Gene Technology Flashcards
1
Q
Recombinant DNA technology
A
2
Q
What is Recombinant DNA Technology?
A
Allows genes to be manipulated , altered and transferred from organism to organism - even to transform DNA itself
3
Q
What is the use of Recombinant DNA Technology?
A
- enable us to better understand organism
- Design new industrial processes
- Medical applications
4
Q
why does Recombinant DNA work?
A
- This is because the genetic code is universal - so the same DNA base triplets code for the same amino acid in all living things
- Also transcription and translation are essentially the same in all living organisms
- so the transferred DNA can be transcribed and translated to produce a protein in the cells of the recipient (transgenic) organism
5
Q
A number of diseases result from being unable to produce various metabolic protein chemicals like insulin, what are some treatments for this?
A
extracting the chemical from a human or animal donor and introducing it to the patient
6
Q
What is a problem with this?
A
this presents problems such as rejection by the patients immune system and risk of infection. The cost is also considerable
7
Q
However there are advantages to this process of making proteins from other sources, what are they?
A
- techniques have been developed to isolate genes, clone them and transfer them into microorganisms
- The microorganisms are then grown to provide a factory for continuous production of a protein
8
Q
The DNA of two different organisms combined this way is called?
A
Recombinant DNA
9
Q
The resulting organism is called?
A
Transgenic or Genetically Modified organism (GMO)
10
Q
What are the number of stages used to make a protein using DNA technology of gene transfer and cloning?
A
- isolation of DNA fragments that have a gene for the desired protein
- insertion of the DNA fragment into a vector
- transformation - transfer of DNA into suitable host cells
- Identification - Of the host cells that have successfully been taken up by the use of gene markers
- Growth/Cloning of the populations host cells
11
Q
What are the three methods for making DNA fragments
A
- Reverse transcriptase
- Restriction endonucleases
- Gene machine
12
Q
Why is it difficult to obtain a DNA fragment containing the target gene?
A
Most cells only contain two copies of each gene, making it difficult to obtain a DNA fragment containing the target gene.
13
Q
Why is mRNA often easier to find?
A
But cells that produce the protein coded for by the target gene will contain many mRNA molecules that are complimentary to the gene. These mRNA molecules can be used as templates to make lots of DNA.
14
Q
Where is reverse transcriptase obtained?
A
Naturally occurring in retroviruses/ viruses such as HIV
15
Q
Why can reverse transcriptase obtained by retroviruses be used to make DNA?
A
the coded genetic information of retroviruses is in the form of RNA, and in a host cell they are able to synthesise DNA from RNA using an enzyme reverse transcriptase.
16
Q
What is the role of Reverse transcriptase?
A
it catalyses the production of DNA from RNA. (reverse of usual transcription)
17
Q
Describe the process which uses reverse transcriptase to make cDNA and dsDNA?
A
- A cell that readily produced the protein is selected (e.g B cells in the islets of Langerhans from pancreas are used to produce insulin)
- These cells have larger quantities of the relevant mRNA, which is therefore more easily extracted (than DNA)
- The isolated mRNA is then mixed with free DNA nucleotides and reverse transcriptase.
- the reverse transcriptase uses the mRNA as a template to synthesise new strands of complimentary DNA (cDNA)
- to make the other strand of DNA, the enzyme polymerase is used to build up the complimentary nucleotides on the cDNA template. This double strand of DNA is the required gene.
18
Q
What is an advantage of making DNA this way?
A
the cDNA made is intron free because it is based on the mRNA template, and virus cells do not have any introns
19
Q
What is a disadvantage of making DNA this way?
A
More steps so more time consuming and technically more difficult
20
Q
What are restriction endonucleases?
A
Naturally occurring enzymes in bacteria, in order to protect them from invading virus. Some bacteria produce restriction enzymes to cut up viral DNA. these are called restriction endonucleases.
21
Q
Why are restriction endonucleases specific?
