Molecular Biology Techniques

• Recombinant DNA technology is possible because the genetic code is universal
• In bacteria, we use PCR to amplify the gene of interest
• We need to use the mature mRNA transcript for recombinant technologies, but you cannot use PCR on mRNA
• Each cell expresses different mRNA based on their functions
• cDNA reflects the expression pool of the cell
• Making cDNA

  1. Use a chemical detergent to lyse cells
  2. Isolate mRNA by applying an immobilised poly(T) tail and washing off tRNA, small RNAs and immature RNAs
  3. Synthesise the first strand of DNA from RNA using an RdDp
  4. PCR
• To synthesise DNA from RNA, we need an RNA-dependent DNA-polymerase (RdDp)
• You must have the appropriate promoter for the organism or cell/tissues in which you want to express the target sequence
• Plasmids are a naturally occurring extra-chromosomal piece of DNA in bacteria
• Plasmids store genes that are not frequently used, such as genes that confer resistance
• Plasmids are a vector for horizontal gene transfer
• Features of plasmids

  • Origin of replication
  • Antibiotic resistance gene
• Properties of plasmids

  • Selection pressure
  • Self-replicating
  • Transferable
• Extracting Plasmid DNA

  1. Cell walls are lysed with high pH
  2. Cell components are neutralised to extract plasmid DNA
• If more plasmids are required, then more bacteria needs to be lysed
• Restriction enzymes/restriction endonucleases are used to cut in the middle of the plasmid
• Restriction enzymes come from bacteria
• Since plasmids are circular, they are not susceptible to exonucleases
• You want to make directed and limited cuts to the plasmid DNA
• Restriction enzymes cut within palindromic sequences
• palindromic sequences = identical inverted sequences
• How is DNA of bacteria protected from restriction enzymes?

  The DNA of the host bacteria is methylated so that it is not susceptible to endonucleases
• Restriction enzymes were originally used as a host defence mechanism against bacteria
• Restriction enzymes can leave sticky ends of the DNA, meaning that there is a 5' or 3' overhang
• Sticky ends are more specific since annealing can only happen through base pairing
• Restriction enzymes can leave blunt ends of the DNA, meaning that there are no overhangs
• Blunt ends anneal to another strand with blunt ends
• Most plasmids have been heavily genetically engineered to have:

  • Unique restriction sites
  • MCS
• MCS = multiple cloning site
• MCS: Many different cut sites that are not present in other parts of the plasmid
• Adding tails to primers to ensure that they will bind to the restriction site
• The added bits at the 5' end do not anneal to the original template, but will still allow DNA  polymerase to synthesise the new strand
• Adding tails to PCR primers produces millions of DNA copies with the correct restriction site and one copy without the restriction site
• Taking up foreign DNA = transformation
•  Transformation

  • Growing cells in calcium reduces the charge on DNA, increasing the chances of transformation
  • Applying heat or electricity puts small holes in the cell wall and membranes, allowing it to take up plasmids
• To selecting for transformants, the bacteria can be grown on media containing antibiotics
• Only the transformed cells will have the resistance gene, allowing them to grow on antibiotic media
• Non-recombinant molecules can arise if:

  • The plasmid was cut and then re-ligated without the gene of interest
  • The plasmid was not cut at all
• Growing on antibiotic media can select for transformants, but cannot differentiate non-recombinant and recombinant molecules
• Reporter Genes: A gene that you use as a way to report on an activity