3.8.4.1 Recombinant DNA Technology (C21) Flashcards
What is meant by the term ‘recombinant DNA technology’?
transferring DNA fragments from one organism or species to another.
Why does recombinant DNA technology work so well?
the genetic code is universal as are the processes transcription and translation mechanisms.
What does universal mean?
all organisms have the same amino acid coded for by the same DNA codon (triplet code).
How are DNA fragments obtained?
- conversion of mRNA to DNA (cDNA) using reverse transcriptase.
- cut out of DNA using restriction enzymes.
- creating a new gene in a ‘gene macnine’.
Via which two methods can DNA be amplified?
in vivo and in vitro
What does in vivo mean?
carried out inside a living body
What does in vitro mean?
carried out outside of a living body e.g. in a test tube
What does PCR stand for?
polymerase chain reaction
Is PCR in vivo or in vitro?
in vitro
When obtaining DNA fragments using restriction enzymes what must be added to the start and end of the desired gene?
promoter and terminator regions
When obtaining DNA fragments using restriction enzymes what is the name of the item that transports the desired gene into the host cell?
vector (plasmids for bacterial host cells)
When obtaining DNA fragments using restriction enzymes what enzyme must be used to bind the phosphate-sugar framework of the desired gene into the DNA (preferable with ‘sticky ends’)?
DNA ligase
What is the name given to DNA which consists of genetic material from two different organisms?
recombinant DNA
What is the name given to an organism which contains genetic material from two different organisms?
GMO (genetically modified organism) and transgenic as it has genetic information from more than one sources.
What are the stages involved in making a GMO?
- isolation
- insertion
- transformation
- identification
- growth / cloning
When making a GMO explain what happens in stage 1. (isolation)
The gene is isolated using 1 of 2 methods:
- reverse transcriptase is used to obtain DNA from mRNA
- restriction endonuclease enzymes are used to cut the desired gene from the DNA
When making a GMO explain what happens in stage 2. (insertion)
- DNA fragment inserted into vector DNA (normally plasmids)
- Vector DNA is cut open using same restriction endonuclease that was used to isolate DNA fragment of the target gene (so sticky ends are complementory)
- Vector DNA + DNA fragment are mixed together with DNA ligase - which joins the sticky ends of DNA fragment to sticky ends of vector DNA
- The new combination of bases in DNA is the recombinant DNA
When making a GMO explain what happens in stage 3. (transformation)
- Vector with recombinant DNA used to transfer the gene into host cell
- If plasmid vector is used, host cells has to be persuaded to take in plasmid vector + its DNA
- If use bacteriophage it will infect the host bacterium by injecting DNA into it
Why are all of the vectors not successfully taken up by the host cell?
- only some bacterial cells take up the recombination DNA (e.g bacterial cells taking up the plasmid).
- some vectors (e.g plasmids) have not taken up the desired gene.
- sometimes multiple fragments join together to form their own plsmids
When making a GMO explain what happens in stage 4. (identification)
- Marker genes are inserted into vectors whilst the gene is being cloned
- Host cells grown on agar plates - each cell divides + replicates its DNA - creates a colony of cloned cells (all containing the cloned gene + marker gene)
- Marker gene can code for antibiotic resistance - host cells are grown on agar plates containing specific antibiotic so transformed cells have the marker gene will survive + grow
- Identifed transformed cells are allowed to grow more - producing lots of copies of cloned genes
How does stage 4 (identification) work with the fluorescence method?
- Jellyfish plasmid is used which produces a green fluorescent protein (GFP).
- Marker gene can code for fluoresence
- When agar plate placed under UV light only transformed cells will glow
Where is the reverse transcriptase used in obtaining a DNA fragment (stage 1. isolation) come from?
Retroviruses such as HIV
Describe the use of promoter + terminator regions
- They are inserted into vector if you want the transformed host cells to produce the protein coded for by the DNA fragment
- Promotor regions: DNA sequences that tell enzyme RNA polymerase when to start producing mRNA
- Terminator regions: tell it when to stop
- If the correct promotor isn’t added then the DNA fragment won’t be transcribed
Describe how you use reverse transcriptase to isolate a gene?
- mRNA is isolated from cells
- Mixed with free DNA nucleotides + reverse transcriptase
- Enzyme uses mRNA as template to synthesis new DNA called cDNA (complementory DNA)
Why do bacterial cells have restriction endonuclease enzymes naturally?
- Often invaded by viruses (bacteriophages) which inject foreign DNA into their cells
- So the bacteria have the enzymes so they can cut up the viral DNA
What is another name for the recognition site that the restriction endonuclease cut at?
cleavage site
Once all of the stages for producing a GMO with recombinant DNA have been completed, there is only one thing left to do. Grow or clone the new organisms. If your GMO is a bacteria what must you do to ensure maximum growth?
Ensure the conditions for bacterial growth are optimum (e.g. suitable temperature, water availability, Oxygen and a nutrient source).
What is ‘nick name’ for the machine used to create genes in the laboratory?
the gene machine
Before we started to use gene technology to create new organisms, how did breeders and farmers alter their livestock?
selective breeding over many generations.
Give some uses of DNA technology
Examples:
* Increased yield of farm animals and crops.
* Improved nutrient content of food.
* Introducing disease and pest resistance to animals and plants.
* Making crop plants herbicide tolerant.
* Developing tolerance to environmental conditions.
* Making vaccines.
* Producing medicines and treatment of disease.
What items/apparatus are needed to perform PCR?
- The DNA fragment to be copied.
