Topic 8B: Genome Projects and Gene Technologies Flashcards
1
Q
What is gene sequencing and how does it work?
A
- Only works on fragments - to sequence a whole genome it must be cut into smaller pieces - each piece is sequenced then put back in order to sequence the whole genome
2
Q
What was the human genome project?
A
- Completed 2003
- Mapped the whole human genome for the first time
3
Q
What are features of sequencing simple organisms?
A
- Helps to identify their proteins
- Prokaryotes don’t have introns - much easier
- Useful for medical research and development e.g. identifying protein antigens on pathogenic bacteria helps vaccine development
4
Q
What are features of sequencing complex organisms?
A
- More difficult - has large sections of non coding DNA
- Have regulatory genes - determine when genes are on or off
- Hard to translate the genome to proteome - need to find the coding sections
- Work on human proteome happening - found 3000 protein codes
5
Q
What were sequencing methods like in the past?
A
- Labour intensive
- Expensive
- Small scale
6
Q
What are sequencing methods like now?
A
- Automated
- Cost effective
- Larger scale
7
Q
How are DNA fragments made with reverse transcriptase?
A
- DNA produced from mRNA - used as a template
- DNA produced is cDNA
- mRNA isolated from cells, mixed with free nucleotides and reverse transcriptase to synthesise new DNA strands
- cDNA has no introns
8
Q
What are advantages of using reverse transcriptase?
A
- mRNA is present in cells from actively transcribed genes - lots available to make cDNA
9
Q
What are disadvantages of using reverse transcriptase?
A
- More steps to using it so more time consuming and technically difficult
10
Q
How are DNA fragments made using restriction endonuclease?
A
- Enzymes recognise specific palindromic sequences and cut there
- Different enzymes recognise different sequences as they have specific complimentary active sites
- Leaves blunt / sticky ends - Sticky -> small tails of unpaired bases used to bind to another complimentary strand
11
Q
What are advantages of using restriction endonuclease?
A
- Sticky ends make it easier to insert the gene to make recombinant DNA
12
Q
What are disadvantages of using restriction endonuclease?
A
- Fragments still contain introns
13
Q
How are DNA fragments made using a gene machine?
A
- Database has info to produce the sequence so DNA does not have to exist naturally - any sequence can be made
- Sequence is designed
- 1st nucleotide is fixed to a support (e.g. a bead)
- Nucleotides added in sequence with protecting groups - ensure the nucleotides join at the correct points and prevent branching
- Short DNA sections (oligonucleotides) - roughly 20 nucleotides long are produced
- Broken off the support, protecting groups removed, oligonucleotides joined to make longer DNA fragments
14
Q
What are advantages of using a gene machine?
A
- Can design exact DNA fragments wanted with sticky ends, labels and preferential codons
15
Q
What are disadvantages of using a gene machine?
A
- Need to know the sequence of amino acids or bases
16
Q
What is recombinant DNA?
A
- DNA from one organism which has foreign DNA inserted
17
Q
What is a transgenic organism?
A
- Organism containing a foreign gene in its DNA
18
Q
How is making a transgenic organism possible?
A
- DNA code is universal
- Transcription and translation very similar in most organisms
19
Q
What does in vivo mean?
A
- Inside a living organism
20
Q
How is a gene inserted into a vector?
A
- Plasmid is cut open by the same restriction endonuclease as DNA so they have complimentary sticky ends
- DNA fragment and plasmid mixed with DNA ligase which joins the sticky ends of the fragment to the vector - ligation
- This forms recombinant DNA
21
Q
How is the vector inserted into a host cell?
A
- Plasmids mixed with bacteria in a medium containing Ca2+ making the bacteria more permeable to allow the plasmid to enter
22
Q
What are bacteria with recombinant DNA called?
A
- Transformed
23
Q
What happens to transformed bacterial cells?
A
- Grown on large scale - useful protein removed and purified
24
Q
How are transformed host cells identified?
A
- Marker genes - inserted in vectors too - only transformed cells have the gene
- The marker can code for antibiotic resistance - only transformed cells are resistant so survive
- Can be fluorescent - identified by UV light
25
What is needed for proteins to be produced by transformed cells?
- Needs specific promotor and terminator regions
- Promotor tells RNA polymerase where to start
- Terminator region signals where to stop
26
What does in vitro mean?
- Outside a living organism
27
What is mixed in PCR?
- Mixture of DNA sample, free nucleotides, DNA polymerase
28
How does PCR occur (heating and cooling)?
- Mix heated to 95°C to break H bonds between DNA strands
- Cool to 50-65°C allowing primers (complimentary to bases at the start of the fragment) to bind
- Heated to 72°C so DNA polymerase can work
- DNA polymerase lines up free nucleotides alongside each template strand and joins nucleotides
29
What is produced from PCR?
- Base pairing forms complimentary strands - 2 new copies of DNA formed and cycle complete
- Cycle starts again - now all 4 strands templates
- Each PCR cycle doubles the amount of DNA
30
How can plants be genetically engineered?
- Gene inserted into a plasmid, added to a bacterium, inserted into a plant
- If correct promotor region present, cells produce protein
31
How can animals be genetically engineered?
- Gene inserted into early embryos - all body cells have the gene
- Insert into egg cells - all offspring cells have the gene
32
- What are benefits of genetic engineering in agriculture?
