Flashcards in 3.8.4 rDNA technology, probes and genetic fingerprinting Deck (75)
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?
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')?
What is the name given to DNA which consists of genetic material from two different organisms?
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?
5. growth / cloning
When making a GMO explain what happens in stage 1. (isolation)
The gene is isolated using 1 of 2 methods:
1. reverse transcriptase is used to obtain DNA from mRNA
2. restriction endonuclease enzymes are used to cut the desired gene from the DNA
When making a GMO explain what happens in stage 2. (insertion)
RE's cut at specific recognition sites to make either blunt or sticky ends. Sticky ends are better as there are bases which will H bond the bases together, as well as the phosphate-sugar connection (made by DNA ligase enzyme). Promoter and terminator bases sequences are also added to initiate start and end of the gene being transcribed. The desired gene is then inserted into a vector (most commonly a plasmid) which has had its DNA cut with the same RE so that there is a palindrome.
When making a GMO explain what happens in stage 3. (transformation)
once the desired gene has been successfully incorporated into the vector it must be reintroduced to the host cell (e.g. plasmid into bacteria). Calcium ions and temperature changes are used to make the membrane permeable to allow the vector to pass into the cell.
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. (isolation)
marker genes are used to identify which vector has successfully taken up the desired gene, and which host cell has successfully taken up the vector. Markers used are anti-biotic resistance, enzyme and fluorescence.
How does stage 4 (identification) work with the anti-biotic resistance method?
-The cells that survived the first antibiotic have taken up the plasmid.
-Those cells are cultured by spreading them thinly on nutrient agar plates.
-Each cell on the plate will become a culture of clones.
-A small sample from each colony is transferred to another plats (the replica plate) and placed in the same position as on the original plate.
-The replica plate contains the second antibiotic against which the bacterial cells will have no resistance if they have the plasmid with the DNA fragment (as the resistance gene will have been removed).
-Colonies killed on the replica plate are the ones with the required gene.
-The same ones on the original plate are alive and can be cultures further.
How does stage 4 (identification) work with the enzyme method?
-the enzyme gene marker codes for the production of the protein enzyme lactase.
-Lactase turns a certain substance from clear to blue.
-the desired gene is placed in the centre of the lactase coding gene.
-If the plasmid with the required gene is present in the bacterial cells the colony grown from it will not produce lactase and therefore not cause the colour change to blue whilst the undesired cells will cause a colour change.
How does stage 4 (identification) work with the fluorescence method?
-A more recent and faster method is the transference from a jellyfish plasmid which produces a green fluorescent protein (GFP).
-The desired gene to be cloned is transferred to the centre of the GFP gene so any bacterial cells which have taken up the desired gene will not express the GFP so they will not fluoresce whilst the other cells without the desired gene will.
-There is no need for replica plating as the desired cells have not been harmed. All is required is to retain the cells which do not fluoresce by viewing them under a microscope.
Where is the reverse transcriptase used in obtaining a DNA fragment (stage 1. isolation) come from?
Retrovisuses are human viruses of which human immunodeficiency virus (HIV) is the best known.
They have RNA as their genetic information but they can synthesise DNA from the RNA using REVERSE TRANSCRIPTASE (RT) (an enzyme).
How does using reverse transcriptase to isolate a single gene work?
-A cell that produced the protein is selected.
(e.g. Beta-cells of the islets of Langerhans in the pancreas produce insulin)
-The mRNA is extracted from the cells.
-RT is used to make DNA from RNA. This is known as COMPLIMENTARY DNA (cDNA) as the DNA bases are complimentary to the RNA bases.
-To make the other strand of DNA the enzyme DNA POLYMERASE is used to build up the complimentary nucleotides on the cDNA template.
-The resulting double strand of DNA is the required gene!!!
Why do bacterial cells have restriction endonuclease enzymes naturally?
Bacteria are often invaded by viruses (bacteriophages) which inject foreign DNA into their cells and so the bacteria have enzymes which cut up the viral DNA. These are RESTRICTION ENDONUCLEASES (RE).
There are many different RE’s. Each cuts the DNA at a different sequence of bases called the recognition sequence.
What is another name for the recognition site that the restriction endonuclease cut at?
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