1
Q
why are bioinformatics useful?
A
- format of information is universal
- facilitates access to large amounts of data
- genes can be put into clusters which show the same pattern of gene expression
2
Q
what is a genome?
A
all the DNA of an organism including the genes that carry all the info for making a protein
3
Q
what is a PCR? (polymerase chain reaction)
A
a biomedical technology that can be used to amplify a short length of DNA into thousands of millions of copies.
4
Q
outline the stages of PCR
A
- Add DNA template to nucleotides, primer, and taq polymerase, and add to thermocycler.
- Mixture is then heated to 95C to separate two DNA strands.
- Mixture is cooled to 68C to allow primers to anneal.
- Mixture is heated to 72C to allow Taq Polymerase to add nucleotides to the new strand of DNA.
- Process is repeated until required amount of DNA is obtained.
5
Q
why is the PCR heated to 95C?
A
expose the bases by separating hydrogen bonds.
6
Q
why is the PCR heated to 68C
A
allows the primers to anneal and tells polymerase which parts need to copy.
7
Q
why is the PCR heated to 72C?
A
optimum temperature for taq polymerase to function
8
Q
Why is Taq polymerase used in the PCR?
A
- Adapted to live in hot environments.
- Enzymes do not denature at hot temperatures.
- Allows PCR to continue as a cycle without having to add more enzyme.
9
Q
Why is PCR difficult to use?
A
- Too little DNA
- DNA damaged/fragmented
- DNA sample is contaminated
10
Q
how is PCR different to natural DNA replication
A
- natural DNA works at 37C, PCR works at 95C
- DNA polymerase VS Taq polymerase
- PCR is smaller (has 10,000 base pairs)
11
Q
how is PCR used in tissue typing
A
reduce risk of rejection of transplanted organs
12
Q
how is PCR used in detection of encogenes
A
finding a particular mutation that has resulted in cancer to give tailored medicine
13
Q
how is PCR used in forensics
A
amplifying small quantities found at crime scenes for genetic profiling
14
Q
how is PCR used in identifying viral infections
A
amplifying small quantities of viral DNA in patient samples
15
Q
Explain how PCR is used in genetic finger printing
A
PCR allows amplification of small quantities of DNA from fingerprinting which can be used for genetic profiling
16
Q
which way will DNA move in electrophoresis and why?
A
it will move towards positive anode because DNA is always slightly negative
17
Q
what is electrophoresis?
A
separation of DNA fragments depending on size, used to separate proteins by their size.
18
Q
outline the steps of electrophoresis
A
- Restriction endonuclease cut DNA samples at specific recognition sites.
- Agarose gel is set up with alkaline buffer (provides resistance to movement)
- Dense loading dye is used to help add the digested DNA to the wells.
- Make sure not to pierce the bottom of the well with pipette!
- DNA samples are put in place and gel is left.
- DNA fragments move through the gel (smaller ones travel faster) towards the anode as DNA has an overall negative charge.
- Buffer is poured away, and dye is added to stain the fragments.
19
Q
suggest 2 uses of electrophoresis
A
genetic finger printing
forensics
20
Q
why do smaller fragments travel faster through the gel in electrophoresis?
A
lower molecule mass because lower number of base pairs, therefore less resistance to flow.
21
Q
what is the function of the loading dye in electrophoresis?
A
help add digested DNA to the wells.
22
Q
what is the problem of using electrophoresis?
A
not all proteins have a negative surface charge
23
Q
what is the solution to the fact that not all proteins have a negative surface charge in electrophoresis?
A
heat protein to denature them and expose hydrophobic region. Use a charged detergent to equalise the surface charge to make sure all proteins are negatively charged. Separation will therefore be down to mass/length, and not charge.
24
Q
what is DNA profiling?
A
the creation of a unique genetic finger printing of an individual
25
what are minisatellites (variable number tandem repeats VNTRs)
a sequence of 20-50 base pairs repeated hundreds of times.
26
how many base pairs are micro-satellites? what are they also referred to as?
2-4 base pairs - short tandem repeats (STRs)
27
state the (one word!!) procedure of DNA profiling
1. extraction
2. digestion
3. separation
4. hybridisation
5. development
27
if restriction enzymes are cut at specific recognition sites, how does the number of VNTRs affect the size of DNA fragments produced after being cut at restrictions enzymes?
more VNTRs = larger fragments.
27
satellites always appear at the same position in the...
chromosome
28
describe extraction process of dna profiling
DNA is extracted from sample
29
describe separation process of DNA profiling
first, fragments separated using gel electrophoresis.
second, DNA fragments transferred from gel to nylon membrane in process of southern blotting.
30
describe digestion process of DNA profiling
restriction endonucleases cut DNA into fragments
31
describe hybridisation process of DNA profiling
DNA probes added to label the fragments - these radioactive probes attach to specific attachments.
31
describe development process of DNA profiling
membrane with radioactively labelled DNA fragments is placed on X ray film.
development of X ray film reveals dark bands where the radioactive/fluorescent DNA probes are attached.
32
what is southern blotting
transferring single stranded fragments of DNA from the agarose on to a nylon membrane.
33
what is a DNA probe?
single short strand of DNA that is complimentary to a section of DNA being investigated.
34
how does a fluorescent marker work?
emits colour on exposure to UV light when it binds to complimentary DNA.
35
how can the use of probes be applied to society?
can be used to identify disease alleles leading to personalised medicine and genetic counselling.
