UNIT 8 – DNA Technology
Warm-up Questions / What is Chargaff’s Rule?What are the three parts of a nucleotide?
What is the significance of Purines and Pyrimidines?
What is DNA’s backbone made of?
/ Remember DNA…
- Contains genetic info (heredity)
- Made of 4 ______(ATGC)
- Purines & Pyrimidines
- Double Helix
- ______Strands
- Codes for ______
Genetic Engineering
AKA: Gene Modification; the manipulation of an organism’s ______by use of ______.
Traditional (older) methods of gene modification include:
Inbreeding and ______breeding
DNA Technology Overview
- Isolation – of the DNA containing the targeted gene.
- Insertion – of the DNA into a vector.
- Transformation – Transfer of DNA into a suitable host.
- Identification – finding host organisms containing the vector and DNA (by use of gene markers).
- Growth/Cloning – of successful host cells.
Stage 1 –Producing DNA Fragments / How is complementary DNA made using reverse transcriptase?
How are restriction endonucleases used to cut DNA into fragments?
Reverse Transcriptase
A group of viruses called ______(e.g. HIV) contain an enzyme called reverse transcriptase.
It is used to turn viral RNA into ______so that it can be
transcribed by the host cell into ______.
Reverse Transcriptase makes DNA from an RNA template – it does the ______of transcription.
Using Reverse Transcriptase
You ______a gene that you want.
______mRNA from a cell that is making the gene you want.
A single-stranded complimentary______of DNA (cDNA) is
made using reverse transcriptase on the mRNA template.
______-stranded cDNA is used to make double stranded
DNA using DNA polymerase.
This forms a double stranded copy of the gene you wanted.
/ Restriction Enzymes
Bacteria contain restriction enzymes which help to ______themselves from invading viruses.
Restriction enzymes ______up viral DNA at specific sites – this property can be useful in gene technology.
There are 2 ways that restriction enzymes make cuts.
- Blunt Ends
- Sticky Ends
Blunt Ends
Some restriction enzymes cut ______across both DNA chains, this forms what are called blunt ends.
Sticky Ends
Some restriction enzymes make a ______cut in the two DNA chains, forming sticky ends (uneven).
Sticky ends have a strand of single stranded DNA on one side which are ______to another single strand.
They will join with another sticky end only if it has been cut with the ______restriction enzyme.
Meaning, that its letters will match up with the other side.
/ Restriction Enzymes
Are also know as ______.
- Hint: all enzymes end in “ase”.
Ex: GAATTC and the complement CTTAAG
Warm-up Questions / What type of virus can perform transcription backwards?
What type of molecule ends in “-ase”?
What is the difference between blunt and sticky ends?
Stage 2 –
Insertion into a Vector / What is the importance of “sticky ends”?
How can a DNA fragment be inserted into a vector?
The Improtance of Sticky-Ends
DNA from different source can be joined together IF they have the same ______ends = the same recognition site.
- In order to have the same sticky ends they must have been cut using the ______restriction endonuclease.
The new DNA molecule is now called ______DNA, because it was made by re-combining different DNA together.
Insertion of DNA into a Vector
VECTOR = something used for ______.
PLASMID = the vector used to transport ______in DNA.
A plasmid is a circular piece of DNA found in bacteria (______the chromosome). Plasmids are useful because they contain antibiotic resistance genes.
The Bacteria Plasmid
Sometime an antibiotic ______gene is broken/cut-into when the restriction enzyme cuts open the plasmid ______to make room for inserting the targeted gene. There are many other antibiotic resistant genes that are not cut, they stay intact and are used to ______select the correct host cells that the plasmid will be transferred into.
If you are using a plasmid to make an antibiotic (penicillin for example) then you don’t want to put that plasmid into a cell that will be killed by penicillin, you want to put it into a cell that is resistant to penicillin (antibiotic resistant). Then the cell will produce the antibiotic and ______die. The penicillin can be harvested and used to help someone in need of that medicine.
Insertion into Plasmids
1- ______the gene you want out of the DNA
2- Cut the matching sticky-end area out of a bacterial ______
3- Use DNA Ligase to______the DNA gene into the bacterial plasmid
4- A ______plasmid is made! It contains a new gene!
5- The bacteria makes whatever the new gene coded for.
Warm-up Questions / What type of virus can perform transcription backwards?
What type of molecule ends in “-ase”?
What is the difference between blunt and sticky ends?
What is the molecular glue that binds DNA back together?
Stages 3 & 4 – Transformation & Identification / How is the DNA of the vector introduced into host cells?
What are gene markers and how do they work?
Introduction of DNA into host cells “Transformation” (stage 3)
The DNA plasmid that was removed from a bacteria (and altered) must be put into the new host cell.
- This process is called ______.
- A transformed/hybrid cell has been made.
Identification (stage 4) of bacteria containing the plasmid
Only about 0.001% of bacterial cells ______(take in) these altered plasmids when the two are mixed together.
Because very few transform, we must ______the bacteria containing the altered plasmids – we do this by ______the bacteria on a medium containing an antibiotic.