A
there are may restriction enzymes that have an active site complimentary in shape to a range of different DNA base sequences, describes as recognition sequences, and therefore cuts the DNA at a specific location
22
Q
How can restriction enzymes cut up DNA to produce ‘Blunt Ends’?
A
Some restriction enzymes cut straight across both chains forming Blunt Ends
e.g one restriction endonuclease cuts in the middle of the base recognition sequence GTTAAC
23
Q
How can restriction enzymes cut up DNA to produce ‘Sticky Ends’
A
Some restriction enzymes cut in a staggered fashion, through the two chains. This leaves an uneven cut in which each strand of DNA has exposed unpaired bases (overhang). The exposed staggered ends are palindromic. An example is a restriction endonuclease that recognises a six-base pair AAGCTT.
24
Q
What is meant by Palindromic?
A
some sections of DNA have Palindromic sequences of nucleotides. These sequencs consist of Anti-parallel base pairs (base pairs that read the same in the opposite direction).
25
Each of these sticky ends can be joined together with another sticky end via complimentary bases, but only if..?
but only if the sticky ends were cut up by the same restriction endonuclease.
26
Sticky ends can be joined together if they have the same...?
recognition sites e.g the same sticky ends (and in order to have the same sticky ends they ned to be cut up by the same restriction endonuclease)
27
What enzyme joins sticky ends together?
Sticky ends are joined together using DNA ligase to join the sugar-phosphate backbone together.
28
What is the new DNA molecule called?
The new DNA molecule is called recombinant DNA
29
What is an advantage and disadvantage of making DNA fragments via Restriction endonucleases?
ADV: Sticky ends on DNA fragment make it easier to insert to make recombinant DNA
DISADV: Still contains introns
30
What is the gene machine?
Fragments of DNA can be created in a lab using a computer generated machine, without the need for a pre-existing DNA template, instead a database contains the information needed to produce the DNA fragment.
31
Describe how a gene machine is used to produce DNA fragments?
1. the desired sequence of the nucleotide bases of a gene is determines from the desired protein that we wish to produce. The amino acid sequence of this protein is determined. From this the mRNA codons are looked up and the complimentary DNA triplets are worked out
2. The desired sequence of nucleotide bases for the gene is fed into a computer
3. The sequence is checked for biosafety and biosecurity to ensure it meets international standards as well as various ethical requirements
4. the computer designs a series of small, overlapping single strands of nucleotides, called oligonucleotides, which can be assembled into the desired gene.
5. In an automated process, each of the oligonucleotides is assembled by adding one nucleotide at a time in the required sequence.
6. The oligonucleotides are then joined together to make a gene. This gene does not have introns or other non-coding DNA. The gene is replicated using the polymerase chain reaction
7. The polymerase chain reaction also constructs the complimentary strand of nucleotides to make the required double stranded gene. it then multiplies this gene many times to give numerous copies.
8. Using Sticky ends, the gene can then be inserted into a plasmid. This acts as a vector for the gene allowing it to be stored, cloned or transferred to other organism in the future.
9. the gens are checked using standard sequencing techniques and those with errors are rejected.
32
What are the advantages of this process?
- sequence of nucleotides can be produced in a very short time and with greater accuracy
- The artificial genes are intron free , so can be transcribed and translated by prokaryotic cells (for next process)
33
What is a Disadvantage of this process?
Need to know the sequences of amino acids and bases
34
In Vivo Cloning
35
Describe the role of restriction endonucleases in the production of DNA fragment?
- Restriction endonucleases are used to cut DNA of interest
- these enzymes cut at recognition sites leaving sticky ends (overhang of few bases)
36
What is the importance of sticky ends?
- DNA from different source can be joined
together IF they have the same sticky ends
- if the same restriction endonuclease is used to cut DNA, then all the fragments produced will have ends that are complimentary to one another.
- This means that the single-stranded ends of any one fragment can be joined to the single strand of any other fragment
- Once the complimentary bases of the two sticky ends have paired up,, an enzyme called DNA ligase is used to bind the sugar-phosphate backbone of the two sections of DNA so unite them as one
37
The DNA of two different organism being combined this way is called?
