application of reproduction & genetics Flashcards
(86 cards)
1
Q
define DNA sequencing
A
- reading the base sequence of a length of DNA and determining the order of bases in the sequence.
- originally done through Sanger sequencing but Next Generation Sequencing is much quicker and more powerful
2
Q
define 100K genomes project
A
- launched in 2012
- it uses NGS to sequence 100,000 genomes from NHS patients with cancer or rare diseases and from members of their family
3
Q
define polymerase chain reaction
A
a method allowing DNA to be amplified rapidly for analysis
4
Q
define short tandem repeats
A
repeating blocks of introns found in DNA
5
Q
define primer
A
a short, single stranded DNA molecules which is complementary to the start of the sequence you want to amplify in PCR
6
Q
define gel electrophoresis
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a method of separating DNA fragments according to size to produce a genetic fingerprint
7
Q
define DNA profiling
A
the process of determining an individual’s DNA characteristics
8
Q
define genetic engineering
A
a technique allowing DNA to be manipulated, altered or transferred from one form to another
9
Q
define recombinant DNA
A
produced when the donor DNA fragment is spliced into the DNA of another organism
10
Q
define reverse transcriptase
A
an enzyme found in retroviruses which produces a strand of DNA from a strand of mRNA
11
Q
define plasmid
A
a small DNA molecule within a cell that is physically separated from chromosomal DNA and can replicate independantly
12
Q
define restriction endonucleases
A
enzymes which cut DNA at specific base sequences
13
Q
define DNA ligase
A
enzyme which joins DNA, used to splice the sugar phosphate backbones of the donor and vector DNA
14
Q
define gene therapy
A
replacing defective alleles with copies of a new functional DNA sequence
15
Q
define somatic cell therapy
A
targets body cells in affected tissues
16
Q
define germline therapy
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introduces corrective genes into germ line cells (sperms/eggs) so genetic corrections are inherited
17
Q
define tissue engineering
A
biochemistry, cell biology, engineering and material science to repair, improve or replace biological tissues
18
Q
what is the name of repeating blocks of introns found in DNA?
A
hyper variable regions or short tandem repeats
19
Q
define exon
A
regions of DNA that code for proteins
20
Q
define introns
A
between exons are regions of non-coding DNA called introns which contain blocks of repeated nucleotides
21
Q
what are the three stages of PCR?
A
- separation
- annealing
- extension
22
Q
what is made using PCR?
A
copies of specific fragments of DNA
23
Q
describe the process of separation in PCR
A
- the target DNA molecule is dissolved in a buffer and is heated to 95C
- this breaks the hydrogen bonds and denatures the DNA causing the two strands to separate exposing the nitrogenous bases
24
Q
describe the process of annealing in PCR
A
- the sample is cooled to 50-60C to allow the short DNA primers to bind to the DNA strands
- they form hydrogen bonds with complementary bases
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describe the process of extension in PCR
- heating to 70C allows thermally stable DNA polymerase (Taq) to add complementary nucleotides by forming the phosphodiester bonds in the sugar phosphate backbones
- this creates two double stranded molecules
26
Taq polymerase is described as thermophilic. explain what this means and why this is important for the process of PCR
- can withstand high temperatures
- will not denature at high temperature so can use high temperature to separate DNA strand
27
identify the limitations of PCR
- contamination
- error rate
- sensitivity to inhibitors
- limits on amplification
- DNA fragment size
28
describe contamination as a limitation of PCR
- any DNA that enters the system by mistake will be amplified
- sources may be air-borne or come from the previous PCR reactions using the same apparatus
29
describe error rate as a limitation of PCR
- taq polymerase cannot proof-read and correct errors in the base sequence
- each cycle copies and multiplies DNA, so these errors accumulate
30
describe sensitivity to inhibitors as a limitation of PCR
molecules in the sample may act as inhibitors and PCR is very sensitive to them
31
describe limits of amplification as a limitation of PCR
after about 20 cycles, PCR slows down and plateaus because:
- reagent concentrations become limiting
- the enzyme denatures after repeated heating
- DNA in high concentration causes the single stranded molecules to base pair with each other rather than the primers
32
describe DNA fragment size as a limitation of PCR
- PCR is most efficient at making DNA about 1000-3000 base pairs long because taq polymerase cant correct its errors
- however, many genes, including human genes, are much longer than this
33
what can the information (produced by HGP and the 100K genome project) be used for?
- identification of allele sequences has enabled scientists to scan a patient's DNA sample for mutated sequences and to compare the sequence of DNA bases in a patient's gene to a normal version of the gene
- IVF embryos can be screened for the presence of alleles which cause conditions including cystic fibrosis, Huntington's disease and thalassaemia
- genetic screening can be useful in association with genetic counselling, allowing a couple to make informed decisions before having children
34
what ethical issues are there regarding the screening of DNA?