- (Medium = growth substance on a plate).
Gene markers are used to ______which plasmids have taken up the new gene (DNA).Gene markers can be:
- Resistance to an antibiotic
- A fluorescent ______
Gene marker off = yes, has new gene.
Gene marker working = no new gene.
Flourescent Markers
The gene from ______which produces Green Fluorescent Protein (GFP) has been incorporated into many plasmids.
If the new DNA fragment has been inserted into the GFP gene, the bacterial will not ______and can easily be identified.
- These non-glowing bacteria will be used to make whatever the newly inserted gene is for. Ex: making insulin or penicillin.
If the DNA fragment has not been inserted into the GFP gene, the bacteria will glow and would ______be used.
Antibiotic-Resitance Markers
The second Gene Marker is an antibiotic-resistance ______(ex: resistance to ampicillin) is used to identify plasmids with a ______DNA fragment in them.
If the DNA fragment has been inserted into the ampicillin resistance gene it will no longer grow on medium containing ampicillin. In order to identify these bacteria we use a process called______
The ampicillin resistance gene is disrupted when the restriction enzymes ______open the plasmid and adds in the new gene.
So if a bacteria is no longer resistant to this antibiotic and ______, we know it also contained the newly inserted gene.
Replica Plating
Replica Plating is the process of ______which bacteria have or do not have resistance to certain antibiotics.
Here’s how its done:
- Bacteria that were mixed with pasmids ______altered genes are grown on a medium plate that also has antibiotics mixed in.
The bacteria that lived do not have the new gene, because their antibiotic resistant gene is still working.
Warm-up Questions / What makes a plasmid “recombinant”?
List the 2 types of gene markers.
What is Replica Plating used to find?
Stage 5 - Cloning / What are the two types of cloning?
What process can clones hundreds of plasmids?
What machine is used to analyze plasmid production?
Clonging the Bacteria
Following successful identification of the bacteria containing the plasmid ______the DNA fragment, the bacteria are ______.
- As the bacteria are cloned, so is the ______containing the new DNA fragment.
- (Remember, this process is called Transcription!)
/ Type 2 – In Vitro DNA Cloning
This process uses the Polymerase Chain Reaction (______).
The following ingredients are needed for a PCR:
DNA Fragment: serves as a template/model to be ______
DNA Polymerase: to carry out ______(adding nucleotides)
Primers: RNA to initiate synthesis,______the DNA to be copied
DNA Nucleotides: match up between DNA and RNA primer
Thermocycler: machine to do reaction, and to check the progress
Uses of Recombinant DNA Technology
Genetic Modification (GM): Transgenic ______
Goal of GM is to increase:
- Disease resistance
- Fast growing animals
- Herbicide resistance
- Plant crops increase yields
- Increased nutrient content
- More medicine made at a time
Genetically Modified Organisms (GMO)
Examples helpful of GMO microorganisms:
- Antibiotics: produced quickly by ______
- Enzymes: ______industry (ex: lipase for cheese making)
- Hormones: insulin is made by ______
GM plants include:
Soybeans Canola Alfalfa
Corn Tomato Cotton
Wheat Squash Rice
Beets Papaya Potato
Beneficial Gene Combinations
Herbicide Resistance: crops ______killed when herbicide (weed killer) is sprayed. Lower cost to farmer. Less traffic in fields so soil is not compacted and time is not wasted.
- Pest Resistance: ______from insect attacks.
Plants that produce plastic: plants produce ______used for production of biofuels.
(GM) Transgenic Animals
Transgenic means ______genes were transferred into an animals DNA.
Sheep – genes to growing more wool
Cows – genes to produce ______in their milk
Goats – genes to producing spider silk in their milk
/ Why are these modifications an advantage?
1. Agriculture: GM improves animals ______than selective breeding alone. Allows for more exact changes in traits.
2. Medicine: xenotransplantation (ex: human gets a pig heart transplant). Nutritional supplements by food/milk.
3. Industry: goats producing spider silk – can be extracted from milk then woven together to make ______/rope (ex: military uniforms).
How do you clone an animal?
Reproductive cloning uses a technique called: Somatic Cell Nuclear Transfer.
- Take a somatic cell (any cell in the ______other than a sperm or egg) from the animal you plan to clone.
- Extract the cell's ______, which contains the cell's DNA, and discard the rest of the somatic cell.
- Then remove the nucleus of an egg cell (from a ______animal) and insert the somatic cell’s nucleus in its place.
- Treat the reconstructed egg with chemicals/electricity to ______cell division. If the egg divides normally and forms a blastocyst (a small clump of cells that form after an egg is fertilized), scientists will ______it into a surrogate mother to grow and develop into a new animal.
Any animal created using this technique is not actually an identical clone of the donor animal. Only the clone's ______DNA is the same as the donor. A small portion of the clone's genetic information comes from the egg cell's ______(small organelles that help make energy the cell).
- Dolly the sheep was the first______ever cloned (1996 in Scotland).