6.1.3 manipulating genomes Flashcards
(52 cards)
1
Q
DNA sequencing is
A
working out the sequence of nucleotides
2
Q
purpose of PCR
A
amplify (increase the length of ) DNA sample hwen there is not enough to be analysed
3
Q
differences between natural DNA replication and PCR
A
- PCR requires addition of primer molecules to make it start
- only short sequences can be replicated
- a cycle of heating and cooling is neded
4
Q
what do u need for PCR?
A
- DNA sample
- free nucletodies
- primers
- taq DNA polymerase
5
Q
PCR 3 steps (and temperatures)
A
- Denaturation 95 degrees
- Annealing 68 degrees
- elongation 72 degrees
6
Q
describe steps of PCR
A
- DNA smaple is mixed with free nucleotides, primers, TAQ DNA POLYMERASE
- DENATURATION: heat to 95 degrees to break the hydrogen bonds between CBP. U now have 2 separate strands
- ANNEALING: cool to 68 degrees so that primers can ANNEAL (bind by hydrogen bonding) to one end of each DNA strand. so now u have a small sectino of double stranded DNA at the end of each strand
- EXTENDING: taq DNA polymerase binds to the double stranded end, temperature is raised to 72 degrees , and the DNAP catalyses addition of free nucleotides to the single stranded. moving in the 5 to 3 direction
- repeat !
7
Q
what is special about taq DNAP
A
- taken from thermophilic bacteria
- optimum temperature is 72 degrees
- means that cycles can be repeated as wont denature in the denaturation section
8
Q
why do u need a primer for PCR?
A
- to CBP to dna
- to provide a starting point for taq DNA polymerase to bind to
- as taq dna polymerase cannot bind to single stranded DNA
9
Q
USES OF PCR
A
- forensic science. amplifying small sections of DNA for further DNA profiling. criminal, parentage
- tissue typing, reduce risk of rejection
- identifying viral infections
10
Q
purpose of electrophoresis
A
used to separate different sized fragments of DNA based on length
11
Q
describe breifly how electrophoresis works and its cmponent
A
- agarose gel plate covered by a buffer solution. electrodes at each end of tank so a current can flow
- DNA samples pre-digested with restriction endonuclease enzymes
- add the DNA
- DNA is negatively charged due to the phosphate groups, so is attracted to positive electrode
- smaller fragments travel faster so move further
- at the end, remove buffer and to vidualise u can use:
- RADIOACTIVE DNA PROBES and x rays
- OR remove buffer and add dye to stain the fragments
12
Q
electrophoresis can alaso be used for
A
proteins. eg hameoglobin
13
Q
describe sanger sequencing [for short strands]
A
- PCR: 4 dishes containing the free nucleotides, DNA polymerase, primers, and a DDNTP fluroescent chain terminator
- thousands of varying lengths are generated due to the adding of the fluroescent base at random positions
- electrophoresis, ethidium bromide and uv to see
- the samller ones travel further
- use it to sequence a genome; what u read from the smallest to the largest is the complementary
14
Q
improved merthod to sanger
A
-high throughput
-pyrosequencing
- whole genome sequencing
15
Q
applications of gene sequencing
A
- HGP
- genome wide comparisons between individuals and species
- !!!!evolutionary relationships
- !!!!genotype phenotype relationships
- !!!!epidemiology (genome of pathogens, allele for a disease)
- predict the amino acid seequence of proteins
16
Q
somatic cell therapy
A
- insert gene into affected body cells
- only affects some cells
- short term (cells die ), must be repeated
17
Q
germ line therapy
A
- insert gene into gamete (egg cell)
- long term sokution, all cells and offspring have it
- designer babies
18
Q
what can u use for somatic and germline to insert the gene
A
- viral vectors
- liposomes
19
Q
what do u use as markers in genetic engineering
A
antibiotic resistant
20
Q
FARMER probelm with genetic engineering
A
patented seeds can only be used for the year, have to buy new seeds eery year
21
Q
how does dna sequencing allow for predictino of amino acid
A
- sequence DNA nucleotides of a gnee
- 3 base pairs = 1 amino acid
22
Q
bioinformatics
A
- access to large amounts of data online
- data on DNA sequences AND PROTEIN structures!!!!
- with a universal format
waht can u do?
- rapid cmpairosn of sequences
- it has amino acid sequences held in database
- computer modelling of new protein structure from base sequence
23
Q
how can DNA sequencing help with a viral outbreak?
A
- sequence of DNA base pairs = codes for aa sequence
- base pairs of antigens on sufrace
- can create vaccine with specific antigen
24
Q
Suggest how the interdisciplinary field of bioinformatics may be useful in determining whether a
newly-sequenced allele causes a genetic disease.