- ownership of genetic information that could lead to potential discrimination e.g insurance or job application, social stigmatisation and misuse of the data
- there are a number of concerns regarding the possibility of routine screening for adult onset disorders such as Alzheimer's disease and some cancers. some people do not want to learn this information about themselves and it could cause anxiety
- concerns have arisen over embryo screening, choosing alleles to ensure specific characteristics
35
outline the solutions provided using sequenced genomes, regarding the control of malaria
- the DNA sequence of the anopheles gambiae genome was completed in 2002 and is allowing scientists to develop chemicals that could render the mosquito susceptible to insecticides again, preventing it from transmitting malaria
- the genome of plasmodium sp. was sequenced in 2002. a better understanding of genetical control of plasmodium infection will allow the development of more effective drugs
36
state some uses of DNA profiling
- forensic use
- paternity testing
- phylogenetic study to determine relatedness
- siblings : people who have been adopted may wish to determine blood relatives
37
outline the process of gel electrophoresis
- DNA fragments are loaded into wells at one end and a voltage is applied across the gel
- DNA fragments have a negative charge (on the phosphate group) and so are attracted to the positive electrode. smaller fragments will move further because they move through the pores in the gel more easily
- a DNA ladder can be run alongside the sample. a DNA ladder contains DNA fragments of known length. this can be used to determine the length of DNA in the sample being analysed
38
what are the applications of genetic engineering?
- transfer genes into bacteria to make useful products, such as human insulin
- transfer genes into plants and animals so they acquire new characteristics (e.g disease resistance)
- transfer genes into humans so they no longer suffer from genetic disease (e.g cystic fibrosis)
39
outline the stages of genetic engineering
1. identify and isolate DNA fragments from donor organism using restriction enzymes or reverse transcriptase
2. insert DNA fragments into a vector
3. transfer the recombinant DNA into suitable host cell
4. identify host cells which have taken up and are expressing the recombinant DNA
5. clone the host cells
40
define donor DNA
a DNA fragment containing desired gene from donor
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define vector
transfer host DNA into a suitable host cell
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define clone
genetically identical copy
43
what are the 5 steps of the formation of recombinant DNA?
1. identify and isolate DNA
2. inserting the DNA into a vector
3. transfer of recombinant DNA into a host cell
4. identify host cells which have taken up the recombinant DNA and are expressing it
5. clone the host cells
44
in what two ways can the desired gene be isolated
1. locate it on the donor DNA and cut it out using restriction endonuclease (enzymes that cut DNA as specific base sequences)
2. extract mRNA from a cell actively synthesising the required protein/polypeptide and use reverse transcriptase and DNA polymerase to produce a double stranded cDNA fragment
45
what is the source of reverse transcriptase enzyme?
retrovirus
46
what is the role of reverse transcriptase enzyme?
- can be used to produce a single strand of DNA (cDNA) from this mRNA
- DNA polymerase enzymes can then add free DNA nucleotides to the template strand to produce a double-stranded section of cDNA which codes for the desired protein
47
outline the production of cDNA
1. mRNA is extracted from donor cells e.g from beta cells in the pancreas (for the gene for insulin production)
2. reverse transcriptase enzymes are used to make a DNA copy of the mRNA
3. the newly synthesised single strand of DNA is called cDNA (complementary DNA). many copies of cDNA are made
4. DNA polymerase enzymes catalyse the addition of free DNA nucleotides, this converts the cDNA into a double strand
5. DNA is copied many times using the PCR.
48
what are the benefits of using reverse transcriptase to produce cDNA?
- overcomes the problem of locating the gene on DNA
- avoids restriction enzymes cutting the desired gene into non-functional fragments
- no introns present in cDNA
- no need for post-transcriptional processing to produce functional mRNA
49
describe the process of inserting the DNA into a vector
- cut the isolated donor DNA using restriction endonuclease (restriction enzymes)
- these cut the DNA between specific base sequences (restriction sites) such as GAATTC
- many restriction enzymes cut DNA in a staggered pattern and leave overhanging ends with unpaired bases exposed, known as "sticky ends"
- the SAME restriction enzymes are then used to cut the plasmid DNA at the same base sequences
- DNA ligase enzymes are used to splice (join) together the sugar-phosphate backbones of the donor and vector DNA together
- the new plasmid is now known as recombinant DNA
50
why should the same restriction enzyme be used on the DNA isolated for insertion and the vector (plasmid) DNA?
- to ensure the sticky ends of the donor DNA fragments and plasmid are complementary
- this means complementary bases can form hydrogen bonds
51
describe the process of transferring recombinant DNA into a host cell
- recombinant DNA mixed with bacterial cells and calcium chloride in the hope that bacteria will take up the plasmid
- however, usually only 1% of bacteria take up the plasmid and become transformed
52
how do scientists identify which bacterial cells have taken up the plasmid?
by using a marker gene
53
outline the pros of genetic engineering of bacteria
- allows the manufacture of medical productions e.g insulin to treat diabetes and human clotting factors to treat haemophilia
- prevention and treatment of disease e.g produce vaccines
- enhancing crop growth - modified bacteria secrete molecules toxic to pests
54
outline the cons of genetic engineering of bacteria
- plasmids are easily transferred. genes may be exchanged with other bacteria and antibiotic resistance marker genes could be transferred to pathogenic bacteria
- a new microorganism with a new gene may become a threat if released into the environment
- the possible transfer/activation of oncogenes by using fragments of human DNA
55
describe GM soy beans
- soy beans are an important source of food
- used to produce a wide range of products e.g. flour, protein, oil, bread and soya milk.
