Genetic engineering 1

Molecular Revolution

The era beginning when scientists learned to isolate, sequence, and manipulate DNA.

Genomics

The sequencing, interpreting, and comparing of whole genomes.

Recombinant DNA Technology

Lab techniques allowing scientists to mix and match DNA sequences from any organism to create new combinations not found in nature.

DNA Cloning

The process of producing many identical copies of a DNA segment or gene.

Plasmid

A small, circular DNA molecule found in bacteria that replicates independently of chromosomal DNA.

Restriction Endonucleases

Enzymes that cut DNA at specific sequences, creating specific fragments.

Sticky Ends

Short single-stranded overhangs of DNA that can pair with complementary sequences.

DNA Ligase

An enzyme that seals sticky ends of DNA segments to create stable recombinant DNA.

Pituitary Dwarfism Type I

A condition caused by a defective GH1 gene, leading to no growth hormone production.

cDNA (Complementary DNA)

DNA synthesized from an mRNA template using reverse transcriptase.

cDNA Library

A collection of recombinant plasmids, each containing a unique cDNA copy from a cell's mRNA.

DNA Probe

A single-stranded, labeled piece of DNA complementary to a target sequence used for screening.

Transgenic Organisms

Organisms that have foreign DNA inserted into their genome, also called GMOs.

Agrobacterium tumefaciens

A bacterium used in genetic engineering to insert DNA into plant chromosomes.

Golden Rice

A genetically engineered rice that expresses β-carotene to prevent blindness due to vitamin A deficiency.

Bt Corn

Corn that contains a gene from Bacillus thuringiensis for insect resistance.

Roundup Ready Soybeans

Soybeans that have a gene conferring tolerance to glyphosate herbicide.

Site-Directed Mutagenesis

A method used to introduce a specific mutation into a gene.

CRISPR-Cas9

A gene editing technology that uses a guide RNA and the Cas9 enzyme to cut DNA at specific sites.

Ethical Concerns in Genetic Engineering

Issues arising from the use of recombinant technologies, such as safety and fairness questions related to GMOs and enhancements.

Genomics definition

The sequencing, interpretation, and comparison of whole genomes

Molecular revolution

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

Recombinant DNA technology

Techniques allowing mixing and matching DNA from different organisms

Purpose of recombinant DNA technology

Study gene function and engineer organisms for useful traits

DNA cloning

Producing many identical copies of a DNA segment or gene

Meaning of “cloning a gene”

Isolating a gene, inserting it into a plasmid, and replicating it in bacteria

Plasmid definition

Small circular DNA in bacteria that replicates independently

Plasmid use in biotech

Acts as a vector to carry foreign DNA into host cells

Selectable marker in plasmids

Antibiotic resistance gene used to identify transformed cells

Methods of plasmid transfer

Conjugation, transformation, viral delivery

Transformation definition

Uptake of foreign DNA by bacteria

Restriction enzyme function

Cuts DNA at specific palindromic sequences

Restriction site

Specific DNA sequence recognized and cut by restriction enzyme

Sticky ends

Single-stranded overhangs that can base-pair with complementary sequences

DNA ligase role

Seals sugar–phosphate backbone to create stable recombinant DNA

Steps to insert gene into plasmid

Cut plasmid + DNA → sticky ends pair → ligase seals → transform bacteria

Pituitary dwarfism type I

Caused by defective GH1 gene → no growth hormone production

Early sources of HGH

Animal HGH or cadavers; unsafe or ineffective

Reverse transcriptase

Synthesizes DNA from an mRNA template → creates cDNA

Why use mRNA instead of genomic DNA?

cDNA has no introns → allows expression in bacteria

cDNA definition

Complementary DNA made from mRNA; contains only expressed sequences

cDNA library

Collection of plasmids with cDNA copies representing active genes in a cell

DNA probe

Single-stranded, labeled DNA that hybridizes to complementary sequences

Probe use

Identify colonies containing target gene within a cDNA library

Screening a library

Colonies transferred to filter → probe binds target sequence → signals match

How HGH is produced in E. coli

Clone GH1 cDNA into plasmid → transform bacteria → express hormone

Ethical issues with HGH use

Non-medical use in children/athletes; unknown long-term safety

Transgenic organism

Organism with foreign DNA inserted; also called genetically modified organism (GMO)

