Genetic engineering 1

🧬 Biol 112 — Genetic Engineering I (Detailed Exam Notes)

(Covers Chapter 20: The Molecular Revolution — Biotechnology and Beyond)

🧩 1. Overview: The Molecular Revolution

(Slides 7–10)

Molecular Revolution:

• Began when scientists learned to isolate, sequence, and manipulate DNA.

• Revolutionized biology: we can now remove, analyze, and reinsert genes.

• By 2001, the first human genome sequence was completed; by 2021, over 417,000 genomes had been sequenced.

• Genomics: sequencing, interpreting, and comparing whole genomes.

Possible Exam Question 💭

⚙ 2. What Is Recombinant DNA Technology?

(Slides 11–13)

Definition:
A collection of lab techniques that allow scientists to mix and match (recombine) DNA sequences from any organism to create new combinations not found in nature.

Uses:

• Study gene function

• Modify genetic traits in crops, animals, and microbes

• Produce medicines (e.g., insulin, HGH)

Recombinant DNA = cornerstone of biotechnology
→ Manipulating DNA for scientific or practical purposes.

Goals:

1. Understand how genes work.

2. Engineer organisms for useful outcomes (biotech, medicine).

🧬 3. DNA Cloning

(Slides 14–20, Fig. 20.2)

DNA cloning:
= Producing many identical copies of a DNA segment or gene.

🧠 If a scientist says they “cloned a gene,” it means:

• They isolated that gene.

• Inserted it into a plasmid.

• Replicated it many times inside bacterial cells.

🔹 4. Plasmids

(Slides 15–16, Fig. 19.3, 26.7)

Plasmid:

• Small, circular DNA molecule found in bacteria.

• Replicates independently of the chromosome.

• Often carries antibiotic resistance genes (used as markers in cloning).

Transfer methods:

Function:

• Used as vectors to carry foreign genes into host cells.

• Contain an origin of replication, selectable marker, and multiple cloning site.

Possible Exam Question 💭

🧩 5. Restriction Enzymes & DNA Ligase

(Slides 17–20, Fig. 20.2)

Restriction Endonucleases:

• Enzymes that cut DNA at specific sequences (palindromic sites).

• Example: EcoRI → makes staggered cuts producing sticky ends.

Sticky Ends: short single-stranded overhangs that can pair with complementary sequences.
DNA Ligase: enzyme that seals sticky ends → creates stable recombinant DNA.

Steps to Insert a Gene into a Plasmid:

1. Cut plasmid and foreign DNA with same restriction enzyme.

2. Mix them → sticky ends base-pair.

3. Use DNA ligase to seal sugar–phosphate backbone.

4. Transform recombinant plasmid into E. coli.

5. Grow and replicate cells → clone gene.

⚙ 6. Pituitary Dwarfism and Human Growth Hormone (HGH)

(Slides 22–25, Fig. 19.1)

Pituitary Dwarfism Type I:

• Caused by defective GH1 gene → no growth hormone.

• Leads to slow growth, delayed puberty, short stature (~120 cm).

Early HGH Sources:

• From animals (ineffective or infectious).

• From human cadavers (scarce, risk of CJD prions).
  → Banned in 1984.

Solution:
Use genetically engineered E. coli to produce HGH.

🧬 7. Producing HGH via Recombinant DNA

(Slides 25–34, Figs. 19.2, B8.1, 19.6)

Step 1: Use Reverse Transcriptase

• Enzyme that synthesizes DNA from mRNA template → makes complementary DNA (cDNA).

• cDNA = copy of expressed gene without introns → ideal for cloning in bacteria.

Possible Exam Question 💭

Step 2: Create cDNA Library

Definition: A collection of recombinant plasmids each containing a unique cDNA copy from a cell’s mRNA.

Steps:

1. Isolate mRNA.

2. Reverse transcribe to cDNA.

3. Make cDNA double-stranded.

4. Insert each cDNA into plasmid.

5. Transform E. coli.

6. Library = collection of clones representing active genes in that cell.

Step 3: Identify the Desired Gene

DNA Probe:

• Single-stranded, labeled piece of DNA complementary to target sequence.

• Hybridizes to matching DNA in the library (fluorescent or radioactive signal).

Screening:

• Filters from bacterial colonies are probed → colonies with the target gene light up.

