4.4 Introduction to biotechnology

• Recombinant DNA= the joining of two separate pieces of DNA (comes from a different source)
  • joined together in a non-naturally occurring way
  • e.g. plasmid cloning

1. identify gene of interest (GOI)

2. replicate the gene using PCR

3. transfer GOI into the recipient organism 

• involves integration of the amplified GOI into plasmid vector
• transform plasmid into host cell

4. have a method to check successful transformants

1. GOI is amplified by PCR (read how PCR works)

• amplification is in vitro
• catalysed by DNA polymerase -> synthesizes a new DNA strand complementary to template strand 
• primer is needed -> DNA polymerase can only add nucleotides from the 3' -OH end (aka lagging strand)

After two separate fragments are produced from PCR restriction digest ligation turns them into recombinant plasmid. [2]

2. Integrate amplified GOI into plasmid vector

• Restriction endonuclease enzymes cleave DNA at specific sequences (restriction sites)
  • restriction sites produce "sticky ends"
  • sticky ends can ligate with complementary sticky ends of other fragments
    • DNA ligase seals the ligation

• Restriction sites are added to 5' end of primers
  • become integrated into PCR product at each end

3. Transform recombinant plasmid into host cell

• Methods: heat shock and electroporation
• Heat shock:
  • cells are made chemically compentent through treatment of buffers (contains calcium-> produces repulsion form the charge Ca²⁺, neutralises charge)
  • temperature induces pores in cell membrane where it promotes DNA uptake

• Selectable marker - antibiotic resistance
  • agar plate will contain the antibiotic which will have the bacteria containing plasmid with resistance
  • only cells that take up a plasmid will survive

4. Validate successful transformants

• Gel electrophoresis
  • electrical current passes through gel
  • DNA fragments move towards positive pole -> small fragments move faster up
  • a dye specific for nucleic acids is added to gel
  • DNA fragments appear as bands on gel

PCR STEPS:

1. Denaturation (95): separation of 2 nucleotide strands of template DNA

2. Annealing (55-> based on primers): primers bind to the single strands

3. Extension(72): nucelotides are added to primers from 5' to 3' by DNA polymerase -> forms double stranded copy

Components of PCR:

• Template DNA (DNA containing GOI)
• Primers (amplifies target region)
• DNA polymerase (active at 72)
• Buffer (contains confactor for DNA pol. and dNTPs)

• Molecular cloning= assemble recombinant DNA and direct their replication within host organisms/ creates multiple identical copies of DNA segments
  • Vector= section of DNA that can carry another DNA fragment without losing the capacity for self replication
    • Hybrid vector= vector containing an additional DNA fragment
  • Gene cloning= fragment of DNA includes one/+ genes
• Plasmids replicate separately from bacterial chromosome
  • Highly mobile (very small DNA molecule)

• Sanger sequencing: same as PCR, ATGC has a fluorescent marker attached
  • markers used to identify final bases

• https://quizlet.com/130540065/f215-5-genomes-and-gene-technologies-flash-cards/?funnelUUID=79c7fab1-efb1-4204-94b3-14ec4e73a642 (Outline how genetic markers in plasmids can be used to identify the bacteria that have taken up a
  recombinant plasmid.)

• Gene therapy is the alteration of genes that will replace a defective allele with a normal allele
  • vectors are used for delivery of genes into specific cells (e.g. bone marrow)
  • should be done in dividing cells that are renewing cell population
• Retroviral vector-> carries allele without replicating itself