PLASMID EXTRACTION

• Plasmid


  A small, circular DNA molecule that is separate from the chromosomal DNA and is found in bacteria and some other organisms

  
• Plasmid


  • They are capable of replicating independently within the host cell
  • They often carry genes that provide additional functions to the host organism, eg antibiotic resistance, toxin production, or the ability to metabolize certain nutrients
• Stringent Plasmids


  Replication of the plasmid occurs at the same time and follows a similar pattern to that of the chromosomal DNA

  
• Relaxed Plasmids


  • Replicate and segregate independently of the chromosomal replication cycle
  • They can replicate even when the host cell is not actively undergoing chromosomal replication
• Plasmids can be transferred between bacterial cells, allowing for the spread of genetic traits among bacterial populations

  
• In biotechnology, plasmids are commonly used as vectors for the cloning and expression of foreign genes in recombinant DNA technology
• Plasmid as Vector

  • The Ti (tumor-inducing) plasmid from Agrobacterium tumifaciens is commonly used to produce transgenic plants
  • The T-DNA region of the plasmid can be inserted with target genes through the process of generating recombinant plasmid DNA
• Recombinant DNA


  A molecule that has been artificially created by combining genetic material from different organisms
• Recombinant DNA technology allows scientists to manipulate and modify genetic material, creating organisms with new traits or characteristics
• Applications of recombinant DNA include biotechnology, medicine, agriculture, and research, including the production of genetically modified organisms (GMOs), the development of therapeutic proteins and vaccines, and the study of gene function and regulation
• The use of molecular biology tools such as restriction enzymes (RE), DNA ligase enzyme, plasmid cloning, and plasmid extraction
• Example of Recombinant DNA uses

  • Producing transgenic plants
  • Developing therapeutic proteins and vaccines
  • Studying gene function and regulation
• Alkaline Lysis Method for Plasmid Extraction


  1. Cell culture and harvesting

  2. Cell resuspension

  3. Alkaline lysis

  4. Neutralization

  5. Purification and precipitation
• Cell culture and harvesting


  • Bacteria containing the target plasmid are cultured using media containing selective antibiotics such as rifampicin and kanamycin
  • A bacterial colony is cultured overnight in LB or other suitable broth, typically for 12-16 hours
  • Cells are harvested by separating them from the culture media using centrifugation methods
• Cell resuspension


  • Cells are resuspended in a buffer containing Tris, EDTA, glucose, and RNAse
  • Tris serves as a buffer for pH
  • EDTA acts as a chelating agent for divalent cations, rendering DNases inactive and stabilizing the cell walls
  • Glucose establishes suitable osmotic pressure for the cells
  • RNase A is used for degrading RNA after cell lysis occurs
• Alkaline Lysis


  • The lysis buffer contains NaOH and SDS
  • SDS dissolves cell membranes (lysis) and denatures proteins
  • NaOH is important for disrupting hydrogen bonds between nucleotides, causing dsDNA (chromosome and plasmid) to denature into ssDNA
• Neutralization


  • Potassium acetate lowers the alkalinity, causing ssDNA to revert back to dsDNA
  • Small plasmids are easier to form dsDNA, compared to larger chromosomes
  • Denatured proteins, chromosomes, and tissue debris, as well as SDS, are precipitated
• Purification and precipitation


  • The plasmid DNA has been isolated from most cell debris
  • However, it is still contaminated with salts, EDTA, RNase, and cellular proteins
  • Purification: using phenol
  • Precipitation: using ethanol
• Quality and Quantity


  • The plasmid extraction results are analysed to determine concentration and purity
  • Two methods that can be used are gel electrophoresis analysis and spectrophotometry
  • NanoDrop is a spectrophotometer used to determine the concentration and purity of plasmid
• NanoDrop


  • Provides readings of plasmid concentration in units of ng/µl based on absorption at A260
  • The purity of the plasmid is determined through the absorbance ratio at wavelengths A230 and A280
• Plasmid Purity

  • A260/A280 ratio around 1.8 - 2.0 is considered indicative of pure plasmid obtained
  • A low A260/A280 ratio indicates contamination by proteins
  • A high A230 value indicates contamination by phenol, EDTA, or carbohydrates
• Gel electrophoresis can be performed to confirm the extraction results
• DNA concentration is estimated by measuring the absorbance at 260nm, adjusting the A260 measurement for turbidity (measured by absorbance at 320nm), multiplying by the dilution factor, and using the relationship that an A260 of 1.0 = 50µg/ml pure dsDNA
• Plasmid Quantity and Quality

  • Concentration (µg/ml) = (A260 reading – A320 reading) × dilution factor × 50µg/ml
  • DNA yield (µg) = DNA concentration × total sample volume (ml)
• DNA is not the only molecule that can absorb UV light at 260nm. RNA also has a great absorbance at 260nm
• Plasmid Purity Evaluation

  • The ratio of the absorbance at 260nm divided by the reading at 280nm (A260/A280) provides an estimate of DNA purity with respect to contaminants that absorb UV light, such as protein
  • A ratio of 260nm to 230nm can help evaluate the level of salt carryover in the purified DNA
• Phenol Chloroform Purification Method


  • Involves mixing an aqueous nucleic acid sample with a phenol-chloroform mixture
  • The organic phase then separates from the aqueous phase, taking with it any proteins that were in the original sample
  • Nucleic acids will remain in the aqueous layer of the mixture, along with other contaminants (e.g., salts and carbohydrates)
• Silica Based Purification Methods


  • Employ a simple bind-wash-elute process
  • Nucleic acids bind to the silica membrane in the presence of chaotropic salts
  • Polysaccharides and proteins do not bind well to the column and residual traces are removed during alcohol-based wash steps, along with the salts
  • After binding and washing, nucleic acids are selectively eluted under low-salt conditions, using water or TE buffer
• Silica Based Methods - Disruption of Hydrogen Bonding

  Chaotropic salts, such as guanidine hydrochloride or guanidine thiocyanate, disrupt the hydrogen bonds between complementary bases, causing the DNA strands to denature and unwind
• Silica Based Methods - Disruption of Hydrophobic Interactions

  Chaotropic salts disrupt the hydrophobic interactions between DNA molecules and surrounding molecules, increasing the solubility of the DNA in solution and preventing it from aggregating or reannealing
• Silica Based Methods - Exposure of Silanol Groups

  Chaotropic salts disrupt the hydrogen bonds between water molecules and the silanol (Si-OH) groups on the silica surface, exposing the silica surface for interaction with DNA molecules
• Silica Based Methods - Binding of DNA to Silica

  In the presence of chaotropic salts, the denatured and solvated DNA molecules interact with the exposed silanol groups on the silica surface through electrostatic forces, effectively immobilizing the DNA
• Magnetic Bead Based Purification Methods


  • Employ bind-wash-elute
  • Use magnetic beads or particles functionalized with silica surfaces to allow selective binding of DNA in the presence of high concentrations of salt
  • DNA bound to a magnetic bead can be easily separated from the aqueous phase using a magnet
• Anion Exchange Purification


  • Uses positively charged DEAE functionalized resins to capture the negatively charged phosphate backbone of target DNA molecules
  • Specific salt and pH conditions are used to bind, wash, and selectively elute the purified DNA
• Loading Buffer

  • Bromophenol blue: A tracking dye that migrates ahead of most DNA fragments during electrophoresis
  • Glycerol: Increases the density of the DNA sample, making it easier to load into the wells of the gel
  • EDTA: Chelates divalent cations to prevent nucleic acid degradation
  • SDS: Denatures proteins present in the sample, preventing them from interfering with electrophoresis