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1

 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;
     

2

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

  1. percentage risk is too high for human application;
  2.  incorrect mRNA so tRNA brings incorrect amino acid; 
  3. sequence of amino acids changed so incorrect shape
  4. may produce a harmful protein;
  5.  protein non-functional so chloride ions not transported resulting in thick mucus 

3

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

4

Advantages of genetic engineering over selective breeding

  • much faster and efficient 
  • several genes can be inserted at once

5

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 

6

Describe the polymerase chain reaction

  1. Heat DNA to about 90°C to separate strands   
  2. Mixture is then cooled to 55°C causing the primers to anneal to complementary bases at the end of each fragment 
  3. Add DNA polymerase and nucleotides                     
  4. Primers provide the starting sequence for DNA polymerase
  5. Temp is then raised to 72°C, as optimum for DNA P
  6. DNA polymerase forms new strands by joining nucleotides along each template strand
  7. This is done by comp base pairing
  8. cycle repeated

7

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

8

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;

9

Explain how the polymerase chain reaction and DNA sequencing can be used to compare DNA. (10 marks)

  1. Desribe the PCR (4 marks)
  2. Use Sanger sequencing. Process:
  3. Use restriction endonucleases to make sections of DNA;
  4. Use radioactively labelled (terminator) bases;
  5. DNA replication stopped at base (A,T,G,C) 
  6. Electrophoresis description
  7. Smaller fragments move faster
  8. Fragments can be identified by position on gel
  9. Autoradiograph made using photographic film
  10. If the DNA bands match;

10

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,

11

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

12

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

13

Gene therapy definition

  • replacement of defective genes with healthy genes in order to treat genetic diseases

14

How are 2 polynucleotide chains of DNA held together

  • hydrogen bonds;
  • between bases;

15

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;

16

How can using a gene probe spot a defective gene?

  • fluorescent probe will attach to mutant allele;
  • H-bonds to one DNA strand;
  • as a result of complementary base pairing;
  • probe emitts light so detected

17

How is DNA replicated

  • 2 polynucleotide strands separate as DNA helicase breaks hydrogen bonds
  • Template strands are exposed and free  nucleotides attach 
  • By complementary base pairing (A-T, G-C)
  • Parent strand acts as a template (Semi-conservative replication)
  • DNA polymerase joins nucleotides together;

18

How might enzymes be used to isolate healthy genes that make the correct CFTR protein to insert it into a bacteria?

  • Restriction endonuclease;
  • cuts DNA at recognition site, removing gene;
  • The same RE must be used to cut plasmid of bacteria;
  • This is to produce complementary sticky ends
  • DNA Ligase joins pieces of DNA together

19

Describe a Plasmid

  • circular DNA that contains only a few genes;
  • different from main bacteria DNA;

20

Practical use of polymerase chain reaction

  • provides multiple copies of a DNA fragment;
  • Can be used to analyse in forensic detection

21

Primers Description

  • short, single stranded length of DNA which anneal to the template strand
  • complementary base sequence;
  • indicates where replication starts and stops
  • keeps strands apart

22

Role of vectors?

  • Transfers foreign DNA Into host cell;

23

Use of electrophoresis?

To separate new strands based on lengths

24

Ways in which the DNA structure can be changed as a result of a mutation

  • Addition / deletion / substitution;
  • Of nucleotide

25

What does the CTFR protein normally do?

  • Transports chloride ions across cell surface membrane;
  • Maintains water potential of cells

26

Why are people with cystic fibrosis more likely to develop bacterial infections

  • mucus not removed;
  • mucus traps bacteria allows bacteria to breed;

27

Why do we use viruses to introduce genes into cells

  • they have the ability to inject DNA into cells
  • target specific cells and have the ability to replicate in cells;

28

 Why doesn't implanting embryos into the uterus of sheeps work most of the time?

  • Embryo foreign so rejected (2 Marks)

29

Why is a jellyfish gene that glows attached?

  • It acts as a marker gene
  • Shows that the useful gene has been taken up and expressed
  • We can choose to only impant the DNA that has the correct gene

30

Why is it better to inject a gene into an isolated cell instead of inserting them into cells in an organism

  • isolated cells divide by mitosis;
  • all cells made would express gene whereas only a few cells would