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Biology > Gene Technology Techniques - A2 > Flashcards

Gene Technology Techniques - A2 Flashcards

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1
Q

What is meant by recombinant DNA technology?

A

The transfer of DNA fragments from one organism to another.

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2
Q

Why does recombinant DNA technology work?

A

-The genetic code is universal
-Therefore transcription and translation occur by the same mechanism
- And result in same amino acid sequence across organisms

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3
Q

Explain the process of using reverse transcriptase to produce DNA fragments.

A
  • mRNA (no introns) complementary to target gene is used as a template
  • it is mixed with free nucleotides which match up their base pairs
  • reverse transcriptase occurs which forms the sugar-phosphate backbone
  • creating cDNA (complementary DNA)
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4
Q

Summarise the process of using enzymes to make DNA fragments.

A
  • restriction endonuclease (REs) cut DNA at specific sequences
  • difference REs cut at different points but 1 RE will always cut at the same sequence
  • therefore using particular REs allows you to cut certain gene of interest
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5
Q

In which 2 ways can we amplify DNA fragments?

A

in vitro - polymerase chain reaction (PCR) (lab)
in vivo - using host cells (in something living)

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6
Q

describe the reaction mixture in the first stage of PCR.

A
  • contains the DNA fragments to be amplified
  • primers that are complimentary to the start of the fragment
  • free nucleotides to match the exposed bases
  • DNA polymerase to create the new DNA
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7
Q

Summarise the process of amplifying DNA fragments using PCR. (in vitro)

A
  1. heat DNA to 95 degrees celsius
  2. breaks hydrogen bonds/separates strands
  3. add primers
  4. add nucleotides
  5. cool to 50 degrees celsius
  6. too allow binding of nucleotides/primers
  7. add DNA polymerase
  8. heat to 75 degrees celsius
  9. DNA polymerase joins nucleotides together
  10. repeat cycle many times - new DNA acts as a template for next cycle
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8
Q

summarise the process of inserting a DNA fragment into a vector. (in vivo)

A
  • a plasmid is used as the vector
  • and is cut using the same restriction enzymes as DNA so that ends are complementary
  • DNA ligase join the fragments and plasmid together
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9
Q

Summarise the process of inserting a vector into a host cell.

A
  • known as cell transformation
  • the host cells (bacteria) are mixed with vectors in ice cold solution
  • then heat shocked to encourage cells to take up vectors
  • the cells can then be grown and the DNA fragment will be cloned
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10
Q

Summarise the process of identifying transformed cells.

A
  • marker genes eg. coding for fluorescence can also be inserted into vectors along with DNA
  • when cells begin to grow, UV lights can be used to identify which have taken up the vector and which have not
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11
Q

How can DNA probes be used to locate specific alleles?

A
  • the probe is designed so its sequence is complementary to the specific allele
  • they are labelled
  • amplified using PCR
  • then added to a sample of single stranded DNA
  • the probe will bind if the allele is present
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12
Q

Give some applications of DNA probes.

A
  1. to screen someone’s DNA for a particular heritage health condition
  2. to identify a gene for use in genetic engineering
  3. to predict how someone will respond to a drug
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13
Q

What is the purpose of DNA hybridisation?

A
  • to measure the degree of difference between 2 strands of DNA
  • can be used to compare someone’s DNA to a certain gene to see if they have it
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14
Q

Summarise the process of DNA hybridisation.

A
  • one DNA strand is labelled and mixed with an unlabelled comparison strands
  • the more similar the strands, the more strongly they will bind
  • and more energy will be required to break the strands apart
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15
Q

What are the benefits of genetic profiling?

A
  • can identify heritage diseases very early
  • so can begin to treat them before symptoms develop, reducing impact on individual
  • treatment can also be personalised so more chance of being effective
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16
Q

What is genetic fingerprinting?

A

a technique used to compare 2 DNA samples and determine whether they came from the same individual

17
Q

How does genetic fingerprinting work?

A
  • every organisms genome contains non-coding regions called variable number tandem repeats (VNTR)
  • the probability of 2 people having the same VNTR is very low
  • so we can compare these areas and see if the 2 DNA samples came from the same person
18
Q

Summarise the process of genetic fingerprinting analysis.

A
  • DNA extracted from sample
  • DNA cut into segments using restriction endonuclease
  • must leave mini satellites intact
  • DNA fragments separated using electrophoresis
  • mixture put on wells in gel and electric current passed through
  • immerse gel in alkaline solution / 2 DNA strands separated
  • southern blotting / absorbent paper to absorb DNA
  • DNA fixed to nylon/membrane using UV light
  • radioactive marker / probe added - complementary to mini satellites
  • areas with probe identified using x-ray film
19
Q

Outline a method for in vivo cell cloning.

