Lecture Notes: PCR and Recombinant DNA Technology

Polymerase Chain Reaction (PCR)

• Definition: PCR is an enzymatic method of amplifying a specific DNA region.
• Process:
  • Enzymes: Use of enzymes to amplify target DNA, involving cycles of denaturation, annealing, and extension.
  • Primers: Requires synthetic oligonucleotide primers, designed based on target DNA sequences.
  • Automation: Utilizes thermostable DNA polymerase for process automation.
  • Exponential Amplification: A single DNA copy becomes over 2 million copies after 20 cycles.
  • Analysis: Products analyzed via agarose gel and ethidium bromide staining.
• Applications:
  • Detection of disease genes, gene expression analysis (reverse transcription PCR).
  • Forensic medicine, detection of infectious agents, prenatal genetic diagnosis, transplant tissue typing, evolutionary studies.

Mechanism of PCR

• Mnemonic "D-A-E":
  • Denature: Heat to separate DNA strands.
  • Anneal: Cool to allow primers to bind to the target sequence.
  • Extend: DNA polymerase adds nucleotides to elongate the DNA strand.
• Preparation:
  • Mix template, primers, dNTPs, polymerase buffer, and thermostable DNA polymerase.
  • Use of thermal cycler to vary temperatures.

Reverse Transcription PCR (RT-PCR)

• Purpose: Amplifies mRNA by converting it to DNA before PCR.
• Process:
  • RNA converted to cDNA using reverse transcriptase.
  • cDNA amplified via regular PCR.
• Applications:
  • Detection and study of RNA viruses like coronavirus.
  • Quantitative RNA analysis.

Applications of Recombinant DNA Technology

• Gene Mapping:
  • Use of FISH (Fluorescence In Situ Hybridization) for chromosomal abnormalities.
• Protein Production:
  • Production of human proteins like insulin, growth hormones, and vaccines.
• Disease Analysis:
  • Detection of polymorphisms, deletions, insertions, and point mutations.
• Agricultural Applications:
  • Engineering plants for resistance and nutritional improvements.

Genetic Mutations and Diseases

• Sickle Cell Anemia:
  • Caused by a missense non-conservative mutation in beta-globin chain.
• Detection:
  • Use of recombinant DNA technology to detect mutations.

DNA Polymorphisms

• SNPs (Single Nucleotide Polymorphisms):
  • Definition and detection using PCR.
• RFLP (Restriction Fragment Length Polymorphism):
  • Detection of inherited variations in DNA sequences.
  • Applications in paternity tests and criminal investigations.
• Microsatellites and VNTRs (Variable Number Tandem Repeats):
  • Used in DNA fingerprinting and identity testing.

Sequencing Technologies

• Next-Gen Sequencing (NGS):
  • Reduces cost of DNA sequencing.

Gene Therapy

• Definition: Insertion of genes into cells to treat diseases.
• IPSCs (Induced Pluripotent Stem Cells):
  • Conversion of somatic cells for potential therapies and disease models.