• Cells containing the human GH1 gene are isolated and grown → mass HGH production.

⚠ Ethical Concerns (Slides 35–36)

• Used beyond therapy → “cosmetic height enhancement.”

• Athletic doping → muscle & bone density increase.

• Raises safety, fairness, and medical ethics questions.

Possible Exam Question 💭

🌾 8. Adding Genes to Organisms (GMOs)

(Slides 37–46)

🧩 Transgenic Organisms

= Organisms with foreign DNA inserted into their genome.
Also called Genetically Modified Organisms (GMOs).

Applications:

• Research (e.g., GFP mice).

• Agriculture (Bt corn, Golden Rice, Roundup Ready soybeans).

⚙ Creating Transgenic Plants

(Fig. 19.20)

• Uses Agrobacterium tumefaciens — a bacterium that naturally inserts DNA into plant chromosomes.

• Gene of interest is cloned into Ti plasmid (tumor-inducing).

• Infected plant cells integrate the foreign gene → grown into adult GM plants.

🌾 Golden Rice (Slides 40–42, Fig. 19.21)

• Engineered to express β-carotene (vitamin A precursor).

• Prevents blindness due to vitamin A deficiency.

• Introduces 3 enzymes from other species into rice genome.

Possible Exam Question 💭

🧬 Bt Corn & Roundup Ready Crops

• Bt Corn: Contains Bacillus thuringiensis toxin gene → insect resistance.

• Roundup Ready Soybeans: Contain gene for glyphosate tolerance → survive herbicide spray.

🧩 9. Removing or Editing Genes

(Slides 47–56)

🔹 Site-Directed Mutagenesis

Developed by Michael Smith (UBC) — 1993 Nobel Prize winner.

Purpose:
Introduce a specific mutation into a gene.

Steps:

1. Insert normal gene into plasmid.

2. Make plasmid single-stranded.

3. Add short synthetic DNA with one incorrect base.

4. DNA polymerase copies → produces mutated plasmid.

5. Transform into bacteria → half of plasmids carry mutation.

🧠 Allows targeted changes in genes → study function or create variants.

⚙ CRISPR–Cas9 Gene Editing

(Slides 54–55)

Definition:
CRISPR = Clustered Regularly Interspaced Short Palindromic Repeats.
Cas9 = bacterial enzyme that cuts DNA.

Mechanism:

1. Design an RNA guide complementary to target DNA sequence.

2. Insert RNA + Cas9 gene into cells via plasmid.

3. Guide RNA binds target DNA → Cas9 cuts both strands.

4. Cell repairs DNA → new sequence can be inserted or deleted.

Advantages:

• Fast, precise, inexpensive.

• Works in almost any organism.

• Used for gene therapy, crop engineering, disease research.

Possible Exam Question 💭

🧬 10. Summary

(Slide 57)

Genetic Engineering Steps:

1. Reverse transcriptase → make cDNA.

2. Plasmid → clone DNA.

3. Transformation → introduce plasmid into host.

4. Create cDNA library → store genes.

5. Screen library → find target gene.

6. Mass-produce protein (e.g., HGH, insulin).

7. Add or remove genes (transgenics, mutagenesis, CRISPR).

Applications:

• Medicine (insulin, HGH, vaccines)

• Agriculture (GM crops)

• Research (gene function, disease models)

🧠 Exam Tip Checklist

✅ Define: recombinant DNA, plasmid, restriction enzyme, transformation, cDNA, probe, transgenic organism, CRISPR.
✅ Know steps for cloning a gene (restriction enzyme → ligase → transformation → screening).
✅ Explain why cDNA is used instead of genomic DNA.
✅ Understand how to identify a gene in a library (DNA probe).
✅ Be able to compare Bt corn vs Golden Rice.
✅ Describe site-directed mutagenesis and CRISPR-Cas9 mechanisms.
✅ Discuss ethical issues with GMOs and recombinant hormones.

✅ This fully covers all slides and learning objectives:

• DNA technology & recombinant DNA

• Plasmids, restriction enzymes, transformation

• Reverse transcriptase, cDNA, libraries, probes

• Transgenic organisms & GMOs

• Site-directed mutagenesis & CRISPR

• Ethical considerations

Would you like me to now turn this into a Quizlet-style flashcard set (term → definition; semicolon-separated) so you can memorize it efficiently for your exam?