| What is a gene? | - Section of DNA that code for specific polypeptides and RNA |
| Genetic modification | - Process of inserting a gene into the genetic makeup of another organism |
| Transgenic organism | - Organism that has had genes inserted in order to make specific polypeptides |
| Reverse transcriptase | - An enzyme that carries out transcription in reverse
- Transcription = DNA to mRNA
- Reverse transcription = mRNA to DNA |
| Restriction enzyme | - An enzyme that digests DNA
- They obtained from bacteria that use them to defend against viruses
- They recognise palindromic(can read it either way is same) sequences of 4-6 bases
- Make blunt or sticky ends |
| Blunt ends | - Only cut phosphodiester bonds |
| Sticky ends | - Cut phosphodiester and hydrogen bonds |
| How do sticky ends stick? | - Sticky end pairs with comp. base, DNA ligase cements the bonds between nucleotides by condensation
- DNA ligase used in repairing broken DNA
- The DNA is now called recombinant DNA
- If the plasmid is cut using the same restriction enzyme it will have complementary sticky ends to the gene |
| Recombinant DNA | - Where the DNA of the 2 different organisms is combined, the product is known as recombinant DNA |
| GM process (1) producing DNA fragments | - One method -> use of reverse transcriptase
- mRNA -> DNA
- Forms comp. strand of DNA called complementary DNA
- Forms another strand using DNA polymerase |
| GM process (2) producing DNA fragments | - Another method of producing DNA fragments is to use restriction enzymes
- Named source of DNA (hair follicle...)
- Digested/hydrolysed using restriction enzymes
- Use a thermocycler
- Heat to (95) to break h bonds
- Cool to (55) allow primers to attach
- Function of primers (allow polymerase to attach, locate specific seq)
- Heat to (72) optimum temp for DNA polymerase
- Free nucleotides bind with comp. nucleotides
- Repeated many times |
| GM process (3) producing DNA fragments | - Some of these leave fragments with 2 blunt or sticky ends
- The sequences of bases on uneven ends are palindromic? |
| A vector | - A gene carrier that the gene is spliced into
- Plasmids are vectors, they are small pieces of circular DNA found in bacteria |
| Transformation | - Ice cold
- Incubate at 42oC for 2 minutes
- Ice cold |
| Finding the bacteria | - Splice in 2 genes - the genes we want and a gene marker
- Gene markers can be :
- Fluorescent 'genes'
- Antibiotic resistant genes
- A particular enzyme (may cause a colour change) |
| Explain how modified plasmids are made by genetic engineering and how the use of markers enable bacteria containing these plasmids to be detected. | - Isolate wanted gene using restriction enzymes to get DNA and produce sticky ends
- Use ligase to join wanted gene to plasmid
- Also include marker gene e.g. antibiotic resistance
- Add plasmid to bacteria to grow (colonies) then (replica) plate onto medium where the marker gene is expressed
- Bacteria not killed have antibiotic resistance gene and (probably) the wanted gene |
| Why are gene markers necessary in gene cloning | - To know what plasmids with the genes were taken up by the bacteria |
| One advantage of using fluorescent markers rather than antibiotic gene markers | - Results can be obtained more easily and more quickly
- Because with antibiotic resistance markers, the bac cells with the gene are killed, so replica plating is necessary to obtain cells with the gene
- With fluorescent gene markers, the bac cells are not killed and so there is no need to carry out replica plating |
| Strengths of GM | - GM bacteria can be used to produce insulin
- GM used to make vaccines
- Genes could be introduced for gene therapy |
| Weaknesses of GM | - GM crops may contain allergens
- Introducing genes could lead to eugenics
- Could decrease biodiversity |