Recombinant DNA
38
Why does the DNA fragment need to be modified?
The DNA fragment needs to be modified (before being added into a vector) to ensure transcription of these genes can occur
39
How is The DNA fragment modified?
- Promoter region is added at the start of the DNA fragment. This is a sequence of DNA which is the binding site for RNA polymerase bind (and transcriptional factors), to initiate transcription to occur
- terminator region is added - this is added at the end of a gene, it causes RNA polymerase to detach and stop transcription, so only one gene at a time is copied into mRNA.
40
Why does the DNA fragment need to be inserted into a vector?
The DNA fragment can not be injected into the host cell, as it is negatively charged, and the phospholipid membrane is negatively charged (both negative phosphate groups repel), Therefore, we need a vector to carry the DNA fragment into a host cell.
41
What is a vector?
used to carry/ carries isolated (and modified) DNA fragments into a host cell
OR
carries isolated DNA fragments from one organism into another
42
What is the most commonly used vector?
the most commonly used vector is a plasmid. Plasmids are circular lengths of DNA found in bacteria., which are separate from the main bacterial DNA.
43
What makes plasmid useful as vectors?
Plasmids are useful because the nearly
always contain antibiotic resistance genes.
44
What happens when the plasmid is cut by the same restriction enzyme used to cut DNA?
- One of the antibiotic resistant genes is disrupted.
- the other antibiotic resistant gene is used in selection of the correct host cells
45
Describe how the DNA fragments is incorporated into the plasmids
-the plasmid is cut open using the same restriction endonuclease
- this creates the same sticky ends
- therefore the DNA fragment sticky ends exposed nucleotides are complimentary to the sticky ends on the plasmid
- the DNA fragment and cut plasmids combine, and enzyme DNA ligase anneals them together
- DNA ligase catalyses the condensation reaction to form phosphodiester bonds between nucleotides
- the plasmids now have recombinant DNA
46
What is Transformation?
The vector (plasmid combined with recombinant DNA) need to be inserted into the host cell, where the gene will be expressed to create the protein required
47
How is transformation achieved?
To do this, the cell membrane of the host cell must be more permeable, by mixing the host cells with Calcium ions (Ca2+) and heat shocked
- this enables the vector/plasmids with recombinant DNA to enter the host cells cytoplasm
48
Why do vectors need to be made permeable?
Vectors are large, so cells have to made permeable
49
What are the many ways a vector can be transferred into a host cell?
- heat shock
- electroporation
- viruses
50
What is a downside of transformation?
Not all bacterial cells will posses the DNA fragments with the desired gene for the desired protein
51
Why don't all bacterial cells possess the DNA fragments with the desired gene for the desired protein?
This is because 3 issues can occur:
- Only few bacteria (as few as 1%) take up the plasmids when the two are mixed together
- the recombinant plasmid doesn't get inside the bacteria cell
- the plasmid re-joins before the DNA fragment is incorporated
- the DNA fragment sticks itself and forms it's own loop, rather than inserting into the plasmid
52
What is the next step?
the next step is that we have to identify which bacteria have taken up the plasmid (with recombinant DNA) before we grow the bacteria on mass
53
How can we identify which bacteria cells have taken up the plasmid with recombinant DNA?
using a marker gene
54
What is a marker gene?
Marker genes are within the plasmid, and can be used to identify which bacteria successfully too up the recombinant DNA
55
What are the three different marker genes?
1. Antibiotic-resistance gene
2. Genes coding for fluorescent proteins
3. genes coding for enzymes
56
Why do plasmids naturally have Antibiotic-resistant genes?
Bacteria have naturally evolved mechanisms for resisting the effects of antibiotics, typically by producing an enzyme that breaks down the antibiotic before it can destroy the bacterium
57
Give one plasmid which is resistant to more than one antibiotic?
Which two antibiotics is the gene resistant to?
