DNA Technology Flashcards
1
Q
What does gene technology allow?
A
- manipulation, alteration and transfer of DNA across species
2
Q
Define recombinant DNA
A
DNA that is made from 2 different organisms that has been combined
3
Q
Define genetically modified organism
A
Organisms that contains recombinant DNA
4
Q
Describe the 5 stages involved in making a protein from DNA
A
- Isolation of desired gene
- Insertion of gene into vector
- Transformation; transfer if DNA into suitable host cells
- Identification of host cells that have taken up gene
- Growth of these host cells
5
Q
What is reverse transcriptase used for?
A
- to isolate the gene before it is placed in a vector
6
Q
How does reverse transcriptase work?
A
- Cell that produces desired protein is identified (will contain large amounts of mRNA)
- mRNA is extracted, reverse transcriptase is used to produce cDNA
- DNA polymerase is used to produce complimentary DNA strand from cDNA (acts as a template)
7
Q
What are restriction endonuclease enzymes used for?
A
To cut at specific recognition sites on DNA sequence
8
Q
What do restriction endonucleases produce?
A
- blunt ends, when DNA is cut vertically
- sticky ends, when DNA is cut in staggered fashion
9
Q
How is DNA inserted into a vector in In-Vivo cloning?
A
- gene and plasmid are cut with same restriction endonuclease enzymes, therefore ‘sticky ends’ are complimentary
- DNA fragments are mixed with opened-up plasmids, and some will be incorporated
- DNA ligase permanently joins plasmid and DNA
10
Q
How are the plasmids reintroduced into bacterial cells?
A
- plasmids and bacterial cells are mixed in medium containing calcium ions.
- ions and temp changes make bacteria permeable, allowing plasmids to pass over membrane
11
Q
Why will DNA of desired gene only be in some bacterial cells?
A
- bacterial cells did not take up plasmid
- plasmid closed up before gene was incorporated
12
Q
How are bacterial cells that have taken up plasmid identified?
A
- all bacterial cells are grown on medium containing antibiotic
- cells that have taken up plasmids will have resistance
- all cells that have not, will die
13
Q
What are gene markers used for?
A
To identify cells that contain plasmids, that have taken up DNA
14
Q
How do antibiotic-resistance markers work?
A
- bacterial cells are grown in medium with antibiotic B, gene for resistance has been made useless if gene has been taken up
- cells that have taken up plasmid will die
- replica plating
15
Q
How do fluorescent gene markers work?
A
- gene is inserted into centre of GFP (green fluorescent protein)
- any bacterial cell that has taken up plasmid will not produce fluorescence
16
Q
How do enzyme gene markers work?
A
- desired gene is inserted into centre of gene that codes for enzyme lactase, which turns colourless solution blue
- if plasmid has taken up gene, will not turn solution blue
17
Q
What is purpose of polymerase chain reaction (PCR)?
A
- automated, rapid method of producing cloned fragments of DNA
- allows amplification of small sample
18
Q
What does PCR require?
A
- DNA fragment
- DNA polymerase
- primers
- nucleotides
- thermocycler
19
Q
Give method of PCR
A
- Temp is increased to 90 degrees, DNA strands are separated
- Temp is decreased to 55 degrees, primers attach to strands and act as starting point for DNA polymerase
- Temp is increased to 72 degrees, optimum temp for DNA polymerase to join complimentary nucleotides to strand
20
Q
What are the advantages of in-vitro cloning?
A
- automated, very rapid
- can produce many clones from small sample
- does not require living cells
21
Q
What are the advantages of in-vivo cloning?
A
- no contamination
- very accurate
- useful when introducing genes into a different organism
- cuts out specific genes
22
Q
Describe how genetic modification can benefit humans
A
- increases yield from animals/crops
- improves nutrient content of food
- producing medicines
- producing plants that are resistant to disease
23
Q
Give 3 substances produced using GM microorganisms
A
- Antibiotics
- Hormones
- Enzymes
24
Q
How can genetic modification improve yield of crops?
A
- can be made herbicide, pest, disease resistant
25
How can genetic modification improve animals?
- genes for resistance can be transferred to different organisms
- can be given growth hormones
- used to treat inherited disorders
26
Define gene therapy
When a defective gene that causes disease is replaced/supplemented with a healthy, cloned gene
27
What is germ-line gene therapy?
