Flashcards in 16. DNA Technology Deck (79)
Explain how enzymes and vectors can be used to isolate genes and insert them into another organism.
-Cut DNA at specific base sequences
-Same (restriction) enzyme also cuts DNA into which gene is inserted/plasmid/virus/Agrobacterium
-Joins two pieces of DNA together/ forms recombinant DNA
-Vector needed to insert DNA into host/plasmid/second organism
-Correct ref to sticky ends; reverse transcriptase; mRNA-->DNA
Describe the similarities and differences between replication and transcription.
-H bonds break/ DNA unzips
-Between complimentary bases
-Complimentary nucleotides/ bases added/ DNA acts as template
-Same, correctly named, enzymes eg polymerase
-Uracil/ thymine used
-All copied or only section copied respectively
-One strand used in transcription, two in replication
-Enzymes that are different, correctly named
Describe how the new protein is made once the gene has been inserted into the cell.
-Unwinding/ unzipping DNA
-Involving breaking hydrogen bonds
-Assembly of mRNA nucleotides
-Complementary base pairing
-Role of polymerase enzymes
-mRNA enters ribosome
-Specific tRNA molecule associated with specific amino acid
-Formation of peptide bonds
-Specific role of ATP/ energy
-Reference to gene switched on
What is DNA from two different organisms combined called?
What are the five stages involved in making a protein using the DNA technology of gene transfer and cloning?
1) isolation - DNA fragments that have the gene for the desired protein are isolated
2) insertion - fragment of DNA inserted into a vector
3) transformation - transfer of DNA into suitable host cells
4) identification - see if host cells have successfully taken up the gene by using GENE MARKERS
5) growth/cloning - cloning of population of host cells
What is a retrovirus and in what form is its genetic material? What enzyme can we use?
A retrovirus is a group of viruses, a notable example is HIV, which have their genetic information stored as RNA.
Therefore we can use reverse transcriptase to form DNA from RNA
Give an example to explain how a specific protein can be harvested using reverse transcriptase
Cell that readily produces the protein is selected (eg if you want insulin then use beta cells in the pancreas)
They will produce lots of the relevant mRNA which is extracted
Reverse transcriptase is used to make the mRNA into DNA (called cDNA, complementary DNA)
DNA polymerase is used to build up complementary nucleotides on the cDNA template- the double strand of DNA is the required gene
What is the DNA formed from RNA using reverse transcriptase called?
cDNA, complimentary DNA
What does a restriction endonuclease do?
It recognises DNA at a specific sequence of bases and cuts it.
These sequences occur in the DNA of all species of organisms, but not in the same places!
What is a natural use of restriction endonucleases?
Some virus's inject their DNA into bacteria in order to take over the cell. Some bacteria defend themselves by producing enzymes that cut up the viral DNA- restriction endonucleases!
What are blunt ends and what are sticky ends?
Blunt ends occur when a restriction endonuclease cuts at a recognition site and cuts between two opposite base pairs.
Sticky ends occur when restriction endonucleases cut the DNA in a staggered fashion, leaving each end of the strand of DNA with exposed, unpaired bases.
What is a typical feature of sticky ends when cut by restriction endonucleases?
Sometimes the recognition sequence is a, for example, a six base pair palindromic sequence
Palindromic sequences are typical because
What are the two ways of cloning fragments of DNA that have been obtained?
-in vivo- transferring the fragments into a host cell using a vector
-in vitro- using the polymerase chain reaction
What is the importance of sticky ends?
It means DNA can be inserted into, for example, a plasmid because the same restriction endonuclease is used to cut both DNAs (the recognition site is the same as you use the same R.E) so the nucleotides of one sticky end are complimentary to the place in the host DNA where you want to insert it
How is the sugar phosphate backbone joined once the sticky ends have formed complimentary base pairing?
What is a plasmid and why is it useful?
They are circular lengths of DNA, separate from the main bacterial DNA
They almost always contain genes for antibiotic resistance so restriction endonucleases can be used at one of these genes to brisk the plasmid loop
They are useful as vectors
What is a vector?
Something that carries DNA into a host cell
Describe the process of transformation (getting a plasmid with the gene into the host cell, bacteria)
Mixture of plasmids and bacterial cells are mixed together in a medium containing calcium ions and heated.