R-plasmid - which carries genes resistant to two antibiotics, ampicillin and tetracycline
58
describe the process in which Antibiotic resistant genes help to identify Plasmids with recombinant DNA?
- The DNA fragment is inserted in between the ampicillin resistant gene
- All bacterial cells are grown on a medium that contains the antibiotic ampicillin
- Bacterial cells that have taken up the plasmids will have acquired the gene for ampicillin resistance
- These bacterial cells are able to break down the ampicillin and therefore survive
- The bacterial cells that have not taken up Plasmids will not be resistant to ampicillin and therefore die
59
What technique can be used to identify plasmids that have been taken up the new gene.
Replica plating
60
Describe how replica plating involving Anti-biotic resistant genes is used to identify the bacteria cell which has taken up the plasmid with the required gene?
- The bacterial plasmid contains the desired DNA fragment that we want to clone and also the genes resistant to Tetracycline and Ampicillin.
- The DNA fragment is deliberately inserted/ incorporated in between the gene for resistance to tetracycline, disrupting this antibiotic resistant gene.
- Tetracycline is no longer able to produce the enzyme/protein that breaks down tetracycline, so the bacteria that have taken up the required gene will no longer be resistant to tetracycline (but will still be resistant to ampicillin)
- We can therefore identify these bacteria by growing them on a culture that contains ampicillin, until they grow colonies
- We can use a sterile velvet block, so bacteria can stick on, so we can stamp it onto another petri-dish with Ampicillin antibiotic dissolved within the agar and leave colonies to grow
- The colonies that have survived and grown, have the plasmid in it, because it has the gene resistant to ampicillin
- We can then repeat the replica plating, but using a culture containing tetracycline antibiotic
- the colonies that grew on ampicillin and tetracycline, must be the original plasmid.
- the colonies that have been killed contain the Bacterial plasmid with recombinant DNA
61
Why is Replica Plating used/ what is an advantage of replica plating?
treatment with tetracycline will destroy the very cells that contain the required gene. However using replica plating, it is possible to identify living colonies of bacteria containing the required gene
62
What are the two other marker genes that do not require replica plating?
- Fluorescent markers
- Enzyme Markers
63
Fluorescent Markers - what are they?
Some Jellyfish contain a gene that codes to create a green Fluorescent protein (GFP)
64
Describe how Fluorescent markers are able to identify the bacterial plasmids with the required gene for cloning?
- GFP can be inserted into the bacterial plasmid
- The DNA fragment can be deliberately inserted in between this gene, which disrupts the gene and prevents GFP production and the ability to fluoresce
- Bacteria which take up the recombinant DNA plasmid will not be able to produce GFP, because the gene is disrupted
- then we can grow bacteria on a culture, allowing colonies to grow
- We can simply identify by viewing these colonies using UV light
- Non-glowing colonies contain the recombinant Bacterial Plasmid.
65
Enzyme Markers - what are they?
Another gene that produces an enzyme called lactase can turn a substance colourless to blue.
66
Describe how enzyme markers can help identify the bacterial plasmid with recombinant DNA?
- the Enzyme lactase is inserted into a plasmid
- The DNA fragment is inserted in between the Gene coding for lactase enzyme
- this disrupts the enzyme and and prevents lactase production
- Bacteria can be left to grow on a colourless substrate.
- the bacteria that does not change colour contains the plasmid with recombinant DNA
67
What is an advantage of these two processes?
do not require use of replica plating. Results can be obtained simply by viewing cells (e.g under a microscope). This makes the process more rapid.
68
Give one advantage of using fluorescent gene markers rather than antibiotic gene markers. Explain your answer?
- Results can be obtained more easily and more quickly
- because with antibiotic resistance markers, the bacterial cells with the required gene are killed, so replica plating is necessary to obtain the cells with the gene
- with fluorescent gene markers, the bacterial cells are not killed and so there is no need to carry out replica plating.
(more detailed answer than previous)
69
What is the final step now that we have obtained the bacterial plasmid with the recombinant DNA?