A
- large volume of data hled in computers about DNA sequences and protein structures in a universal format
- holds info about allele and its variations
- rapid computational analysis to compare the allele to existing info
- modelling of protein structure from sequence
25
Explain why only selected sections of non-coding DNA are used when profiling a human.
- alot of the genome is very similar
- so using CODING sequences would not allow for unique identification
26
importance of using taq DNA polymerase?
- DOES NOT DENATURE AT 95 DEGREES
- allows for the PCR to be cycled many times without reloading the enzymes
27
4 ways to visualise dna after electrophoresis
1. visible stain
2. fluorescent tag
3. radioactive tag
28
what must you do to proteins before protein electrohporesis?
- heat
- denature
- expose charged region
29
what must you add to proteins before protein electrophoresis?
- something negatively charged
- as all proteins are diff charges. this ensures thyere all negative
- so all travel in same dirction, all attracted to anode, can be separated by mass
30
describe how DNA can be visualised after electrophoresis has been completed? (2)
- add ethidium bromide
- UV light
31
restriction enzymes
- specific enzymes that cut DNA at specific seqeucnes (phy hydrolysiing sugar phosphate backbone phosphodiester bond)
- can cut to blund ends, or sticky ends (overhanging single strands)
- sceintists will analyse the DNA and then look at the recognition sites on either side to choose the right restriction enzyes
32
how to do dna profiling
- collect dna sample
- extract DNA
- amplify using PCR
- restriction enzymes to cut into variable lengths
- separate using gel electrophoresis
- then remove buffer and use (radioactive probe and x rays) OR ethidium bromide and UV light to visialose
-
33
describe GE for BACTERIA
- GENE probe complementary to the gene u desire, identifies your gene
- use restriction enzymes to remove gene u desire, creates sticky ends
- SAME restriction enzymes to cut plasmid to produc complmentary sticky ends
- CBP of sticky ends
- DNA LIGASE phosphodiester bonds
- to form the recombinant dna
- (can also insert gene for antibiotic resistance with it so u can add antibiotics and then see which have succesfuly taken up the gene)
- recombinant plasmid mixed with bacteria and put into an ELECTROPORATOR: uses electricity to make bacteria csm more permeable
- bacteria are then grown in large fermenters to make lots
34
GM plants
- all the same just after, tje bacteria acts as a vector
- so can infect a plant cell and insert its dNA into the genome of a plant cell
35
advantage of GMO
- eg insulin, no allergic reaction
- increase crop yield, make food more nutritious
- pest resistance, save money on pesticide and porbs eg eutrophication less deep
-
36
disadvantage gmo
- patented and expensive, farers
- can cross pollinate and contaminate wild species
- ethical
- long term
37
problems of gene therapy
- ethical
- body may identify viral vector carrying gene as foreign and trigger immune response
- gene could be inserted into wrong place in genome
38
examples of GOOD things from gm
-insect resistance in soya => higher yield, less use of pesticides so cheaper
- genetically modified pathogens for research
- 'pharming' => genetically modified animals to produce pharmaceuticals
39
(3) why if they have the same adaptation does it not nec mean thyer eclosely related?
- convergent evolution
- adaptatoin
- as subject to the same selection pressure
40
3 ways to get your gene before genetic engineering
1. gene probe with dna sequence comp to that of hte gene u want, restriction enzymes to cut out desired gene
2. obtain mRNA for the gene you want, use reverse transcriptase to make single stranded DNA, then use dna polymerase for double strands
3. sequence the gene, work out base sequence, make that DNA sequence
41
differences somatic and gl terapy
somatic is
- body cells vs egg(gamete)
- only some cells have allele vs all cells
- not inherited vs is passed onto offspring
- not permanent, short term needs repeating vs permanent
42
sanger sequencing
- extract DNA
- cut into fragments into various lengths using restriction enzymes
- use PCR to amplify
43
how can this help with epidemiology
- idnetify pathogen
- identify vulnerable populations
- design vax programs to target certain individuals
44
dna profiling use
- crime scene test
- paternity test
- genetic screening (analysis of disease risk)
45
recombinant dna
combines DNA from 2 sources
46
plant vectors
-plasmids
- virus
- - agrobacterium tumefaciens
47
bacteria waht vector to use
- bacteriophage
- virus
- BAC
48
animal vector
- plasmid
- virus
- BAC
49
GMO pathogens could be used for
+ research
- biological warfare
50
pharming
genetically engineering animals to have a desired gene
+ less disease, faster growth rate
- unknown impacts of GM, welfare of animal
51
germ line
- allele passed to offspring
- long term cure
-BUT, cannot target a specific tissue
ethical implications
52
somatic +-
+
targets specific tissues
extends life span
-
temporary so repeat tratments needed
allele not passed to offspring
viral vector could trigger immune response
ethical issues