A
  1. cut desired gene from DNA of desired organism
  2. using restriction endonuclease
  3. make artificial DNA with correct sequence of bases
  4. using DNA polymerase
  5. cut plasmids open
  6. with same restriction endonuclease
  7. sticky ends/unpaired bases attach
  8. use DNA ligase to join
  9. return plasmids to bacterial cells
20
Q

How does gel electrophoresis work?

A
  • DNA fragments are placed at one end of a slab of gel
  • an electrical current is applied causing the DNA fragments to move to the other end of the gel
  • shorted fragments travel further
  • the pattern of bands created is unique to every individual
21
Q

Give some applications of genetic fingerprinting.

A
  1. forensics - to identify suspects
  2. medical diagnosis - to identify type of haemoglobin produces by an individual to diagnose sickle cell anaemia
  3. animals and plant breeding eg. breed out harmful alleles, ensure pedigree
22
Q

How can target genes be isolated using restriction enzymes?

A

-DNA contains specific pallindromic sites called restriction sites
-Restriction enzymes can cut DNA at these restriction sites (active site complementary to specific base sequence of DNA)
-If there is is a restriction site either side of the target gene
-Restriction enzymes can be used to cut the target gene out
-Restriction enzymes leave the DNA with sticky ends (unpaired bases)

23
Q

How does genetic engineering occur?

A
  1. DNA extracted from donor cell and cut into fragments by restriction endonuclease
  2. fragments separated using gel electrophoresis
  3. DNA fragment containing gene for insertion is identified using DNA probe
  4. vector (plasmid) extracted from cell using same restriction endonucleases
  5. bacterial plasmids and DNA fragments are mixed
  6. some plasmids take up foreign DNA fragment
  7. DNA ligase used to join plasmid and DNA fragment together forming recombinant plasmid
  8. plasmids are mixed with bacteria, some bacteria take up by RP
  9. these bacteria are selected and cultured to produce many bacteria containing RP-DNA
  10. using marker gene such as antibiotic resistance
  11. bacteria expresses new gene and protein is extracted
24
Q

Benefits of genetic engineering.

A

Agriculture - crops can be GE to give higher yield, more nutrients and pest resistance + resistance to higher temps and drought - helps meet growing populations food demands
Industry - GE organisms produced to make large n. of enzymes quickly and cheaply for industrial processes
Medicine - GE organisms can make large quantities of drugs or vaccines quickly and cheaply

25
Q

Concerns of genetic engineering.

A
  1. use of antibiotic resistance marker genes within GE could lead to transfer of these genes to pathogens
  2. inserting new genes into plants could alter normal genes function and create toxic products within GE food sources
  3. introducing herbicide resistance genes to crop plants could transfer to wild species when interbreeding, producing weeds resistant to herbicides
26
Q

Gene therapy.

A

Insert a gene of functioning protein into the cells of person with genetic disease

27
Q

What is Somatic gene therapy?

A
  • DNA transfers to our normal body tissue
  • cannot be inherited
  • mutation in tumour only eg. breast
    however
  • not all cells take up new DNA/express allele
  • only some tissue types accessible and need to be repeated (when cells replace themselves, faulty DNA back)
  • body can produce immune response to vector
28
Q

Germ line gene therapy.

A

-DNA transferred to sex cells (sperm and egg)
- allows correction of disease causing mutation that are certain to be passed on (passes on to offspring)
however
- effects on gene transfer is unpredictable (could defect the embryo)
- denial of human rights, offspring would have no say in whether their genetic material would be modified
- potential abuse - could enhance favourable characteristics and supress others - could result in eugenics (manipulation of genetic properties of a population).

29
Q

Why are primers added in PCR?

A
  • to prevent strands from re-joining each other
  • create starting point for DNA polymerase to add new DNA nucleotides
30
Q

What is meant by agglutination?

A

antibody sticks to antigen

31
Q

Describe how bacteria containing the insulin gene are used to obtain sufficient insulin for commercial use?

A
  • use of ferments
  • provides nutrients plus suitable conditions for optimum growth
  • reproduction of bacteria
  • insulin accumulates and is extracted
32
Q

Give 2 advantages of using a gene therapy.

A
  1. can target specific cells
  2. can replicate into cells

Biology (27 decks)

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