Genetic Transfers & DNA Technology

• Biotechnology uses ___ (2)

  • to make ___ (4)

• microorganisms & cells

  • food

  • enzymes

  • antibiotics

  • vitamins

Examples of where rDNA can be used 

• plant pest resistance 🌱

• clear toxic waste  ☢

• clothing fabric 👕

• human growth hormones 👦🏻♂

What is recombinant DNA (rDNA) technology?

inserts/modifies genes to make desired proteins

What is a plasmid

A small circular DNA molecule

What are the 5 steps to make a recombinant plasmid? 

1. Isolate plasmid vector

2. Cut it using restrictive enzymes ✂

3. Insert desired gene into plasmid

4. Seal gene & plasmid tugetha (w DNA ligase) → recombinant plasmid.

5. Bacteria divide & reproduce new gene.

What do restriction enzymes do?

Cut DNA at specific sequences.

What does DNA ligase do and make?

• Glues plasmid to gene

• to make recombinant plasmids.

blunt vs staggered cut ends 

• blunt: smooth ends 

• staggered: sticky ends 

Which enzyme makes blunt (smooth) cut

HaeIII, all others are staggered

Enzyme BamHI bacterial source

Bacillus Amyloliquefaciens 

Enzyme EcoRI bacterial source

Escherichia coli 

Enzyme HaelIII bacterial source

Haemophilus aegyptius 𓁖🇪🇬

Enzyme HindilII bacterial source

Haemophilus influenzae

What does PCR (Polymerase Chain Reaction) do

makes multiple copies of DNA outside a cell

tool used in PCR 

Thermocycler🔥

What 5 tools needed for PCR 

• T → Template of DNA

• P → Primers

• D → dNTPs/nucleotides

• T → Taq polymerase

• B → Buffer

*the primer does-not truly blend

What are the 3 steps of PCR, & their temp

• Denaturation 94°C

• Annealing 60°C

• Extension 72°C

PCR Denaturation

• temp

• describe

• 94°C: DNA heated to separate its strands 🧬

PCR Annealing

• temp

• describe

• 60°C

• Primer binds to DNA’s parental strands 🧑‍🧑‍🧒

PCR Extension

• temp

• describe

• (72°C):

• TAQ Polymerase synthesizes new DNA daughter cells👧🏻

5 uses for PCR

1. 🧑‍🤝‍🧑 Clones DNA

2. 📣 Amplifies DNA

3. 🔢 Sequences DNA

4. 🩺 Diagnoses Genetic Diseases

5. 🦠 Detects pathogens

Gene Therapy: Transformation 4 steps

1. 👽 Cells take up foreign DNA

2. 🥵 Turn competent w Calcium Chloride & heat shock

3. 🧫 Add Recombinant Plasma

4. 🧊 Test tube put back on ice to close bacterial pores

In DNA manipulation transformation, what is the temp & time heat shock is used 

• 60 °C

• 30-40s 

Gene Therapy: Electroporation steps

Same steps as transformation,

except it uses electrical shock⚡💥🔌

Protoplast Fusion 5 steps

1. 🍔🌱 Bacteria cell wall enzymatically ingested → protoplasts

2. 🧴 Proplasts treated with Polyethylene Glycol (glue) 

3. 🫂 Protoplasts fuse 

4. 🧬 2 chromosome segments recombine 

5. 🧱🌱 Recombinant cell grows new cell wall

Gene gun:

• Does _

• Into _

• Using _

• Shoots DNA-coated micro particles made of tungsten/gold🥇

• into plant cells 🪴

• burst of helium pressure🎈

• What is DNA microinjection?

• Tool used

• injects DNA directly into a cell’s plasma mem

• glass micropipette under microscope

Give 4 examples of therapeutic uses of recombinant DNA.

• Proteins

• Vaccines

• Human enzymes

• Hormones like insulin

Subunit Vaccine: 

• has 

• made by 

• ex 

• only a pathogen’s protein

• genetically engineered yeast 

• Hepatitis B. vaccine 🐀

• What do DNA vaccines have

• 4 examples of disease treated by DNA vaccines

• Plasmids carry non-pathogenic virus genes

  • SARS 🦠

  • HIV 💋

  • Influenza 🤒

  • Malaria  🦟

What does Gene Therapy do?

replaces mutated/missing genes from cells

2 Examples of Diseases treated w Gene Therapy 

• Hemophilia B.

• SCID

RNAi

• function

• how it works

• silences a gene:

• sirNA binds to mRNA to block translation so protein cannot be made

What disease does RNAi treat in mice?

Hepatitis B. (liver inflamation) 🐀ྀིྀིྀིྀིྀི

Type of gene transfer from parent to offspring

Vertical gene transfer

gene transfer btwn cells of same generation.

Horizontal gene transfer

Exchange of genes between two similar DNA sequences (crossing over).

Homologous Recombination 

Example of bacteria that uses Homologous Recombo

Streptococcus Pneumoniae 🫁

What do virulent bacteria have that non-virulent don’t

capsules that cause disease & possible death

Conjugation:

• define

• how differ from genetic transformation

• what it needs

• transfer DNA of one bacterium to another

• needs direct cell-cell contact

• opposite mating types (F+ donor, F- recipient) 

Transduction

• what it is

• by a _

• transfers bac DNA from donor to a recipient

• by a bacteriophage/virus

What is a transposon/jumping gene?

DNA segment jumps from one region of DNA to another.

Transposons:

• What do they carry

• Have _

• what allows them to cut & reseal DNA anneal?

• Other genes (in addition to ones moving)

• inverted/mirror reflection of nucleotides

• Insertion sequences

  • code for→ enzyme transposase.