36
what is cystic fibrosis
- lack of functional CFTR gene
- incorrect chloride channel proteins
symptoms: thick mucus, inflammation, reduced breathing
37
what is gene therapy
Inserting a functional allele into cells that contain a mutated and non-functional allele. The person can then produce functional proteins, alleviating symptoms of their genetic disorder.
38
How could gene therapy help treat cystic fibrosis
functional chloride channels could be produced alleviating symptoms.
39
what is germ line gene therapy
inserting functional alleles into gametes or zygotes
40
what are strengths of germ line therapy
- Treatment permanent
- All cells will be replaced with functional gene
- Will pass onto offspring
41
what are weaknesses of germ line therapy
- Ethical objections because of possible side effects to future generations…
- Offspring can’t consent
- Concern if they couldn’t disrupt the expression or regulation of other genes = cancer
- Could it lead to parents altering their kids and choosing desirable characteristics.
41
what is somatic cell gene therapy
inserting functional alleles in body cells.
42
how does somatic cell gene therapy work on liposomes
Functional alleles placed in a phospholipid sphere. Liposomes are placed in a nasal spray to treat cystic fibrosis sufferers.
43
what is the weakness of somatic gene therapy on liposomes?
- DNA less likely to be incorporated into nuclear DNA and not able to merge with nuclear membrane.
- Less efficient at introducing a gene.
44
general downsides to somatic gene therapy
- As epithelial cells die and are required to be replaced regularly.
- only helps lung problems caused by cystic fibrosis.
44
how is somatic gene therapy work on viruses?
replace genome with functional allele and virus injects it into the cell and it made to be non-virulent.
45
What is a weakness of somatic gene therapy on viruses?
- Although its safe there is possibility of immune response.
- Patient could become immune to these viruses stopping gene delivery.
- Viruses could insert the gene into part of the genome which is important e.g. controls cell division = mutation/cancer.
- Interferes with normal gene expression.
46
cons of genetically modified bacteria
- antibiotic resistance
- could mutate and become pathogenic
- lead to people creating agents for bio warfare
46
pros of genetically modified bacteria
- treat diseases
46
differences between somatic and germ line
- somatic cannot be inherited, germ line can be inherited
- somatic gene introduced to non-reproduction cell, whereas in germ line introduced to sex gametes
- somatic is short term, germ line is long term.
47
pros of genetically modified plants
- produce toxins which kill insects therefore there is a reduced need to spray pesticide.
- Reduces health problems in farmers who have to spray pesticides.
48
pros of genetically modified animals
- Silk from goat milk can be used for bulletproof vest.
- Genes for human pharmaceutical proteins used to treat hereditary emphysema.
49
cons of genetically modified bacteria
- Toxic to pests and other organisms/species = interferes with food chains.
- Creates superweeds which can’t be brought under control with herbicide.
- GM organisms could damage their health
- companies will charge farmers for GM seeds each year.
50
cons of genetically modified animals
- Concerns were raised about the welfare of the GM goats. However, these animals are valuable and likely to be well looked after and not be eaten.
51
describe what makes a modified nucleotide unique to that of a normal nucleotide
- has no H group on 3rd carbon
- stops DNA replication
- contains fluorescent label
52
chain termination is exactly the same as PCR, what happens after the primers anneal in this process?
single stranded DNA is mixed with DNA nucleotides + one type of modified nucleotide.
53
once the modified nucleotide is added in chain termination...
Polymerase is released and reaction stops. This continues and many DNA molecules are made of varying lengths - electrophoresis separates them.
54
what is gene sequencing?
working out the sequence of all bases in one strand of DNA.
55
what makes pyrosequencing special?
it detects bases via the emission of light.
56
what is genetic engineering?
process that obtains a specific gene from one organism and place that gene into another organism using vector (transgenic)
57
state the benefits of genetic engineering
- increase yield by introducing traits
- production of medicine
- scientific research
- improve crops e.g. golden rice (vitamin A)
58
what is pharming
production of human medicines
e.g. inserting human gene promoter sequence into fertilised egg = desired protein in milk.
59
GENETIC ENGINEERING step 1: explain two ways you could isolate a desired gene.
1. use fluorescent probe to locate gene then cut out using restriction.
2. using PCR and specific primers to amplify region of genome that contains desired gene.
60
GENETIC ENGINEERING step 2: explain how you would get gene into vector to form recombinant DNA
plasmid = circular pieces of DNA found in some prokaryotic cells e.g. E.coli
insert gene into plasmid so complimentary bases from h bonds. DNA ligase will then form phosphodiester bonds in sugar phosphate backbone = recombinant DNA.
61
GENETIC ENGINEERING step 3: explain how you would get vector into cell
heat shock treatment in the presence of calcium ions.
62
when a gene is genetically engineered, it is called transgenic - what does this mean?
an organism that contains a gene from another species.
63
how can replica plating be potentially dangerous?
it can increase chance of natural bacteria becoming resistant to antibiotics - as replica plating requires the use of antibiotic resistant bacteria.
64
how can soy be genetically engineered?
soy is a plant with added "Bt" gene that comes from bacterium which codes for a protein toxic to insects.
gene and plasmid cut with restriction enzyme inserted into plasmid, this plasmid is then reinserted into plant cell of soy.
65
what are the cons of genetically modifying pathogens
- increases risk of cancer
- interferes with gene regulation
66
what are the pros of genetically modifying pathogens
- make vaccines
- less side effects
module 6 biology
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