Involves replacing or supplementing faulty gene in fertilised egg
- more permanent solution as it affects future generations
28
What is somatic-cell gene therapy?
- temporary therapy that targets the affected tissue
- is not passed on
- needs repeating frequently
29
What are the 2 ways in which cloned CFTR genes are introduced into the epithelial cells of the lungs
1. Using a harmless virus
| 2. Wrapping the gene in lipid molecules
30
Outline method of using viruses to deliver cloned CFTR genes to lungs
1. Virus (adenovirus) is made harmless
2. Are grown in lab with plasmids including healthy gene
3. CFTR gene becomes incorporated in DNA of virus
4. Virus is given through nostrils
5. Inserts DNA (including healthy gene) into epithelial cells of lungs
31
Outline method of using lipid molecules to introduce CFTR genes into lungs
1. Healthy genes are inserted into vectors, reintroduced to host cells and cloned
2. Plasmids are extracted and wrapped in lipid molecules to form lysosomes
3. Are given by aerosol, lysosome crosses membrane (lipid soluble) and delivers healthy gene into cells
32
Why is somatic-cell gene treatment for cystic fibrosis not always effective?
- virus may cause infection
- may build immunity to virus
- gene may be delivered but not expressed
33
What is cystic fibrosis and what are the symptoms?
- disease caused by mutant recessive allele
- CFTR gene is faulty, chloride ions are transported across membrane and water does not follow by osmosis, membrane is dry and mucus is viscous
- mucus builds in lungs, leads to respiratory problems
- mucus in pancreatic ducts
34
What are DNA probes are what are they used for?
- short single-stranded sections of DNA that are identifiable (radioactive or fluorescently labelled)
- hybridise with target allele, so it can be located
35
Outline Sangar method of DNA sequencing
1. 4 test tubes containing 4 diff types of terminator nucleotide, nucleotides, DNA polymerase, DNA fragment and primer are set up
2. Binding of normal nucleotides and terminator nucleotides to DNA strand is random, will produce strands of different lengths
3. Fragments placed onto agar gel and voltage is applied, strands move (larger+further due to resistance of gel)
4. Strands can be identified and sequenced due to radioactivity of DNA
36
What is restriction mapping and what is it used for?
When genes are too large for Sangar method of sequencing, DNA is cut using different combinations of restriction endonucleases, and put back together using restriction mapping
37
Outline method of restriction mapping
1. DNA is cut into smaller sections with a series of different restriction endonuclease enzymes
2. Fragments are separated using gel electrophoresis
3. Distance between recognition sites is determined by pattern of fragments produced
38
What can genetic screening be used for?
- to identify inherited conditions
- to help determine how well a patient will respond to treatment
- help identify health risks
39
Give outline of how genetic screening works
1. Sequence of faulty gene is determined, fragment of DNA with bases complimentary is produced
2. Fragment is radioactively labelled (becomes a probe) and is cloned using PCR
3. Probe is added to single stranded DNA fragments of person
4. If person has faulty gene, sequence will be complimentary to probe, will show on x-ray film
40
What is genetic counselling?
- advising patients and relatives about the risks of genetic disorders
41
What might genetic counselling give people advice on?
- if they are carriers of disease
- the type of mutated gene
- the best treatment
42
What does genetic fingerprinting rely on?
- in genome that are many repetitive, non-coding sequences of DNA (core sequences) which are unique to everyone
- more similar, more closely related you are
43
What are the 5 stages of genetic fingerprinting?
1. Extraction of DNA
2. Digestion
3. Separation
4. Hybridisation
5. Development
44
What does 'separation' in genetic fingerprinting involve?
1. fragments are separated using gel electrophoresis
2. Immersed in alkali to produce single strands
3. Thin nylon membrane laid over gel
4. Membrane is covered with absorbent paper which draws liquid up
5. DNA fragments are transferred to same relative position
6. Fixed to membrane by ultraviolet light
45
What does 'hybridisation' in genetic fingerprinting involve?
- DNA probes are complimentary to core sequences, bind in certain conditions
- different probes that complimentary to different core sequences are used
46
What does 'development' in genetic fingerprinting involve?
- x-ray film is put over nylon membrane and is exposed by radiation for radioactive probes
47
What is genetic fingerprinting used for?
- in forensic science
| - paternity
