Manipulating genomes Flashcards
1
Q
What are VNTRs?
A
- Variable number tandem repeats.
- Sections of satellite DNA.
2
Q
What is a microsatellite?
A
- A smaller region of just 2-4 bases repeated 5-15 times.
- Also known as short tandem repeats.
3
Q
How can DNA be profiled?
A
- Extraction of DNA from sample. Amplification with PCR.
- Digestion using specific restriction endonucleases - each one has a specific restriction site. Produces STR mixture.
- Gel electrophoresis
- Transfer into nitrocellulose/nylon membrane w/ blotting buffer.
- DNA probe has a fluorescent/radioactive marker. Will hydridise with ssDNA (single stranded).
4
Q
Describe the process of PCR
A
95C: strands separate by denaturation.
55C: Temp. lowered. upstream (3’ -5’) and downstream (5’ -3’) primers anneal.
72C: Taq DNA polymerase attaches to primers binding sites and extends the new SP backbone.
5
Q
What are the uses of DNA profiling?
A
- Forensics
- Paternity testing
- Phylogeny
- Breeding programmes
- Disease risk
6
Q
Describe the process of DNA sequencing.
A
- DNA strand chopped up, mixed with primers, bases, DNA polymerase and terminator bases (ddNTPs).
- Put in thermal cycler.
- Each time a terminator base is added a strand terminates until all possible chains produced.
- Readout from capillary tubes: DNA fragments separated by electrophoresis in capillary tubes by mass and lasers detect the colour of the ddNTP and the sequence.
- Results analysed by computer.
7
Q
What is bioinformatics?
A
- Development of the software and computing tools needed to organise and analyse raw biological data. E.g constructing algorithms, models etc
- Huge amounts of data.
8
Q
What is computational biology?
A
- Computational biology uses data to build theoretical models of biological systems, which can be used to predict outcomes in certain circumstances.
9
Q
How can DNA sequencing be used to idnetify species?
A
- DNA barcoding
- Comparison of highly conserved regions of DNA, e.g cytochrome C in mitochondrial DNA, found in all species.
10
Q
How can DNA sequencing be used in phylogeny?
A
- Used to track evolutionary history.
- Use basic rate of mutation to find how long ago divergence occurred.
- Find most recent common ancestor
11
Q
What is synthetic biology?
A
- Redesigning existing biological systems for useful purposes.
12
Q
Describe the process of genetic engineering.
A
- Isolation of desired gene
- Insertion into vector
- Transformation of organism (becomes transgenic)
- Identification (check take up)
- Growth/cloning
13
Q
What is isolation in G.E?
A
- Isolating the desired gene.
- Uses endonucleases which cut sticky ends at their specific restriction site.
- Alternatively, the gene can be isolated using reverse transcrition. A section of mRNA produced by the gene can be converted into complementary DNA (cDNA) using reverse transcriptase. DNA polymerase can then be used to make this double stranded.
14
Q
What can be said about the endonuclease enzymes used on the vector and then gene?
A
- They must be the same.
15
Q
What vectors can be used in humans? (G.E)
A
Viruses, liposomes
16
Q
What are marker genes?
A
Genes that code for identifiable characteristics e.g GFP production or antibiotic resistance.
Can be used to identify whether uptake has been successful.
17
Q
What enzyme connects the ‘sticky ends’ in G.E?
A
DNA ligase
18
Q
What is a synthetic way to isolate a desired gene (G.E)?
A
- Create the gene chemically using nucleotides
19
Q
How can transformation (take up of recombinant plasmid) be encouraged?
A
- Mixing high conc. of plasmids with bacterial cells.
- Add calcium salts and heat
- Electroporation: electrical currents make bacteria more porous.
- Electrofusion: plasmid in vescile, electric current, fuses with cell.
20
Q
How are plants genetically engineered using A. tumefaciens?
A
- Cut a leaf. Sterilise.
- Expose the leaf to a bacterial suspension containing a Ti plasmid (from agrobacterium tumefaciens) with genes for desired trait and antibiotic resistance.
- Expose leaf to an antibiotic to kill cells which lack new genes. Callus forms from gene-altered cells.
- Allow callus to sprout shoots and roots.
- Transfer to the soil to grow.
21
Q
How can plants be genetically engineered using electrofusion?
A
- Cell walls must first be removed by cellulases.
- Cells of different types can be fused, using electricity, to form a polypoid, transgenic cell.
- Hormones used to grow new cells walls, callus forms.
22
Q
What is pharming?
A
- The generation of pharmaceuticals using genetically engineered plants or animals.
23
Q
What is an example of pharming?
A
Creating animal models e.g knockout mice to model cancer, they have genes deleted.
24
Q
What is somatic cell gene therapy?
A
- Replacing a mutant allele with a healthy one in the somatic cells.
- Viral vectors or lyposomes are often used.
25
What are the problems with somatic cell gene therapy?
- Temporary.
- Will still pass on faulty gene to offspring.
26
What is germ line gene therapy?
- Insertion of healthy allele into germ cells (usually eggs) or into embryo immediately after fertilisation.
27
Define DNA sequencing
Working out the sequence of bases/nucleotides
28
Why is gene therapy less successful with dominant disorders?
- A remaining dominant gene will still code for the faulty protein causing the disorder.
29
What are the concerns about germline therapy?
- Could lead to designer babies
- Unknown long-term effects
- No consent from embryo
30
What are the ingredients for PCR?
- DNA sample
- XS of DNA nucleotides
- Taq DNA polymerase
- Primers
31
What occurs at 95C? (PCR)
- Denaturation of DNA double helix into ssDNA.
32
What occurs at 55C? (PCR)
- Annealing of primers to the DNA strand.
33
What are the upstream and downstream directions? (PCR)
- Upstream: 3' --> 5'
- Downstream: 5' --> 3'
34
What occurs at 72C? (PCR)
Taq DNA polymerase attaches to primers binding site and extends the new S-P backbone.
35
What ingredients are required for DNA sequencing process?
- primers
- Taq DNA polymerase
- DNA sample
- ddNTPs (terminator bases)
- Free DNA nucleotides
36
What occurs in the thermal cycler in DNA sequencing?
- 96C: ssDNA formed (denaturation)
- 50C: primers anneal to DNA strand
- 60C: DNA polymerase starts to build up new DNA strand by adding complementary nucleotides.
37
How are the different lengths of DNA fragments separated in DNA sequencing?
- Capillary electrophoresis/electrophasis
- The fluorescent markers on the terminator bases are used to identify the final base on each fragment. Lasers detect the different colours and thus the order of the sequence.
- Produces electropherogram
38
How can the order of bases in the capillary tubes be used?
- The order of bases in the capillary tube is the order of bases from the new, complementary DNA strand.
- This can be fed into a computer which can reassemble the original DNA strand.
39
What things can happen which reduce the number of DNA fragments produced by PCR?
- Temp damage to strand/template/fragment
- strands may rejoin
- lack of primers/nucleotides
- primers fail to anneal
40
What is the role of the buffer in PCR?
- Carry charge, allowing fragments to separate.
41
Why is Taq polymerase used?
- It is thermostable; does not denature at 95C
42
What must be added to proteins to allow them to be separated by electrophoresis?
- SDS, adds a uniform negative charge
43
Why must proteins be denatured before electrophoresis?
- Remove all layers of structure other than primary.
- Ensure all are being separated by size alone, not charge etc.
- Exposes charges/hydrophobic regions.
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