DNA technology 1 Flashcards
ways in which the PCR differs from transcription
- transcription uses RNA polymerase instead of DNA P;
- in transcription, only one strand acts as a template whereas there are 2 in PCR both strands;
20% of the DNA produced by the PCR is copied inaccurately. why it is unsafe to use the PCR to clone the CFTR gene for use in treating cystic fibrosis
- percentage risk is too high for human application;
- incorrect mRNA so tRNA brings incorrect amino acid;
- sequence of amino acids changed so incorrect shape
- may produce a harmful protein;
- protein non-functional so chloride ions not transported resulting in thick mucus
Advantages and disadvantages of replacing defective gene in gamete instead of body cells
Advantages:
- only has to be treated once as effect is permanent;
- all cells in body have replaced gene;
- can be passed to offspring;
Disadvantages:
- changes to genetic make-up of individuals may affect future generations
- may affect normal development
Advantages of genetic engineering over selective breeding
- much faster and efficient
- several genes can be inserted at once
Describe how we radioactively label fragments to make them visible following electrophoresis.
- transfer DNA fragments onto nylon sheet
- add radioactive probes so they anneal to DNA on nylon sheet (comp), labelling
- probes show up as bands on photographic film
Describe the polymerase chain reaction
- Heat DNA to about 90°C to separate strands
- Mixture is then cooled to 55°C causing the primers to anneal** to complementary bases at the **end of each fragment
- Add DNA polymerase and nucleotides
- Primers provide the starting sequence for DNA polymerase
- Temp is then raised to 72°C, as optimum for DNA P
- DNA polymerase forms new strands by joining nucleotides along each template strand
- This is done by comp base pairing
- cycle repeated
Disadvantages of using viruses to introduce genes into cells
- may cause disease and harm patient
- immunity may develop to the virus so they may be destroyed by white blood cells
Disadvantages of using gene therapy to introduce functional gene
- long-term adverse effects not known;
- genes introduced which may have damaging effect on other genes;
Explain how the polymerase chain reaction and DNA sequencing can be used to compare DNA. (10 marks)
- Desribe the PCR (4 marks)
- Use Sanger sequencing. Process:
- Use restriction endonucleases to make sections of DNA;
- Use radioactively labelled (terminator) bases;
- DNA replication stopped at base (A,T,G,C)
- Electrophoresis description
- Smaller fragments move faster
- Fragments can be identified by position on gel
- Autoradiograph made using photographic film
- If the DNA bands match;
Explain how to distinguish bacteria who have assimilated the DNA and those who have the plasmid without the new Gene
- replica plating: use of pad** surface to transfer bacteria to **agar plate containing ampicillin
- all bacteria that have taken in a plasmid will survive
- use of agar plate containing tetracycline;
- in bacteria with human DNA, tetracycline gene no longer functional
- bacteria with human DNA grow on plate with ampicillin but are killed by tetracycline;
- bacteria without the gene in plasmid not killed,
Explain why a person cured by gene therapy may still have children who suffer from the abnormality
- gamete cells don’t have healthy allele;
- so the parents are still able to pass on the defective allele to offspring
Explain why some nucleotide substitution have no effect on the protein coded by the gene?
- Silent mutation
- Degenerate nature of the genetic code
- Different codon codes for one type of amino acid
Gene therapy definition
- replacement of defective genes with healthy genes in order to treat genetic diseases
How are 2 polynucleotide chains of DNA held together
- hydrogen bonds;
- between bases;
How can a virus with the CFTR gene be used to insert the gene to a cystic fibrosis sufferer
- virus is inhaled into the lungs as it is sprayed ;
- it then gets into cells, inserting the healthy gene;