Calcium ions and increased temperature make the bacteria permeable, allowing the plasmids to pass through the cell membranes.
After the process of transformation, why do not all of the bacteria contain the DNA fragments?
-only a few bacterial cells (as few as one percent) take up the plasmids when they are mixed together
-some plasmids will have closed up again without incorporating the DNA fragment
How do you identify which bacterial cells have taken up the plasmid with the inserted gene?
-use replica plating (antibiotic resistance gene markers)
-put bacterial colonies on plates with different antibiotics that you know the plasmid with the inserted gene has resistance against
-colonies that grow on the plates with the antibiotic have the plasmid because they have antibiotic resistance, those that die are the ones that did not absorb a plasmid
How do you identify which bacterial cells have taken up plasmids
-with no extra DNA
-with the gene successfully inserted?
>antibiotic resistance marker
Describe how antibiotic resistance markers work. (Plasmids that have taken up genes will have non functional tetracycline resistance gene)
-use replica plating
-bacterial cells that survived the ampicillin antibiotic are known to have taken up the plasmid
-these cells are cultured by spreading them thinly on nutrient agar plates
-each separate cell will grow into a genetically identical colony
-tiny sample of each colony is transferred onto a second (replica) plate in the same position as the original colonies on the plate
-second replica plate contains the second antibiotic, tetracycline, against which the antibiotic resistance gene will have been made useless if the new gene has been taken up
-colonies killed by the antibiotic must be the ones that have taken up the required gene- these colonies therefore are made up of bacteria that has been transformed (has been genetically modified)
Describe how fluorescent markers are used to identify whether or not the plasmid has been taken up by host.
-transfer jellyfish dna for the production of a green fluorescent protein (GFP) into the plasmid
- the gene to be cloned is put in the middle of the GFP gene, meaning that those cells that have taken up the gene will not flouresce because the gene will have been inserted into the middle of the GFP gene, making it non functional
-you can therefore find the cells that have the desired gene by looking under a microscope and seeing which cells do not fluoresce.
Describe how enzyme markers are used to find out if cells have taken up the plasmid with the desired gene. give an example.
-one example of a gene marker is lactase which turns a particular colourless substrate blue.
-required gene is transplanted into the gene that makes lactase
-if the gene is inserted correctly then no blue colour will be formed as lactase cannot be produces as the gene has broken the lactase gene
-blue colour will appear if the plasmid taken up has not had the desired DNA inserted into it
What five things does the polymerase chain reaction require?
-the dna fragment to be copied
-dna polymerase (obtained from bacteria in hot springs so it is not denatured by the heat)
-primers- short sequences of nucleotides with sets of bases complimentary to one end of each of the two dna fragments
-nucleotides - which contain each of the 4 bases found in DNA
-thermocycler- a machine that varies temperature precisely over time
Describe the series of events that occur in the polymerase chain reaction.
-dna strands heated to SEPARATE the two strands (95degrees celsius)
-PRIMERS are annealed- the mixture is cooled to 55 degrees celsius which causes the primers to anneal. The primers provide the starting sequences for DNA polymerase
>primers provide the starting sequence for dna polymerase to begin dna copying because dna polmerase can only attach nucleotides to the end of an existing chain
>primers also prevent the two separate dna strands from rejoining
-SYNTHESIS of dna- temperature increased to 72 degrees so that dna polymerase can add complementary nucleotides to each of the separate dna strands
What are the advantages of in vitro gene cloning?
-it is extremely rapid and can be used even when there is very little genetic material available
-does not require living cells- no complex culturing techniques which require lots of time and effort
What are the advantages of in vivo cloning?
- useful when we want to introduce a gene into another organism
-involves almost no risk of contamination- contaminant dna will not be inserted into the plasmid because as they are cut with a certain restriction endonuclease, the sticky ends are complimentary with the desired gene
-it is very accurate- mutations are rare however in in vitro cloning errors in copying dna can be copied may times
-it cuts out specific genes- not just copies of whole dna samples
-produces large quantities of transformed bacteria that can be used to produce large quantities of gene products eg insulin.
How can genetic modification benefit humans?
-increasing yield from animals or plants
-improving nutrient content of food
-introducing resistance to disease and pests
-making crop plants tolerant to herbicides
-developing tolerance to environmental conditions eg extreme temperatures and drought
-producing medicines for treating disease