- 'Roundup ready' soybeans are genetically modified to contain genes for herbicide resistance.
- the crops can be sprayed to remove weeds without inhibiting their growth
56
describe Bt tomatoes
- BT is a bacterium that lives in the soil
- it contains genes that produce protein that acts as an insecticide
- this leads to less crop spoilage, higher crop yield, less tomatoes targeted by insects
57
describe antisense tomatoes
- tomatoes ripen naturally when they produce an enzyme that breaks down their cell wall
- a second copy of this gene was inserted into tomato plant cells to prevent translation and block the production of this enzyme
- this leads to larger shelf life, less food spoilage during transport
58
outline the benefits for GM crops
- substantial reduction in pesticide use on crops engineered for resistance to fungal pathogens and insect attack
- superior keeping qualities
- higher crop yield
59
outline the concerns for GM crops
- reduction in biodiversity such as limited crop varieties and beneficial insects being killed
- it is claimed there may be adverse health effects of eating a crop that is expressing a new gene and making a new protein
- pollen from GM plants engineered for herbicide resistance might transfer genes to wild relatives / contaminate organic crops
60
what is the main aim of gene therapy?
to treat a genetic disease by replacing defective alleles in a patient with copies of a new DNA sequence
61
what methods can be used in gene therapy?
- a virus as a vector
- a plasmid as a vector
- injection of naked plasmid DNA
62
what are the two ways of replacing defective genes and describe them
1. somatic cell therapy
targets body cells in affected tissues
2. germ line therapy
- rare
- introduces corrective genes into germ line cells so genetic corrections are inherited
63
what are the problems with somatic cell therapy?
genetic changes are not inherited and do not appear in future generations
64
suggest why germ line therapy can be controversial
genes interact with one another
influencing genes in the gamete has unpredictable effects in future generations
65
what causes DMD?
caused by one or more deletions in the dystrophin gene.
- deletions in any of the 79 exons alter the reading frame of the dystrophin mRNA
- the ribosome meets a stop codon too soon and the full polypeptide is not translated
- therefore functional dystrophin is not synthesis
66
how can DMD be treated?
drug drisapersen - an antisense oligonucleotide, a sequence of 50 nucleotides that is complementary to the mutated sequence
67
how does DMD treatment work?
what type of treatment is this called?
- treats DMD by acting as a 'molecular patch', binding to the mRNA over the exon with the deletion.
- that portion of the mRNA becomes double stranded, so the ribosome can not translate it
- this restores the reading frame, so that a shorter, partially functional dystrophin protein can be made
- this is called exon skipping
68
disadvantages of gene therapy?
- only a small proportion of the introduced gene are expressed
- problems when using virus as a vector
69
what are the potential problems of using virus as a vector?
- there may be an immune response in the patient
- the virus may invade non target host cell
- the virus could potentially become pathogenic and cause disease
- the virus may affect other genes such as formation of oncogenes
70
what is genomics?
study of the structure, function, evolution and mapping of genomes as exemplified by human genome project and the 100K projects
71
how does genomics improve healthcare?
- more accurate diagnosis
- better prediction of the effect of drugs
- improved design of drugs
- new and improved treatments for disease
72
what is tissue engineering and what is the goal?
- uses biochemistry, cell biology, engineering and material science to repair, improve or replace biological tissues
- goal: to produce 'off the shelf' bio-artificial organs and to regenerate injured tissue in the body
73
what is the role of stem cells in tissue engineering?
- use to regenerate tissues and organs
- to screen new drugs
- to develop model systems to study normal growth and identify the causes of birth defects
- to investigate the events that occur during human development
74
name some example of tissues or organs that have been created using tissue engineering
bladder
trachea
bronchi
75
two types of stem cells?
embryonic stem cells - found in 3-5 day old embryos
adult stem cells: found in some adult tissues like bone marrow. they replace cells that are lost through wear and tear, injuring or disease, but they cannot form all cell types
76
name an ethical issue of cloning of human tissues and organs
there is fear that stem cells may lead to humans being cloned, an act that fundamentally devalues human life
77
what are some requirements for the use of embryos and embryonic stem cells?
- no financial reward for any development or discovery made using them
- stem cells left over from IVF are deposited in the UK stem cell bank so they are available for other research groups. donors must give specific consent to embryos created with their gametes being used in stem cell research
78
what are the possible arguments against the use of stem cells?
- moral status of an embryo. embryos do have moral rights, but not to the same degree as a living person
- fear that the use of stem cells may lead to humans being cloned
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