerase:

   • Linking: It joins free nucleotides attracted to the exposed bases to form a new strand.

   • Catalysis: It catalyses condensation reactions between the deoxyribose sugar of one nucleotide and the phosphate group of another, forming the sugar-phosphate backbone.

   • Directionality: DNA polymerase always builds in the $5'$ to $3'$ direction. It attaches to the $3'$ end of the original template and reads it in the $3'$ to $5'$ direction. Because strands are antiparallel, the new strand is synthesized $5'$ to $3'$.

Gel Electrophoresis

Gel electrophoresis is a technique used to analyze DNA, RNA, and proteins by separating molecules based on their size (mass) and net electrical charge.

Principles of Separation

• Electrical Charge: Positively charged molecules move toward the cathode (negative pole). Negatively charged molecules move toward the anode (positive pole). DNA is negatively charged due to its phosphate groups, so it moves toward the anode.

• Molecular Size: Fragments move through a gel matrix (agarose for DNA; polyacrylamide for proteins). Pores in the gel allow smaller molecules to move faster and further, while larger molecules move slowly.

• Gel Type: Different gels have different pore sizes affecting migration speed.

Procedural Steps

1. Extraction: DNA is collected from sources like hair roots or saliva.

2. Amplification: The quantity of DNA is increased using the Polymerase Chain Reaction (PCR).

3. Fragmentation: Restriction enzymes (DNA-cutting enzymes) chop the DNA into fragments.

4. Gel Preparation: An agarose gel plate is created with wells at one end and submerged in an electrolyte (salt) solution.

5. Loading: DNA is inserted into wells via micropipette.

6. Current Application: A negative electrode is connected to the well-end, and a positive electrode (anode) to the other. Shorter fragments move faster and further.

7. Visualization: Fragments are transferred to absorbent paper or nitrocellulose and heated. Probes are added:

   • Radioactive Labels: e.g., a phosphorus isotope ($^{32}P$), which darkens X-ray film.

   • Fluorescent Stains: e.g., ethidium bromide, which glows under ultraviolet ($UV$) light.

Polymerase Chain Reaction (PCR)

PCR is an in vitro method used to produce large quantities (billions of copies) of specific DNA fragments from a tiny initial sample.

Requirements for PCR

• Target DNA/RNA: The specific section to be amplified.

• Primers: Short sequences that identify the start point for the specific sections of DNA to be copied.

• Taq Polymerase: Derived from the thermophilic bacterium Thermus aquaticus. It is heat-stable and does not denature at high temperatures.

• Free Nucleotides: Building blocks for new strands.

• Buffer Solution: Maintains optimum $pH$.

The Three Stages of PCR

PCR occurs in a thermal cycler. Each cycle doubles the DNA ($2^n$ connectivity):

1. Denaturation ($95^\circ\text{C}$): Heat breaks the hydrogen bonds to separate the double-stranded DNA.

2. Annealing ($50 - 60^\circ\text{C}$): Temperature is lowered to allow primers to bind to the ends of the single strands.

3. Elongation/Extension ($72^\circ\text{C}$): The optimum temperature for Taq polymerase to build complementary strands.

Results: $30$ cycles can produce over $1$ billion copies ($2^{30}$) from a single molecule in a few hours.

DNA Profiling and Applications

DNA profiling involves separating VNTRs (Variable Number Tandem Repeats), which are unique non-coding regions ($20$ to $50$ bases long).

Paternity Investigations

A child inherits half their VNTRs from each parent.

• Comparison requires the DNA profiles of the mother, child, and alleged father.

• Any band in the child's profile must be present in either the mother's or the father's profile. If a band in the child's profile does not match either, the alleged father is not the biological father.

Forensic Investigations

• Samples (blood, saliva, hair, semen) from a crime scene are compared against suspects and victims.

• Profiles can eliminate innocent individuals or identify unidentifiable bodies (e.g., from fire or decomposition).

• Nature of Science (NOS): Reliability is increased by observing more VNTR markers. The more markers used, the lower the probability of a false match.

Worked Example: Paternity

In a case with a child, mother, and four possible fathers (A, B, C, D):

1. Band 1: Present in Child and Mother (Inconclusive).

2. Band 2: Present in Child but NOT Mother. Must come from father. Only Fathers B and D have it. (A and C eliminated).

3. Band 4: Present in Child but NOT Mother. Only Fathers A, B, and C have it. Since A and C were already eliminated, Father B must be the father.

Advanced Mechanism of DNA Replication (HL)

Directionality and Chemical Bonding

Replication must occur in the $5'$ to $3'$ direction. DNA nucleotides have a phosphate group bonded to the $5'$ carbon of the deoxyribose sugar.

• Process: DNA polymerase adds the $5'$ phosphate group of an incoming nucleotide to the free $3' \text{ -OH}$ group of the growing strand.

Leading and Lagging Strands

Because DNA is antiparallel ($5'$ to $3'$ and $3'$ to $5'$), the replication fork opens in one direction, forcing two different strategies:

• Leading Strand: Synthesized continuously toward the replication fork.

• Lagging Strand: Synthesized discontinuously away from the fork in short segments called Okazaki fragments.

• RNA Primers: DNA primase adds RNA primers to provide a binding point. The leading strand needs only one; the lagging strand needs multiple primers (one for each Okazaki fragment).

Full Ensemble of Enzymes (HL)

• Helicase: Unwinds the helix and breaks hydrogen bonds.

• Single-Stranded Binding Proteins (SSBs): Keep strands separated during copying.

• DNA Primase: Generates the RNA primer initiation point.

• DNA Polymerase III: Starts replication at the primer, linking nucleotides $5'$ to $3'$.

• DNA Polymerase I: Removes RNA primers and replaces them with DNA nucleotides.

• DNA Ligase: Joins Okazaki fragments by forming sugar-phosphate bonds.

Proofreading and Mutations

Human replication requires the synthesis of $3 \times 10^9$ base pairs. Errors are called mutations.

• In Prokaryotes: DNA Polymerase III acts as a proofreader. It recognizes incorrect nucleotides, reverses direction to remove the error from the $3'$ end, and inserts the correct base before continuing.