I. Nucleotides: The Building Blocks

• Structure: Nucleotides consist of three components (Fig 1.28):
  • Nitrogenous Base: These are either purines (Adenine (A) and Guanine (G) - two rings) or pyrimidines (Cytosine (C), Thymine (T) - DNA only, and Uracil (U) - RNA only - one ring) (Fig 1.29).
  • Pentose Sugar: Either ribose (RNA) or deoxyribose (DNA).
  • Phosphate Group(s): One to three phosphate groups can be attached, leading to nucleoside mono, di, or triphosphates (e.g., AMP, ADP, ATP). Remember examples like ATP, CTP, GTP, TTP, and UTP.
• Types:
  • Ribonucleotides: Contain ribose sugar and the bases A, C, G, and U; found in RNA.
  • Deoxyribonucleotides: Contain deoxyribose sugar and the bases A, C, G, and T; found in DNA.
• Bonding: The base and sugar are linked by an N-glycosidic bond. Nucleotides link together via phosphodiester bonds between the 3'-hydroxyl (-OH) group of one nucleotide's pentose sugar and the phosphate group of another, forming a polynucleotide chain. This is a condensation reaction.
• Hydrolysis: Phosphatases/nucleotidasess hydrolyze nucleotides into phosphates and nucleosides. Nucleosidases further break down nucleosides into bases and pentoses.

II. Polynucleotides: DNA and RNA

• Formation: Polynucleotides (DNA and RNA) are formed by polymerization of nucleotides through phosphodiester bond formation (condensation reaction). DNA synthesis (replication) is catalyzed by DNA polymerase, while RNA synthesis (transcription) is catalyzed by RNA polymerase. (Detailed in Chapter 3).
• DNA (Deoxyribonucleic Acid):
  • Structure (Watson-Crick Model): A double helix (Fig 1.31) with two antiparallel polynucleotide strands coiled around a common axis.
  • Bases: A, C, G, and T. A pairs with T (2 hydrogen bonds), and C pairs with G (3 hydrogen bonds).
  • Backbone: Deoxyribose and phosphate groups.
  • Stability: Hydrogen bonds between bases, hydrophobic interactions between bases in the center, and surrounding water molecules contribute to DNA's stability.
  • Dimensions: One complete turn of the helix is 3.4 nm long, containing 10 base pairs; diameter is 2 nm.
  • Location: Primarily in the nucleus.
• RNA (Ribonucleic Acid):
  • Structure: A single-stranded polynucleotide.
  • Bases: A, C, G, and U.
  • Backbone: Ribose and phosphate groups.
  • Types: Three main types with distinct roles:
    • rRNA (Ribosomal RNA): Forms the structural and catalytic core of ribosomes (80S in eukaryotes, 70S in prokaryotes). It comprises about 80% of cellular RNA. It has a catalytic role as a ribozyme, forming peptide bonds.
    • tRNA (Transfer RNA): Cloverleaf structure (Fig 1.32), carries specific amino acids to the ribosome during translation. It contains an anticodon that base-pairs with mRNA codons. Makes up about 10-15% of cellular RNA.
    • mRNA (Messenger RNA): Carries genetic information from DNA to the ribosome for protein synthesis. Its sequence is complementary to the DNA template strand and identical (except for U replacing T) to the coding strand. It is relatively short-lived. Makes up about 5% of cellular RNA.

I. DNA Structure: The Double Helix
A. Fundamental Components:

• Double-stranded helix: Two polynucleotide chains intertwine around a common axis (Watson-Crick model). Visualize this as a twisted ladder.
• Deoxyribonucleotides: Building blocks composed of:
  • Deoxyribose sugar: A 5-carbon sugar lacking an oxygen atom compared to ribose (RNA).
  • Phosphate group: Forms the backbone through phosphodiester bonds.
  • Nitrogenous bases: Adenine (A), Cytosine (C), Guanine (G), Thymine (T).
• Base Pairing: Specific hydrogen bonding between bases:
  • A pairs with T (2 hydrogen bonds)
  • C pairs with G (3 hydrogen bonds) This is complementary base pairing.
• Antiparallel strands: The two strands run in opposite directions (5' to 3' and 3' to 5'). Think of the ladder rungs being upside down relative to each other.
• Dimensions:
  • One complete turn of the helix: 3.4 nm (10 base pairs)
  • Diameter of the helix: 2 nm
• Stability: Hydrogen bonds between bases, hydrophobic interactions within the core of the helix (bases), and surrounding water molecules contribute to the stability of the DNA structure.

B. Key Points for Memorization:

• Think "AT" and "GC": This helps remember the base pairing rules.
• "Deoxy"ribose lacks an oxygen: Distinguish DNA from RNA.
• Antiparallel: Imagine the 5' and 3' ends pointing in opposite directions.

II. RNA Structure: The Single-Stranded Variations
A. General Features:

• Single-stranded polynucleotide: Unlike DNA, RNA is typically a single-stranded molecule.
• Ribose sugar: Contains an extra oxygen atom compared to deoxyribose.
• Nitrogenous bases: Adenine (A), Cytosine (C), Guanine (G), Uracil (U) – note the absence of Thymine and presence of Uracil.
• Three main types: rRNA, tRNA, mRNA. Each plays a crucial role in protein synthesis.

