Biotechnology & Recombination DNA Technology

Biotechnology uses ___ to make ___

• microorganisms & cells,

• to make foods, antibiotics, vitamins, & enzymes

What is recombinant DNA (rDNA) technology?

Insertion/modifies genes to make desired proteins.

Examples of where rDNA can be used 

• plant pest resistance 🌱

• clear toxic waste  ☢

• clothing fabric 👕

• human growth hormones 👦🏻♂

What are the steps to make a recombinant plasmid?

• Isolate plasmid vector & cut it.

• Insert desired gene → recombinant plasmid.

• Transform bacteria with recombinant plasmid.

• Bacteria divide & reproduce new gene.

What do restriction enzymes do?

Cut DNA at specific sequences.

Difference between: blunt and staggered cuts & their ends 

• blunt: smooth ends 

• staggered: sticky ends 

Which cut makes blunt (smooth) cut

HaeIII, all others are staggered

Enzyme BamHI bacterial source

Bacillus Amyloliquefaciens 

Enzyme EcoRI bacterial source

Escherichia coli 

HaelIII bacterial source

Haemophilus aegyptius

HindilII bacterial source

Haemophilus influenzae

What does Polymerase Chain Reaction (PCR) do & use?

• Uses thermocycler

• to make multiple copies of DNA outside a cell

What 5 tools needed for PCR 

1. DNA template,

2. primers,

3. nucleotides (dNTPs),

4. Taq polymerase,

5. PCR buffer.

What are the 3 steps of PCR, describe & their temp 

• Denaturation (94°C, 1 min): DNA strands separate.

• Annealing (60°C, 1 min ): Primers bind to DNA parental strands.

• Extension (72°C, 1 min): Taq polymerase synthesizes new DNA daughters cells

5 uses for PCR

1. Clone DNA for recombination

2. Amplify DNA to detectable levels

3. Sequence DNA

4. Diagnose genetic disease

5. Detect pathogens

What does DNA ligase do?

Glues DNA fragments together to make recombinant plasmids.

DNA manipulation transformation steps

• Cells take up foreign DNA from surroundings;

• made competent with Calcium Chloride & heat shock (30-40s at 60 °C)

• Recombinant plasma added,

• Test tube put back on ice to close bacterial pores

What is electroporation?

Same as transformation, except it uses electrical shock

Protoplast Fusion steps

• Bacterial cell walls enzymatically ingested to produce protoplasts

• Proplasts treated with polyethylene glycol (hot glue) 

• Protoplasts fuse 

• Segment of two chromosomes recombine 

• Recombinant cell grows new cell wall, washes with excess enzymes 

What does a gene gun do?

Shoots DNA-coated microscopic particles (tungsten/gold)

into plant cells using helium.

What is DNA microinjection?

Injecting DNA directly into a cell with a micropipette under a microscope.

Give examples of therapeutic uses of recombinant DNA.

Human enzymes, proteins like insulin, vaccines.

Subunit Vaccine: 

• has 

• made by 

• ex 

• only protein portion of pathogen

• made by genetically engineered yeast 

• Hepatitis B vaccine 

DNA vaccine carries _ and 4 examples 

• Plasmid carry some non pathogenic viruses genes

  • HIV,

  • SARS,

  • Influenza,

  • Malaria  

Gene Therapy

• does

• 2 examples where it’s used for treatment

• replaces defective/mutated/missing genes from cells by using transformation

  • Haemophilia B,

  • SCID

What is RNAi used for & example disease

• silences particular gene 

• hepatitis B in mice