Purpose of GMOs

Improve crops, study gene function, produce pharmaceuticals

Agrobacterium tumefaciens role

Naturally inserts DNA into plant chromosomes; used for engineering plants

Ti plasmid

Tumor-inducing plasmid used to deliver genes into plant genomes

Golden Rice purpose

Produce β-carotene (vitamin A precursor) to prevent blindness

Genes added to Golden Rice

Three genes enabling β-carotene biosynthesis

Bt corn

Transgenic corn containing Bacillus thuringiensis toxin gene for insect resistance

Roundup Ready crops

Contain gene for glyphosate resistance to survive herbicide application

Advantages of GM crops

Higher yield, less pesticide use, improved nutrition

Disadvantages of GM crops

Potential gene escape, resistance evolution, public concern

Site-directed mutagenesis

Purposefully introducing a specific mutation into a gene

Michael Smith

UBC scientist who developed site-directed mutagenesis; Nobel Prize winner

Steps of site-directed mutagenesis

Insert gene into plasmid → single-strand plasmid → add mutant primer → replicate → transform

CRISPR–Cas9 definition

Precise gene editing system using guide RNA and Cas9 nuclease

Guide RNA role

Directs Cas9 to complementary DNA sequence for cutting

Cas9 enzyme function

Cuts DNA at target location to enable editing

CRISPR repair outcome

Cell inserts or deletes bases during repair, creating mutation

Advantages of CRISPR

Fast, precise, cheap, works in many organisms

Applications of CRISPR

Gene therapy, crop engineering, research

Recombinant DNA summary

Combines DNA from multiple sources for expression in host cells

Steps of genetic engineering

Reverse transcriptase → clone gene into plasmid → transform → screen → express protein

Why bacteria need cDNA for human genes

Bacteria cannot remove introns; cDNA lacks introns

Transformation purpose

Introduce recombinant plasmid into host cells

Selectable markers importance

Allow survival of only transformed cells

What a probe detects

Complementary sequence in a DNA library

Why Golden Rice helps prevent blindness

Provides vitamin A precursor to populations lacking it

Pros of Bt crops

Reduced insecticides, increased crop survival

Cons of Bt crops

Insects evolve resistance; ecological impact concerns

Transgenic plant creation summary

Clone gene into Ti plasmid → infect plant cells → gene integrates → grow modified plant

Main ethical GMO concerns

Safety, environmental impact, corporate control, labeling

Define recombinant DNA

DNA composed of sequences from different sources artificially combined

Define vector (genetic engineering)

DNA molecule that carries foreign DNA (e.g., plasmid)

Define cloning site

Region in plasmid with multiple restriction enzyme recognition sequences

Define antibiotic selection

Method to identify bacteria with plasmid by growing on antibiotic plates

Purpose of cDNA library

Allows identification of expressed genes without introns

Define hybridization

Formation of complementary base pairing between probe and target

Define expression vector

Plasmid engineered for strong gene expression in host cells

Define GMO

Organism genetically modified by insertion or deletion of DNA

Define gene knockout

Removing or deactivating a gene to study function

Define gene editing

Modifying DNA sequence using tools like CRISPR or mutagenesis

Define transformation efficiency

Number of cells successfully transformed per microgram of DNA

What makes restriction enzymes specific?

Recognize short palindromic sequences

Why sticky ends are useful

Increase likelihood of correct insertion during cloning

Why ligase is required

Forms phosphodiester bonds to stabilize recombinant DNA

Importance of reverse transcriptase

Allows cloning of eukaryotic genes in bacteria

Define probe hybridization

Probe binds complementary DNA sequence, producing detectable signal

Define Ti plasmid function

Transfers genes into plant genome

Advantages of CRISPR over older methods

Higher precision, cheap, easier design, fewer off-target effects

Ethical issue: GMOs

Concerns over biodiversity, contamination, food labeling, corporate patents

Ethical issue: recombinant hormone use

Performance enhancement, unfair advantage, unknown side effects

Whole-genome sequencing importance

Identifies genes, mutations, evolutionary relationships

Define palindromic sequence

DNA sequence that reads the same 5’→3’ on both strands

Define ligation

Joining DNA fragments by ligase