PCR

• Polymerase Chain Reaction (PCR)


  A tool to make numerous copies of DNA by amplifying sequence-specific DNA fragments in vitro
• PCR

  1. DNA denaturation

  2. Primer annealing

  3. Primer extension
• Each PCR cycle theoretically doubles the amount of DNA
• During PCR, an existing DNA molecule is used as a template to synthesize a new DNA strand
• Through repeated rounds of DNA synthesis, large quantities of DNA are produced
• PCR is a repeating cycle of separation of double DNA strands and synthesis of a complementary strand for each
• Theoretically, PCR doubles the amount of DNA in every cycle
• Denaturation


  High temperatures to separate the two DNA strands and unwind the DNA helix
• Primer annealing

  Lower the temperature to allow primers to bind to their complementary sequence
• Primer extension

  Adjust the temperature for optimal thermostable DNA polymerase to synthesize a complementary strand by reading the opposing strand sequence and extending the primers by adding nucleotides in order in which they can pair
• In vivo, there is a start and stop codon, but in vitro PCR there may be the absence of a start and stop codon because the PCR machine is automated and there is allotment of time for the amplification to start and end
• PCR reaction in a test tube

  • DNA source
  • Primers
  • Prepared master mix (nucleotide bases, enzymes, reaction buffer)
  • Magnesium
• PCR machine

  Automates the repeating cycle of DNA separation, synthesis of complementary strands
• Normal procedure for PCR takes approximately three to four hours
• Components needed for PCR

  • DNA source (template, blood, tissues, organs, stool, and others)
  • Primers (starting point for DNA synthesis)
  • Nucleotide bases (A T C G)
  • Enzyme (catalyze the synthesis)
  • Reaction buffer (KCl, Tris HCl, Triton X-100)
  • Magnesium (cofactor of DNA polymerase) Mg+2
• Electrophoresis


  Migration of charged molecules in an electric field to separate DNA fragments or nucleic acid molecules based on their electric charge
• What is needed in electrophoresis

  • Buffer TAE or Trees Acetate EDTA buffer
  • Gel: agarose gel or polyacrylamide gel
  • Loading dye
  • Visualization dye
  • Ladder
• DNA purity/concentration test


  Spectrophotometer to measure optical density (OD) at 260 nm and 280 nm to determine DNA concentration and purity
• Pure preparation of DNA has OD260/OD280 values of 1.8 to 2.0
• A ratio of less than 1.8 indicates there may be proteins, and a ratio of more than 2.0 indicates samples may be contaminated with chloroform or phenol
• How to calculate DNA concentration

  Absorbance at 260 nm x 50 ng/mL x dilution factor = DNA concentration
• DNA isolation techniques

  • Manual method
  • Silica-based
  • Vacuum
  • Centrifugation
  • Magnetic beads
  • Phenol/chloroform method
  • Salting out procedure (or precipitation)
• Steps in DNA isolation

  1. DNA lysis

  2. Binding

  3. Washing

  4. Elution
• Silica-based DNA isolation


  • Fast, easy-to-perform and economical
  • Nucleic acids bind to the silica membrane in the presence of chaotropic agents
  • Polysaccharides and proteins do not bind well and are removed during wash steps
  • Nucleic acids are selectively eluted under low salt conditions
• Magnetic bead DNA isolation


  • Uses 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 magnetic beads can be easily separated from the aqueous phase using a magnet
  • Allows rapid sample processing and fine control of solution volumes
  • May take longer than column-based silica-based purification but has higher purity and advantage of high RNA recovery
• Phenol/chloroform DNA isolation

  • Equal volumes of phenol/chloroform mixture and aqueous sample are mixed, forming a biphasic mixture
  • Organic phase separates and takes with it any proteins, while nucleic acids remain in the aqueous layer
  • Nucleic acids in the aqueous layer can then be separated from other contaminants by ethanol precipitation
• Salting out DNA isolation


  • Rapid, safe, and inexpensive method
  • Involves salting out of cellular proteins by dehydration and precipitation with a saturated NaCl solution
• Kary Mullis developed PCR in 1984, which created a big impact in the field of molecular biology and biotechnology and became the most accessible and widely used tool in genetic and medical research
• Materials needed for DNA/RNA extraction

  • Microcentrifuge
  • Vortex mixer
  • Dry bath
  • Minicentrifuge
  • Pipettors and pipette tips
  • Microtubes
  • Microtube rack
  • Extraction kit
  • 95-100% ethanol
  • 10% bleach
  • 70% ethyl alcohol
  • Gloves
  • Pipette tip bin
  • Garbage bin
  • Paper towels
  • Forceps
• DNA/RNA extraction protocol

  1. Sample preparation

  2. Add proteinase K solution

  3. Add lysis buffer

  4. Incubate at 56°C

  5. Add ethanol

  6. Transfer to spin column

  7. Centrifuge and discard flow-through

  8. Wash with buffers

  9. Centrifuge to dry column

  10. Elute DNA/RNA with elution buffer
• DNA extraction procedure

  1. Add 100uL of the Lysis B Buffer to the sample

  2. Incubate at 56℃ for 10 minutes

  3. Add 200uL of ethanol and mix

  4. Transfer all the mixture to spin column

  5. Centrifuge the mixture at 6000-8000xg for 1 minute

  6. Add 500uL of the WB1 Buffer to the spin column, centrifuge at 10000xg for 30-60 seconds

  7. Add 700uL Wash Buffer to the spin column, centrifuge at 10000xg for 30-60 seconds

  8. Add 700uL Wash Buffer to the spin column, centrifuge at 10000xg for 30-60 seconds

  9. Centrifuge the Spin column at 13000xg for 2 minutes

  10. Add 100uL the Elution Buffer, incubate at room temperature for 1 minute, centrifuge at 13000xg for 1 minute
• The maximal volume of the samples should be within 1000uL, it is necessary to pool leukocytes with erythrocyte Lysis solution or Lymphocytes Separation solution when the sample volume is over 200uL
• The kit is mostly used for human whole blood, or for other mammalian whole blood, and bird whole blood
• The fresh whole blood should be stored at 2-8℃ or 20℃. If the blood was stored at -20℃, isolate the DNA after thawing
• Low amounts of leukocytes in the blood sample (for human or other mammal whole blood) can lead to little or no DNA in the elution
• PK solution did not be stored at 2-8℃, leading to its low activity can lead to little or no DNA in the elution
• Forgetting to add ethanol to WB1 Buffer and Wash Buffer according to the label on the bottle before the first using can lead to little or no DNA in the elution
• For bird blood, after mixing the sample, PK Solution and Lysis B Buffer, the mixture is too ropy. Used too much bird blood, you should decrease the amount of the sample. Usually, bird blood in controlled within 20uL and adjusted to 200uL using PBS Buffer
• Inefficient cell lyses due to insufficient mixing of the sample with LB Buffer can lead to low A260/A280 ratio for purified nucleic acids
• Inefficient cell lysis due to the decreased protease activity can lead to low A260/A280 ratio for purified nucleic acids