- Higher yields, increased nutrition
- Prevent famine, malnutrition
- Pest resistance - less pesticides needed - low cost, less environmental problems with pesticides
33
- What are benefits of genetic engineering in industry?
- Mass production of enzymes as catalysts - reduced cost
34
- What are benefits of genetic engineering in medicine?
- Drugs and vaccine production - quick, cheap, large quantity
35
- What are concerns of genetic engineering in agriculture?
- Monoculture - all vulnerable to 1 disease, reduced biodiversity
- Superweeds - Interbreed with wild plants and spread recombinant DNA
- Organic farms contaminated with recombinant seeds
36
- What are concerns of genetic engineering in industry?
- Globalisation - few companies control genetic engineering - force smaller companies out of business
- Proper labelling needed - choice to consume engineered products
- Some consumer markets won't import GM foods - economic loss for some producers
37
- What are concerns of genetic engineering in medicine?
- Genetic technology companies could restrict access to life saving tech
- Designer babies
38
What hopes to humanitarians have for GM?
- Reduced famine, malnutrition
- Pharmaceuticals available to more people and more affordable
- Gene therapy
39
How does genetic engineering have ownership issues?
- Who owns genetic information once isolated - donor or researcher?
- Patents for seeds - can charge high prices
40
What is the premise of gene therapy?
- Alters defective genes to treat genetic disorders and cancer
- If caused by 2 mutant recessive alleles - add a working dominant allele
- If caused by a mutant dominant allele - silence it by adding DNA in the middle of it
41
How is an allele inserted in gene therapy?
- Uses vectors like in recombinant DNA technology
42
What is somatic gene therapy?
- Alters alleles in body cells - cells affected by the disorder
- Doesn't affect sex cells - can pass it on
43
What is germ line therapy?
- Alters alleles in sex cells - all offspring won't suffer
- Currently illegal in humans
44
What are ethical issues with gene therapy?
- Used for cosmetic reasons e.g. aging
45
What are DNA probes used for?
- To locate specific alleles of genes or see if mutation is present
46
How do DNA probes work?
- Short DNA strand complimentary to target allele
- Probe will bind to target allele if present
- Has a label - radioactive or fluorescent to be detected
47
How are DNA probes used?
- DNA sample cut up by restriction endonuclease and separated by electrophoresis
- Transferred to a nylon membrane and incubated with a probe
- If present, probe binds to allele - when exposed to UV light will fluoresce or on x-ray film if radioactive
48
What is a DNA microarray?
- Microscopic spots of different DNA probes
- Fluorescently labelled human DNA washed over the array - if any sequences match any probes - will stick
- Washed removing any unbound DNA, visualised under UV light, only labelled DNA visible
- Any spot that fluoresces means the person's DNA contains that allele
49
What are uses of DNA probes?
- Screening for inherited diseases, determine how a patient will respond to specific drugs, identify health risks
50
What is genetic counselling?
- Advises patients about risks of genetic disorders
- Advises people about screening - helps identify if you are a carrier of a mutated allele, the type of mutated allele and most effective treatments
- If screening result positive - counselling used to advise patient on prevention and treatment
51
What is personalised medicine?
- Genes determine how your body responds to drugs - some drugs more effective on some people than others
- Medicines made to be tailored to individuals' DNA - the theory is that doctors have your genetic info - predict how you will respond to different drugs and prescribe the most effective ones
52
What are variable number tandem repeats?
- Not all of the genome codes for proteins - some is VNTRs - don't code for proteins and repeat next to each other over and over
53
How can VNTRs be used to differentiate individuals?
- The number of times the sequence repeats differs person to person so length of these sequences is different
- These repeated sequences occur in lots of places in the genome - number of repeats and places can be compared between individuals -> genetic fingerprinting
54
How is a sample for electrophoresis made?
- Isolate sample of DNA
- PCR used to make copies - primers at each end of the repeat so whole repeat amplified
- DNA fragments produced - length corresponds to number of repeats
- Fluorescent tags added to fragments
55
How is electrophoresis carried out?
- Sample placed in a well on a gel slab with buffer solution that conducts electricity
- Electric current passed through the gel - DNA is negatively charged so moves to positive electrode at far end -> small fragments move faster so travel further - separates fragments by size
56
How are the results of electrophoresis read?
- Fragments viewed as bands under UV light to show genetic fingerprint
- Can compare prints
57
How are genetic fingerprints used to determine genetic relationships?
- About 1/2 of the sequences come from each parent - more bands that match - more closely related -> e.g. in paternity tests
58
How are genetic fingerprints used to determine genetic variability in populations?
- Greater number of bands that don't match - more different - can compare number at different places to find genetic variation
59
How can genetic fingerprints be used in forensics?
- Crime scene DNA isolated, amplified by PCR
- Electrophoresis done compared to suspects
- If they match - links a person to a crime scene
60
How can genetic fingerprints be used in medical diagnosis?
- Refers to unique pattern of several alleles
- Used to diagnose genetic disorders and cancer
61
How are genetic fingerprints used in animal and plant breeding?
- Used to prevent inbreeding which could lead to a decreased gene pool and increased risk of genetic disorders
- Breed the least related individuals together
