Polymerase Chain Reaction (PCR)

• Polymerase Chain Reaction (PCR) is a lab technique used to make millions or billions of copies of a specific DNA sequence
• The goal of PCR is to make enough DNA target

Key Players in PCR

• DNA Template serves as the DNA region that will be amplified
• PCR Primers act as short nucleotide sequences, providing a starting point for DNA synthesis
• Nucleotides (dNTPs) are the building blocks for synthesizing new DNA
  • Includes deoxyadenosine triphosphate (dATP)
  • Includes deoxythymidine triphosphate (dTTP)
  • Includes deoxycytidine triphosphate (dCTP)
  • Includes deoxyguanosine triphosphate (dGTP)
• Taq Polymerase is a heat-resistant DNA polymerase from Thermus aquaticus that synthesizes new DNA strands
• Buffer provides a suitable chemical environment for the activity of DNA polymerase

Steps of PCR (Easy to Remember: DAE)

• Denaturation (95°C) is where the DNA template unwind
• Annealing (55-65°C) is where Primers can bind to their complementary sequence on the DNA template
• Extension / Elongation (72°C) is where Taq polymerase synthesizes new DNA strands by adding nucleotides to the primers

Application of PRC

• Medicine uses it to detect genetic disease mutations
• Forensic Science uses it in criminal investigations and paternity tests, and as a tool in genetic fingerprinting
• Research and Genetic uses it to compare genomes of different organisms for gene mapping

Why PCR Important?

• PCR allows scientists to generate several copies of DNA quickly and efficiently
• PCR plays a crucial role in disease detection, forensic investigations, and genetic research

Genetic Engineering

• Genetic engineering (also called genetic modification) is a laboratory-based process that alters the DNA of an organism
• Genetic engineering may involve changing a single base pair, deleting a DNA region, or adding a new DNA segment
• Scientists use molecular biology techniques to insert and express proteins in different organisms
• Genetic engineering allows the transfer of specific traits between species

Classical Breeding vs Recombinant DNA Technology

• Classical breeding involves interbreeding (crossing) of related individuals, transferring traits only within the same species
• Recombinant DNA Technology involves the transfer of specific traits across species, using enzymes and laboratory techniques
• Genetic engineering modifies an organism's DNA using laboratory techniques
• Classical breeding involves selective interbreeding, while recombinant DNA technology allows cross-species gene transfer
• Molecular cloning involves gene isolation, insertion into a vector, and transformation into a host cell

DNA Introduction Methods

• Restriction enzymes are used like molecular scissors to cut DNA at specific sequences
• To insert into a Vector, the DNA fragment is fused into a vector (carrier DNA)
• Transformation is where the vector is introduced into a host cell

Possible Bacterial Clone Products

• Bacteria can be produced without the vector
• Bacteria can be produced with the vector but without the gene
• Bacteria can be produced with the vector and the gene of interest

Methods to Introduce DNA into Cells

• Biolistics (Gene Gun Method) uses DNA-coated particles shot into cells using a gene gun
• Cells that survive the bombardment take up the DNA to express the desired protein
• Heat Shock Treatment uses calcium chloride to neutralize repulsion between plasmid DNA and the bacterial membrane
  • A sudden temperature increase creates pores in the bacterial membrane, allowing DNA entry
  • Transformed cells acquire new traits
• Electroporation uses an electrical pulse to create temporary pores in the cell membrane
  • DNA and other substances can pass into the cell through these pores
• DNA can be introduced into cells using biolistics, heat shock treatment, and electroporation

Miscellaneous

• in vivo means inside the cell
• in vitro means inside the test tubes