Molecular Biology: DNA Replication, Protein Synthesis, and PCR Techniques

DNA Replication: Definition and Mechanism

Definition: DNA replication is the process of producing two identical copies of DNA from one original DNA molecule. It is bidirectional and semiconservative—each daughter DNA contains one parental (old) strand and one new strand.

Steps of DNA Replication

1. Unwinding of DNA: Hydrogen bonds between base pairs break.
   • Two antiparallel strands separate, starting at the origin of replication.
   • The enzyme Helicase unwinds DNA, forming a replication fork.
2. Nucleotide Activation: Deoxyribonucleotides are activated using energy and the enzyme phosphorylase.
3. Strand Cutting and Stabilization: Endonuclease cuts the DNA at A-T rich regions (fewer hydrogen bonds).
   • Topoisomerase and helix destabilizing proteins stabilize the replication fork.
4. Primer Formation: Primase synthesizes an RNA primer at the 3’ end to initiate synthesis.
5. Strand Synthesis: DNA Polymerase adds nucleotides in the 5’ → 3’ direction.
   • Leading strand: synthesized continuously.
   • Lagging strand: synthesized in Okazaki fragments, later joined by DNA Ligase.
6. Primer Removal & Replacement: RNA primers are removed and replaced by DNA nucleotides via DNA Polymerase.
7. Proofreading: Errors are corrected by endonuclease (removal) and DNA Polymerase (correction).
8. Helix Formation: The two strands twist to form a double helix.

Key Concept: Semiconservative Replication means each daughter DNA contains one original and one new strand.

Protein Synthesis

Protein synthesis is the biological process by which cells build proteins, essential for cell growth, function, and survival. It involves decoding the genetic instructions from DNA into a functional protein via two major steps: Transcription and Translation, as outlined in the Central Dogma of molecular biology:

DNA → mRNA → Protein

1. Transcription (DNA → mRNA)

• Occurs in the nucleus.
• RNA Polymerase synthesizes messenger RNA (mRNA) from a DNA template strand.
• This process takes place in the transcription bubble, where DNA strands temporarily unzip.
• mRNA is complementary to the DNA strand, replacing thymine (T) with uracil (U).
• Once formed, mRNA leaves the nucleus and enters the cytoplasm.

2. Translation (mRNA → Protein)

• Occurs in the cytoplasm, utilizing ribosomes and transfer RNA (tRNA).
• tRNA carries amino acids and matches its anticodon with codons on the mRNA.
• Steps of translation:
  • Activation: The correct amino acid attaches to tRNA (tRNA becomes "charged").
  • Initiation: Translation begins at the AUG codon (which codes for methionine).
  • Elongation: tRNAs sequentially add amino acids to the growing polypeptide chain via peptide bonds.
  • Termination: When a stop codon is reached, the completed polypeptide is released.
• After translation, proteins undergo folding and post-translational modifications, often in the Golgi apparatus.

Polymerase Chain Reaction (PCR)

PCR is a laboratory technique used to amplify specific DNA sequences rapidly and accurately.

Key Features of PCR

• Allows millions of copies of a DNA sequence to be made in vitro (outside a living organism).
• Uses Taq DNA Polymerase, a heat-stable enzyme derived from Thermus aquaticus.

Steps in One PCR Cycle

1. Denaturation (~92°C): The double-stranded DNA separates into single strands.
2. Annealing (40–60°C): Short primers bind to the target DNA regions.
3. Extension (72°C): DNA Polymerase synthesizes new complementary DNA strands.

Each cycle doubles the amount of target DNA, leading to exponential amplification.

PCR Applications

• Genetic and forensic analysis
• Diagnosis of infections and genetic diseases
• Ancient DNA analysis in archaeology and paleontology
• Support for the Human Genome Project and evolutionary studies

Bioimaging

Bioimaging is the examination of biological structures and function using various qualitative and quantitative techniques. This field brings together engineers, physicists, biologists, and chemists engaged in developing the methodology for these types of examinations. While several aspects of bioimaging are confined to research, some techniques have been adapted for live studies, such as the detection of biological conditions (diseases and abnormalities) in humans.