We need to make more copies/clones of the DNA of interest
70
How do we do this?
A fermenter is used to grow multiple copies of the host cell, which have been identified as containing the recombinant plasmid.
- this large cloned population of the host cell can then produce the protein coded for by the inserted DNA fragment (e.g bacteria producing insulin from the inserted insulin gene)
71
What are the advantages of In Vivo cloning?
- it is particularly useful where we wish to introduce a gene into another organisms. (As it involves the use of vectors, once we have introduced the gene into a plasmid, this plasmid can be used to deliver the gene into another organism e.g human)
- It involves almost no risk of contamination. This is because a gene that has been cut by the same restriction endonuclease can match the sticky ends of the opened-up plasmid. Contaminant DNA will therefore not be taken up by the plasmid.
- it is very accurate. the DNA copied has few, if any errors. At one time, about 20%of the DNA cloned in Vitro was copied inaccurately, and these errors will be copied into subsequent cycles
- it cuts out specific genes - it is therefore very precise procedure as culturing of transformed bacteria produced many copies of a specific gene and not just copies of the whole DNA sample
- it produces transformed bacteria that can be used to produce large quantities of gene products. the transformed bacteria can produce proteins for commercial or medical use
72
In Vitro Cloning - PCR
73
What is In Vitro cloning?
via PCR
74
What is the Polymerase Chain Reaction (PCR)?
The polymerase Chain reaction is a method of copying fragments of DNA. The process is conducted using an automated machine called a thermocycler, making it both rapid and efficient.
75
What is required for the Polymerase Chain reaction?
- Thermocycler
- DNA fragment to be copied
- DNA polymerase - taq polymerase
- Primers
- Nucleotides - 4 bases IN DNA (AGCT)
76
What is the thermocycler?
a computer-controlled machine that varies temperature precisely over a period of time
77
What is the role of DNA polymerase (taq polymerase) in this process?
This is an enzyme capable of joining together tens of thousands of nucleotides in the matter of minutes
78
Where is taq polymerase obtained, and why is this type of DNA polymerase required specifically in this reaction?
Taq polymerase is obtained from bacteria in hot springs and is therefore tolerant to heat (thermostable) and does not denature during the high temperatures used as part of the process
(Normal DNA polymerase denatures at around 40-50°C,)
79
What is taq polymerase's optimum point?
Taq polymerase optimum point is 72°C and doesn't denature until high temperatures above 100°C have been reached
80
What are primers?
Single - stranded short sequences of nucleotides/DNA that have a set of bases complimentary to those at one end of each of the two DNA fragments
81
What are the three stages in PCR?
1. DNA is separated into two strands
2. Addition (annealing) of the primers
3. Synthesis of DNA
82
Describe how the Polymerase chain reaction occurs?
1. the reactants are mixed together in a PCR vial. The mixture contains the DNA to be Amplified, DNA polymerase, small primer sequences of DNA and a good supply of nucleotides. The vial is then placed in a PCR machine.
1. The temperature is increased to 95°C, this will break the hydrogen bonds and split DNA fragment into two separate DNA strands
2. The temperature of the mixture is then decreased to 55°C, so primers can join (anneal) to their complimentary bases at the end of the DNA fragment. Hydrogen bonds reform. The primers provide the starting sequences for DNA polymerase to begin DNA copying because DNA polymerase can only attach nucleotides to the end of the existing chain. Primers also prevent the two separate strands from re-joining
3. The temperature is finally increased to 72°C. This is the optimum temperature for DNA polymerase to add complimentary nucleotides along each separated DNA strands. It begins at the primer on both strands and adds the strands and adds the nucleotides in a sequence until it reaches the end of the chain.
83
Why are primers required? - MSA
to allow DNA polymerase to attach which enables replication of the gene to begin
84
Why are two different primers required?
Prevents two original strands from annealing because the sequences at the ends of the target sequence are different/ one is at the beginning and one is at the end
85
How many copies are there at the end of the first cycle?
