Genetic EngineeringRecombinant DNA (rDNA) TechnologyGenetic EngineeringRecombinant DNA (rDNA) Technology

• rDNA technology involves cloning DNA by cutting & pasting DNA from different sources

• Restriction enzymes & DNA ligases are important enzymes for this process

• DNA ligases join together adjacent DNA fragments

Genetically Modified Organisms (GMOs)

Genetically Modified Organisms (GMOs)

• GMOs are organisms that have had genetic material removed and/or inserted in order to change a particular trait or traits of the organism.

• The process is called gene splicing or genetic engineering

• Organisms produced by transplanting genetic materials between different types of organisms are called transgenic organisms.

Transgenic Organism ExamplesTransgenic Organism Examples

• Genes from bacteria are spliced into corn and cotton to make them less susceptible to insect damage

• Human growth hormone implanted into mice & other animals so that it can be harvested

• ANDi (first transgenic monkey) is a rhesus monkey carrying GFP protein, showing foreign gene can be inserted into primate chromosome

• May lead to primate models of human diseases

Restriction enzymesRestriction enzymes• Restriction enzymes are DNA-cutting enzymes that are found in bacteria

• They are also called endonucleases (cut within DNA sequences)

• Microbiologists from 1960s discovered that some bacteria are protected from destruction by viruses because they cut viral DNA, restricting viral replication

Restriction enzymes Q & ARestriction enzymes Q & A

In 1970, Hamilton Smith isolated HindIII (1st restriction enzyme well characterized and used for DNA cloning), which comes from Haemophilus influenzae.

They are named based on genus & species of bacteria it was isolated from. (EcoRI = Escherichia coli, RY13).

They cut DNA by cleaving phosphodiester bonds (in sugar-phosphate backbone) that join adjacent nucleotides

Which was the first one well understood?

How are they named?

How do they work?

SpecificitySpecificity

• Restriction enzymes show specificity for certain substrates (DNA in this case)

• They recognize, bind to, and cut DNA at specific sites called restriction sites (recognition site)

• Usually a 4-base pair or 6-base pair cutter

• Restriction sites are palindromes (reads same forward & backwards on opposite strands)

Restriction cutsRestriction cuts

• Some cut DNA to create fragments with overhanging single-stranded ends (sticky ends or cohesive ends), while others create fragments with non-overhanging ends (blunt ends)• Enzymes that create sticky ends are favored for cloning experiments since the DNA fragments can be easily joined together

• DNA from any source can be digested (as long as it has the specific restriction site)

GE ApplicationGE Application

• In 1972, Paul Berg joined DNA from E.coli and a primate virus called SV40

• He cut both with EcoRI (restriction enzyme)

• He then added fragments to tube with DNA ligase

• This became 1st recombinant DNA molecule

PlasmidsPlasmids

• Plasmid DNA is circular form of self-replicating DNA that scientists can manipulate to carry and clone other pieces of DNA

• Found primarily in bacteria

• Considered extrachromosomal DNA because they are present in addition to chromosomes

• They are small (~1000 - 1400 base pairs) in size

VectorsVectors

• Plasmids can be used as vectors (pieces of DNA that can accept, carry, and replicate other pieces of DNA)

• 1st plasmid vector pSC101

(SC = Stanley Cohen, pictured left)

• Contained gene for tetracycline (antibiotic) resistance and restriction sites for several enzymes

• rDNA animation

VectorsVectors

• Cohen & Boyer (pictured left) awarded patents (1980) for pSC101 and gene splicing & cloning technologies

• Major concern at the time was the thought of recombinant bacteria leaving the lab

• Boyer joined forces with Robert Swanson (venture capitalist) to create Genentech in an effort to commercialize these technologies

Vector FeaturesVector Features

Modern plasmid DNA cloning vectors usually consider 6 desirable features:

1. Size (must be small enough to separate easily)

2. Origin of replication (ori) - DNA sequence at which replication is initiated

3. Multiple cloning site (MCS) - a stretch of DNA with recognition sequences for common restriction enzymes (Engineered into plasmid so that digestion does not result in loss of DNA fragment)

Vector FeaturesVector Features

4. Selectable marker genes - allow for selection and identification of transformed bacteria• Most common selectable markers are antibiotic resistance.

• Lac z gene widely used (gene of interest inserted within lac z gene)

• Plated on X-gal (substrate similar to lactose but turns blue when cleaved by ß-gal); so, recombinant bacteria turn blue & nonrecombinant are white

SelectionSelection

• Selection is a screening process designed to facilitate the identification of recombinant bacteria while preventing growth of nontransformed bacteria (or those containing plasmid without foreign DNA)

• Blue-white screening is becoming more popular (uses ß-galactosidase)

Antibiotic selectionAntibiotic selection

• Antibiotic selection uses a plasmid vector with genes encoding resistance to 2 different antibiotics, usually ampicillin (ampR) and tetracycline (tetR)

• Foreign DNA inserted into one of the 2 antibiotic resistance genes (disrupts gene - preventing protein)

• Transformed cells are plated to an agar plate with no antibiotic or plate with one (ampicillin)

Replica platingReplica plating• Replica plating uses sterile pads pressed against colonies on plate (cells adhere to make an exact copy)

• Then pad is placed on 2nd replica plate containing 2nd antibiotic (tetracycline)

• Nontransformed bacteria cannot grow in presence of either antibiotic without plasmid

• Compare plates since recombinant can’t grow on 2nd plate

Replica plating diagramReplica plating diagram

Vector FeaturesVector Features

5. RNA polymerase promoter sequences - place where RNA polymerase binds to begin transcription

6. DNA sequencing primer sequences - known sequence that allows sequencing of cloned DNA fragments that have been inserted into the plasmid

Types of VectorsTypes of Vectors

One primary limitation of bacterial plasmids as vectors is the size of DNA fragments (usually cannot exceed 6-7kb: 6000-7000 base pairs).

• Bacteriophage vectors

• Expression vectors

• Bacterial artificial chromosomes (BACs)

• Yeast artificial chromosomes (YACs)

• Tumor-inducing (Ti) vectors

Gene TransferGene Transfer• Cohen discovered that plasmid DNA enters a bacterial cell (transformation) treated with calcium chloride, chilled on ice, then briefly heated

• A more recent transformation technique is electroporation (brief pulse of high-voltage electricity to create tiny holes in bacterial cell wall allowing DNA to enter)

• Cells that have been treated for transformation (so they are more receptive to take up DNA) are called competent cells

BiolisticsBiolistics• Sometimes, biolistics are used in order to have foreign DNA enter a cell

• DNA is blasted into the cell using tiny bullets composed of tungsten or gold particles with DNA attached

• Done with a gene gun (aka bioblaster)

• Can be used on bacteria, yeasts, & mammalian cell lines

National Institutes of Health (NIH)

National Institutes of Health (NIH)

• Concerns arose because of new techniques

• In 1975, NIH formed the Recombinant DNA Advisory Committee (RAC) to evaluate risks and establish guidelines for rDNA technology