Flashcards in Genetic Technology (Chapter 19) Deck (85)
What is the aim of genetic engineering?
To remove a gene(s) from one organism and transfer it to another so that the gene is expressed in its new host
What is DNA that has been altered by genetic engineering called?
What is recombinant DNA (rDNA)?
DNA made by joining pieces from two or more different sources
What is a genetically modified organism(GMO)/transgenic organism?
The organism which expresses the new gene(s)
What does genetic engineering provide?
A way of overcoming barriers to gene transfer between species
What are the stages of gene transfer?
1) the required gene is identified - may be cut from a chromosome/made by mRNA by reverse transcription/synthesised from nucleotides
2) multiple copies of the gene are made using PCR
3) the gene is inserted into a vector (e.g. plasmid) which delivers the gene to the cells of the organism
4) the vector takes the gene into the cells
5) the cells that have the new gene are identified and cloned
What are the 3 tools needed for genetic engineering?
1) enzymes - restriction endonucleases/reverse transcriptase/ligases
2) vectors - plasmids/viruses
3) genes coding for easily identifiable substances that can be used as markers
What are restriction endonucleases (REs)?
A class of enzymes which recognise and break down the DNA of bacteriophages (viruses) by cutting the sugar-phosphate backbone of DNA at specific places within the molecule
What is the role of REs in bacteria?
To resist a viral infection
How do REs work?
1) they bind to a specific target/restriction site (a sequence of bases) on DNA and cut at that site
2) they either cut straight across the sugar-phosphate backbone to give blunt ends or cut in a staggered fashion to give sticky ends
What are many REs?
How is bacterial DNA protected from REs?
By chemical markers or by not have having the target site
What are sticky ends?
Short lengths of unpaired bases which can easily form H-bonds with complementary sequences of bases on other pieces of DNA with the same restriction enzyme
Why is gel electrophoresis needed to find a specific piece of DNA?
Because when long pieces of DNA are cut with a restriction enzyme, there will be a mixture of different lengths
What is a plasmid?
A small, circular piece of double-stranded DNA
What are 4 characteristics of plasmids?
1) they are used as vectors
2) they occur naturally in bacteria
3) they can be exchanged between different bacteria
4) they often contain genes for antibiotic resistance
How are plasmids obtained from bacteria?
1) the bacteria containing them are treated with enzymes to break down their cell walls
2) the 'naked' bacteria are then spun at a high speed in a centrifuge so that the relatively large bacterial chromosomes are separated from the much smaller plasmid
How is a gene inserted into a plasmid?
1) the circular DNA of the plasmid is cut open with a restriction enzyme
2) opened plasmids and lengths of DNA are mixed together
3) some of the plasmid sticky ends pair up with the sticky ends on the new gene
4) DNA ligase is used to link together the sugar-phosphate backbone of the DNA molecule and the plasmid, producing a closed circle of double-stranded DNA, containing the new gene (recombinant DNA)
Why does the same restriction enzyme need to be used to cut the gene and the plasmid?
So that the sticky ends are complementary
What has to be done if the restriction enzyme used gives blunt ends?
The sticky ends need to be attached to both the gene and the plasmid DNA
What characteristics of plasmids make them good vectors or are modified to make them good vectors?
1) they have a low Mr - are readily taken up by bacteria
2) they have an origin of replication - can be copied
3) they have several single target sites for different REs in a short length of DNA called a polylinker
4) they have one or more marker genes, allowing identification. Of cells that have taken up the plasmid
How else can plasmids be obtained?
They can be made artificially
How are recombinant plasmids inserted into bacteria?
The bacteria are treated by putting them into a solution with a high [Ca2+], then cooled and then given a heat shock to increase the chances of plasmids passing through the cell surface membrane
What happens to the bacteria after they are treated with the plasmids?
Roughly 1% of the bacteria take up the plasmids with the desired gene (are transformed) and the rest take up plasmid without the gene or no plasmid
How are the bacteria containing the recombinant DNA identified? (old method)
1) spread the bacteria on agar plates, each containing an antibiotic
2) any plasmid with a gene won't be able to grow bc the plasmid contains antibiotic resistance genes
3) ∴ only bacteria with the plasmids survive
4) to identify recombinant bacteria, use a replica plate containing agar with second antibiotic
5) the bacteria containing the desired gene will not grow as the 2nd resistance gene has been inactivated by the insertion of the new gene
6) ∴ identify the colonies on the first plate that did not grow on the second plate
What happens once the bacteria with the recombinant DNA are identified?
1) DNA polymerase copies the plasmids
2) the bacteria divide by binary fission so that each daughter cell has several copies of the plasmid
3) the bacteria transcribe the new gene and may translate it to give the required gene product e.g. insulin
What is the concern with using the antibiotic resistance gene as a marker?
The gene could spread to other bacteria, leading to untreatable diseases caused by pathogenic bacteria
What are two alternatives to using the antibiotic resistance gene as a genetic marker?
1) enzymes that produce fluorescent substances e.g. GFP
2) enzyme beta-glucuronidase (GUS) from E.coli
How can the enzyme which makes the protein GFP (green fluorescent protein) be used as a genetic marker?
1) the gene for the enzyme is inserted into the plasmid
2) shine UV light on the bacteria
3) the bacteria which glow green are the bacteria which have taken up the recombinant plasmid