There are now two copies of the original fragment because both strands are copied simultaneously
86
How long does each cycle take?
So how can we produce?
- Each cycle takes approximately 2 minutes
- So over a million copies of the DNA can be made in only 25 Temperature cycles and 100 billion can be produced in in just a few hours
87
What are the advantages of In Vitro cloning/ PCR?
- Automated - more efficient
- Rapid - 100 billion copies of DNA can be made within hours
- Doesn't require living cells - Quicker and less complex techniques required
88
Give two uses of the polymerase chain reaction? - MSA
- used in the replication of DNA from crime scenes -( so there is no loss of valuable time before forensic analysis and matching can take place )
- gene cloning
89
The Applications and Implications Of Recombinant DNA Technology.
90
What are the uses of Recombinant DNA technology?
- Production of medicines - using animals or plants
- Genetically Modified food
- Gene therapy : Somatic and Germ cell Therapy
91
What applications has been made to Recombinant DNA?
- Applications to Agriculture
- Applications to Industry
- Applications to Medicine
(- Applications to Forensic Science)
92
What Benefits has Recombinant DNA technology contributed to Agriculture?
1. Agricultural crops can be GM so they give higher yields and ae more nutritional - e.g Golden rice - genetically engineered to contain beta-carotene, and when digested is converted into Vitamin A. - Therefore can be used to reduce the risk of famine and malnutrition in economically less developed countries.
2. Can be GM so they resistant to pesticides, droughts, herbicides, different diseeases - so reduces cost of pesticides, medicines, and also increases survival in area with little water
3. Plants can also be GM to produce a particular substance, which is then extracted and harvested (pharming)- can be used to combat diseases
93
What are some Concerns when it comes to using Recombinant DNA technology in Agriculture?
1. Monoculture - farmers may only plant one type of GM crop - could make the whole crop vulnerable to the same disease because plants are genetically identical AND also reduce Biodiversity, which is damaging to the environment
2. Possibility of Superweeds these are weeds resistant to herbicides - can occur of GM plants interbreed with wild plants
3. Organic food Farmers can have their crops contaminated by wind-blown seeds from nearby GM crops. - so can't sell crops as organic and loose money
4. Impossible to predict the ecological consequences of GM plants when released into the environment
5. impossible to predict the long-term consequences of GM food
94
What Benefits has Recombinant DNA technology contributed to Industry?
1. industrial processes use enzymes that act as biological catalysts. These enzymes can be produced from Transformed organisms, - so can be produced in large quantities for less money, reducing costs
2. can be used to control pollution, for example, to destroy harmful gases released from factories
95
What are some Concerns when it comes to using Recombinant DNA technology in Industry?
1. without proper labelling, some people won't have a choice whether to consume genetically modified food
2. Biotech companies control some forms of GE. As the use of this technology increases, these companies get bigger and more powerful, Forcing smaller companies out of business.
3. the financial cost of recombinant DNA in industry, could be used for those who need it more, More justified to use it on fighting hunger and poverty
96
What are the benefits when it comes to using Recombinant DNA technology in Medicine?
1. Animals and plants can be modified to produce a range of substances e.g antibiotics, hormones and enzymes that are used to treat diseases and disorders
2. Drugs and vaccines also can be produced from recombinant DNA technology - very quickly, cheaply and in large quantities. This could make them more affordable and therefore more available to people
3. gene-therapy (replacing defective genes can cure certain genetic disorders, such as cystic fibrosis
97
What are some Concerns when it comes to using Recombinant DNA technology in Medicine?