- DNA polymerase to join together the nucleotides in a hot environment.
- Primers with short, set nucleotides complementary to those on the ends of the fragment to join to the ends of the DNA fragment. 15-20 bases long.
- Nucleotides of all ATC and G to bind to complementary bases.
- Thermocycler to control temperature changes during PCR.
What temperature is needed to separate the DNA strands in PCR?
Heat the contents of the thermocycler to 95°C to separate the DNA strands.
What temperature is needed to for primers to anneal in PCR?
Cool the thermocycler contents to 55°C. This causes the annealing primers to attach (anneal) to the start and end complementary bases on the DNA strands. (this also keeps the strands apart).
What temperature is needed for DNA to bind new nucleotides in PCR?
Heat up to 72°C. This is the optimum temperature for DNA polymerase to cause new nucleotides to bind to their complementary base pairs.
Why do we use PCR?
PCR is the basis of Genetic Fingerprinting, where a sample form a known source can be compared against a sample.
It allows even a tiny sample of skin, blood, a hair follicle or semen to contain enough DNA to be amplified in PCR.
Why is a thermocycler used in PCR?
Temperatures to be controlled and timed accurately
Where do we get thermostable enzymes from to use in PCR?
thermostable enzymes are from thermophilic bacteria (e.g. Thermus aquaticus) which survive in hot springs which join nucleotides together.
What are the advantages of in vivo coning?
- Can add a gene to another organism via vectors and plasmids.
- Low risk of contamination as the same RE is used to cut the same codes for all DNA involved so no stray DNA is present.
- Only desired genes are cut out.
- Produces transformed bacteria to produce many products (e.g. insulin).
- No prior knowledge of bases sequences is needed.
What are the advantages of in vitro coning?
- Quick. Can make billions of copies from a small sample in a few hours.
- No living cells are needed.
What are the disadvantages of in vitro coning?
- Cannot add genes to other organisms.
- A very pure sample is needed as contaminants can also be multiplied giving false results.
- The whole DNA fragments are copied.
- Only magnifies DNA sample.
- Prior knowledge is needed to synthesise primers.
What are the disadvantages of in vivo coning?
- Takes days or weeks to make billions of copies from a small sample.
- Requires living cells and growing cultures.
What are primers and how are they used in PCR?
- Short pieces of DNA with set bases complementary to those on DNA sample
- They attach to the start and end of the DNA to provide start and stop instructions for PCR
- DNA polymerase can only attach nucleotides at the end of an existing chain.
- Primers also keep the 2 strands apart.
Why are 2 different primers needed in PCR?
The sequences at the 2 ends of the strand are different
What bond is broken when DNA strands are separated in PCR?
H Bonds
What could happen if contaminant biological material exists in the sample?
Any contaminant would also be copied.
role of DNA/Taq polymerase
joins nucleotides together in DNA (complementary strand)
name 2 enzymes involved in inserting DNA fragments into plasmids
ligase
restriction endonucleases
describe the role of ligase
joins gene/DNA into the plasmid/vector
describe the role of restriction endonuclease when used to add a piece of DNA into a plasmid
cuts the plasmid
What is reverse transcriptase
Enzyme from HIV, it converts RNA into DNA
How can reverse transcriptase be used in genetic engineering?
Mature mRNA is extracted and converted to: cDNA by Reverse Transcriptase
cDNA is converted to double stranded DNA by DNA polymerase
What is an advantage of using reverse transcriptase in genetic engineering?
- mRNA is found in large amounts in cells
- mRNA contains no introns, so the gene products are easily expressed even by bacteria
How is a gene machine used?
- Desired gene sequence is designed using a computer – it is checked for safety and standards.
- Small pieces of DNA (oligonucleotides) are synthesised and joined together to make a sequence of DNA
- Using sticky ends these pieces of DNA can be inserted into a vector that can be used in cloning.
What is the advantage of using a gene machine?
Gene machines are quicker as they don’t need to isolate DNA/mRNA first
Describe the whole PCR cycle
- DNA heated to 90 to 95°C to separate the strands
- DNA is cooled to 55°C so that primers can bind
- Free nucleotides attach by complementary base pairing
- Temperature is increased to 72°C and DNA polymerase joins nucleotides together forming a phosphodiester bond
- Cycle is repeated and DNA is copied at an exponential rate
Why will the PCR reaction eventually plateau/stop
The cycle will eventually stop when the nucleotides and primers run out.
Why is Taq polymerase used in PCR
Taq polymerase is thermostable, it works at high temperatures and doesn’t denature.
What are the applications of PCR
PCR can be used in Crime scene analysis and genotyping (and lots more)
Describe Gel Electrophoresis
- DNA is cut at areas of tandem repeats using restriction endonucleases
- DNA fragments are placed in wells at the top of an agar gel.
- An electric current is applied over it.
- DNA is negatively charged due to the phosphate group
- The DNA moves towards the positive electrode, but at different rates.
- Small fragments move further through the gel
- A ladder/marker can be used to determine the size of the DNA fragments
Describe the process of genetic fingerprinting
- DNA isolated
- Sample replicated using PCR
- PCR products are run on electrophoresis gel
- Genetic fingerprints compared to see if they match
What cells in an animal should you genetically modify?
Early-stage embryos, as the gene will be present in most cells in the organism
Allows all cells to make the protein
Where do you insert the gene and why?
Insert a promoter into genes that are produced and easily extracted e.g. milk
Extracting proteins from other places may harm the organism
Expressing proteins in all cells may cause harm
