19) Genetic Technology Flashcards
1
Q
What are the properties of plasmids that allow them to be used in gene cloning ? (7)
A
- double stranded DNA
- have restriction site for restriction enzyme
- allows gene (for cloning) to be inserted
- small
- plasmid can enter (host) cell / bacterium
- circular
- stable
- contain marker genes / genes for antibiotic resistance
- used to identify recombinant, (bacterial) cells
- replicate, fast / independently
- get many copies of cloned gene
2
Q
Outline the principles of genetic engineering. (6)
A
- add DNA to give new characteristic
- gene obtained using restriction enzyme.
- Reverse transcriptase makes gene using mRNA.
- gene inserted into plasmid using Ligase.
- recombinant plasmid inserted into host cell.
- cloning.
3
Q
Explain the roles of restriction endonucleases/enzyme
A
- cut DNA at restriction site.
- give sticky (staggered cut) /blunt (straight cut) ends.
4
Q
Explain the roles of Ligase
A
- join DNA with plasmid.
(- seal sugar phosphate backbone.
- make phosphodiester bonds.
- make recombinant DNA.)
5
Q
Explain the roles of Reverse Transcriptase
A
- uses mRNA as a template to make cDNA
6
Q
define ‘Recombinant DNA’
A
DNA produced by combining DNA from two or more different organisms BY using enzymes
7
Q
state what is ‘genetic engineering’
A
- manipulation of genetic material
- inserting/deleting/substituting DNA at specific parts of the genome
- used to modify characteristic in host organism
8
Q
name 3 ways genes can be generated for genetic engineering
A
- genes cut from donor DNA9 using restriction endonucleases
- generated from an mRNA sequence using reverse transcriptase
- genes synthesised from nucleotides
9
Q
function of PCR
A
- if only small amount of DNA available so..
- cloning & amplifying a fragment of DNA
10
Q
4 substances required for PCR
A
- DNA fragment to be amplified
- Primers (short nucleotide sequences)
- DNA Tag Polymerase
- Nucleotides
11
Q
describe the steps of PCR (4)
A
- heat to 90C so DNA denatures
- cool to 50/65C so primers bind to DNA
- heat to 68/75C so Taq DNA Polymerase makes new strand of DNA
- repeat cycles to amplify DNA
12
Q
explain the role of Taq Polymerase
A
- copies DNA at high temperatures
- it is resistant to heat
- so does not denature at high temperatures
- does not need to be added again each time/cycle
13
Q
why are primers needed for PCR
A
- provide a starting point for Taq Polymerase
- prevent two DNA strands joining back after they are separated
14
Q
what is the function of gel electrophoresis
A
to separate DNA fragments, nucleic acids and proteins according to size & charge
15
Q
outline the process of gel electrophoresis
A
- cut DNA w restriction enzyme
- DNA mixed w loading dye/staining agent
- gel is covered w buffer solution
- DNA is negatively charged
- moves to anode
- due to electric field (when current applied)
- larger/longer fragments move slowly/less far
- gel impedance causes DNA to separate in order of size
- visualise under UV light, DNA fluoresce
- compare position w DNA ladder to estimate lengths of DNA fragments
16
Q
what is a plasmid
A
- circular DNA structure found in bacteria
- vector
- carries DNA into host cell
- contains DNA for fluorescent protein
- contains promoter
17
Q
how can microarrays be used to assess gene expression
A
- use mRNA to make cDNA
- fluorescent dye attached to cDNA
- probes on microarray
- binding of cDNA to probe
- wash off excess DNA
- use UV light to produce fluorescence
- intensity of fluorescence indicates level of gene expression
18
Q
Benefits of producing human proteins by Recombinant DNA
A
- the protein can be produced, rather than that from an animal (eg: pig)
- rate is faster than other methods
- ca be made in large quantities
- cheap/low cost
- does not harm animals
- no risk of transferred disease
- does not cause immune response
- gene product can be easily extracted & purified
19
Q
why is genetic screening carried out ?
A
- to test/diagnose genetic conditions
- to determine best treatment for particular genotype in personalised medicine
20
Q
what are the ethical considerations of genetic screening?