B. Types of RNA:

1. Ribosomal RNA (rRNA):
   • Abundance: ~80% of cellular RNA.
   • Lifespan: Several days.
   • Structure: Combines intra-chain single-stranded and double-stranded sections bound to proteins to form ribosomes.
   • Ribosome Subunits: Eukaryotes (80S = 60S + 40S); Prokaryotes (70S = 50S + 30S). The numbers represent sedimentation coefficients, not actual sizes.
   • Function: Forms the ribosome, the site of protein synthesis. Recent research suggests it also functions as a ribozyme (catalytic RNA).
   • Location of synthesis: Nucleolus (eukaryotes).
   • Key Point: Remember the ribosomal subunit sizes for eukaryotes and prokaryotes.
2. Transfer RNA (tRNA):
   • Abundance: 10-15% of cellular RNA.
   • Lifespan: Several hours.
   • Structure: Cloverleaf shape due to intra-chain hydrogen bonding.
   • Size: Smallest type of RNA (~80 nucleotides).
   • Types: 61 types (one for each codon except stop codons).
   • Anticodon: A three-base sequence that complements a specific mRNA codon.
   • Amino acid attachment site (CCA): At the 3' end, it binds a specific amino acid.
   • Function: Carries amino acids to the ribosome for protein synthesis.
   • Key Point: The anticodon is crucial for matching the tRNA to the correct mRNA codon.
3. Messenger RNA (mRNA):
   • Abundance: ~5% of cellular RNA.
   • Lifespan: Shortest lifespan (minutes).
   • Structure: Linear; can be long (several genes can be transcribed into one mRNA) and may coil or fold.
   • Function: Carries genetic information from DNA to the ribosome, acting as a template for protein synthesis.
   • Key Point: mRNA is a temporary carrier of genetic information.

DNA vs. RNA: A Comparative Study Guide
This guide summarizes the key differences between DNA (Deoxyribonucleic Acid) and RNA (Ribonucleic Acid), crucial for understanding their distinct roles in cellular processes.

Definition & Principle: 

Electrophoresis is a laboratory technique used to separate charged molecules (like amino acids, proteins, and DNA fragments) based on their differing migration rates in an electric field. This differential movement occurs because charged molecules are attracted to the oppositely charged electrode. 

II. Methodology: 

Medium: The separation takes place within a gel matrix (typically agarose gel, but polyacrylamide gel or even paper can be used). This gel acts as a sieve, slowing down the movement of larger molecules more than smaller ones. 

1. Sample Preparation: The sample (e.g., a mixture of DNA fragments) is mixed with a buffer solution (often acidic) to ensure the molecules carry a net charge. A tracking dye is added to visually monitor the progress of the electrophoresis. The sample is then loaded into wells at one end of the gel. 

1. Electrophoresis Setup: The gel is placed in a chamber filled with buffer solution. Electrodes are positioned at opposite ends of the chamber. Applying an electric current causes the charged molecules to migrate through the gel towards the electrode with the opposite charge. Smaller, more highly charged molecules move faster and further. 

1. Visualization: After electrophoresis, the separated molecules are visualized. This might involve staining the gel with a dye that binds to the molecules of interest or using fluorescent labels incorporated during sample preparation. ​
   
2. III. Applications: 
3. Protein Separation: Electrophoresis is ideal for separating proteins due to its gentleness; separated enzymes often remain active. ​
4. Disease Diagnosis: Analyzing blood plasma proteins can reveal the presence of antibodies produced in response to pathogens. Comparison with standard antibodies aids in disease identification.Forensic Science (DNA Fingerprinting): DNA is cut into fragments of varying lengths, and these fragments are separated to create a unique banding pattern for each individual. 
   
   DNA Sequencing: Electrophoresis is used to separate DNA fragments of different lengths, allowing scientists to determine the order of nucleotides in a DNA sequence. 
   
   IV. Limitations: 
   
   Sample Size: Electrophoresis is only suitable for separating small quantities of material. 
   
   Charge Limitations: Uncharged molecules or molecules with very similar charges will not separate effectively using this technique. 
   
   V. Key Terms: 
   
   Anode: The positive electrode. 
   
   Cathode: The negative electrode. 
   
   Agarose gel: A common gel matrix used in electrophoresis. 
   
   Buffer solution: Maintains pH and provides ions for conductivity. 
   
   Tracking dye: A dye added to the sample to monitor its progress. 
   
   Kilobase pairs (kb): A unit of measurement for DNA fragment length. 
5. VI. Study Questions: 
6. Explain the principle behind electrophoresis. Why do molecules move in an electric field? 
7. Describe the components of an electrophoresis apparatus. 
8. What are the advantages of using electrophoresis for protein separation? 
9. How is electrophoresis used in forensic science and disease diagnosis? 
10. What are the limitations of electrophoresis, and why do these limitations exist? 
11. What factors influence the rate of migration of a molecule during electrophoresis? (Consider size, charge, and gel matrix).