A
- psychological burden to make decision
- can find out sex / has disease or not
- can choose to abort baby
- method of obtaining fetal DNA can cause miscarriage
21
Q
Suggest why a promoter & desired gene is transferred into an organism
A
- for transcription factors to bind to promoter
- for RNA polymerase to bind to promoter
- so gene is expressed at right time
- to control transcription of marker gene
22
Q
Explain how DELLA proteins act as repressors preventing gene expression
A
- (DELLA proteins) bind to transcription factor
- transcription factor, cannot bind to, DNA / promoter
- RNA polymerase cannot bind to promoter
- no transcription / mRNA not made
23
Q
how does gene editing stop certain genes from being expressed
A
- insert/delete/substitute DNA
- so transcription of gene prevented
- replaces mutated DNA w correct sequence
24
Q
2 domains that are a source of restriction endonucleases
A
- bacteria
- archaea
25
Steps to carry out new method for large-scale production of a specific restriction endonuclease
- obtain specific restriction endonuclease gene
- PCR to make more copies of the gene
- cut a plasmid with restriction enzyme to give sticky ends
- join gene with plasmid using ligase
- add plasmid to bacteria
- use marker gene to select bacteria w recombinant plasmids
- use fermenter
26
advantages of database for study/use of restriction endonucleases
- data stored is large amount of DNA sequences
- compare sequences
- share data
- use to predict structure of enzyme
27
outline the procedure of gene therapy
- give a drug to increase no. of stem cells in bone marrow
- vector containing normal allele
- remove stem cells
- mix stem cells w vector
- radiotherapy to make space in bone marrow to kill stem cells
- transduced stem cells injected into blood
28
ethical/social implications of gene therapy
- expensive
- long term treatment
- no donor needed
- no need for regular treatment
- religious objections
- may cause cancer
29
outline ways in which genetic technology can be applied to medicine [7]
- genetic engineering
- make insulin
- genetic screening
- detect number of alleles for Huntingtons
- detect allele for cystic fibrosis
- gene therapy
- insert normal allele into cell
- to treat SCID
30
explain how a marker gene can show the expression of genes
- marker gene added to plasmid/vector
- use SAME promoter
- so both genes expressed
- both proteins produced
- use UV light to detect fluorescence
- fluorescence shows transcription/expression of gene of interest
31
advantages of genetic engineering resistance
- higher yield
-increased income
32
Explain why DNA hybridisation occurs between the probe DNA and the added DNA in microarray analysis
complementary base-pairing between DNA strands
33
State the name of the type of proteins that control gene expression in plants.
transcription factors
34
outline the steps required to prepare a genome for analysis using microarray
- extract DNA from cells
- cut DNA into small fragments using restriction enzyme
- denature into single stranded DNA
- add fluorescent dye
35
social/ethical considerations for genetic testing
-cost/availability of test
- informed reproductive decision
- reduced worry/ you can tell if child is negative or positive
- plan ways to care for child
36
Name the disease that is treated using recombinant human factor VIII
haemophilia
37
Explain why a single infusion of gene-corrected stem cells is enough to cure the disease.
- stem cells multiply
- correct gene is passed onto/inherited by new cells
38
State two ethical considerations of using a retrovirus for gene therapy
- retrovirus / new gene, could / must not, insert in wrong place / disrupt other genes
- could / must not cause cancer
- could / must not cause, infection / disease
- could / must not, cause, immune / allergic response
39
Explain why gene editing is more suitable as a potential cure for Huntington’s disease
- HD is caused by a dominant allele
- adding a ‘normal’ recessive allele would not work
- gene editing can delete mutant DNA
40
Some individuals taking part in gene therapy trials have been naturally exposed to the
virus carrying the functional gene, so that their blood already contains antibodies to
the virus.
Predict how this will affect the success of the gene therapy treatment.
decrease success because:
- antibodies / immune response, may destroy vector
- (so) limiting / stopping delivery of gene / allele /DNA, to cells
- destroys cells that have successfully taken up the virus
41
advantages of gene editing
- its accurate
- patients own gene can be edited/no introduction of gene
- no need to introduce promoter
- less risk of cancer/immune response
42
use of genes for fluorescence using marker genes
- insert marker gene to vector/plasmid
- gene of interest inserted close to marker gene
- marker gene protein emits light
- visible colour change
- use UV light
- easy to identify transformed bacteria
43
Explain why electrophoresis produces a DNA banding pattern on a gel.
- negative DNA moves to positive electrode
- smaller/lighter fragments move faster/longer
44
what does DNA polymerase do
makes complementary DNA strand