1. companies that own genetic engineering companies may limit their use of the technologies they have a hold that could be saving lives
2. May be used unethically or unjustifiably for example introduce genes for intelligence, cosmetic improvements etc. - may get into the wrong hands and be used to control
98
Gene Therapy
99
What is gene therapy
Gene therapy involves replacing the defective genes (mutated allele) inside cells to treat genetic disorders and cancer
100
What does gene therapy being carried out depend on?
depends on whether the disorder is caused by a mutated dominant allele or two mutated recessive alleles
101
How is gene therapy carried out if the disorder is caused by two mutated recessive allels?
if it's caused by two mutated recessive alleles you can supplement a working dominant allele to make up for them - you 'supplement' the faulty ones
102
How is gene therapy carried out if the disorder is caused by a mutated dominant allele?
if it's caused by a mutated dominant allele you can 'silence' the dominant allele by sticking DNA in the middle of the alle so it does not work anymore
103
In order to insert DNA fragment within the body cells, what is needed?
A vector
104
What main vectors can we use?
LIPOSOMES - (spheres made up of lipids) Plasmids and altered viruses
105
What are the two types of gene therapy?
- Somatic therapy
- Germ Line Therapy
106
What is somatic therapy?
This involves altering the alleles in body cells, particularly the cells that are most affected by the disorder
107
Give an example of a disease that somatic therapy has been used to treat?
Cystic Fibrosis - somatic therapy targets the epithelial cells lining the lungs
108
Why can offspring still inherit the disease?
any offspring can inherit the disease, as somatic therapy does not affect the individuals sex cells (gametes) so sperm or egg cells
109
What is Germ Line therapy?
this involves altering the alleles in sex cells. This means that every cell of every offspring produced from these cells will be affected by gene therapy and they won't suffer from the disease.
110
What isn't Germ Line Therapy used? - MSA
Currently ILLEGAL To Conduct On Humans
changes to genetic make-up of individual/species/genome/future
generations/germ line;
may affect normal development;
111
What are the ethical issues surrounding gene therapy?
- some people are worried that the technology could be used in ways other than medical treatment , such as treating cosmetic effects of aging
- other argue that there is more potential to do harm than good
- e.g risk of overexpression of genes - gene may produce too much of the missing protein
112
What are the advantages of Gene therapy?
- may be used to prevent, treat, cure genetic/inherited disorders
- may also be used to treat cancer, infections like HIV
- Relatively safe
113
What are the disadvantages of gene therapy?
- risk of cancer, allergic reactions, damage to tissue, organs if an injection is involved
- risk of immune reaction (rejection)
- Expensive, only for the rich
- Open to misuse
114
DNA/Genetic Fingerprinting/ profiling
115
What is genetic Fingerprinting?
Is the analysis of VNTR DNA fragments, and this can be used to determine genetic relationships and the genetic variability within a populaion
116
What are VNTR's?
Variable Number Tandem Repeats - bases sequences of DNA which are repetitive and non-coding (introns)
117
How much of our DNA consists of Non-coding DNA, (so VNTR's)?
95% of human DNA is currently not known to code for any characteristic but may yet to be found functional
118
Why are VNTR;s specific to each individuals/ different in each individual?
For every individual the number and length of VNTR's has a unique pattern
119
Which to individuals are VNTR's the same in?
Identical twins
120
What is the probability of two individuals having the same identical sequences of VNTR's?
is extremely small - 1 in 30,000 million
121
the more closely related two individuals are, the more...?
similar the VNTR's will be
122
What are the 6 stages involved in Genetic Profiling?
1. Collection and Extraction
2. Digestion
3. Separation
4. Hybridisation
5. Development
6. Analysis
123
Describe what happens in the Collection and extraction phase?
- DNA is extracted from a sample, e.g blood, body cells, hair follicles, semen, spit
- If only a small amount of DNA is found/available it can be amplified using PCR
124
Describe what happens in the Digestion phase?
- restriction endonucleases are added to cut DNA into smaller fragments
- The endonucleases are chosen for their ability to cut close to, but not within the target DNA
- the sections of DNA that are cut out are called Restriction fragments and all range in size because the base sequences being cut may be far apart
125
Describe what happens in the separation stage?
- the fragments of DNA are placed into small wells in agar gel and are separated according to size by Gel Electrophoresis under the influence of electrical voltage
- DNA is negatively charged so it is attracted to the positive end of the gel
- the smaller the DNA fragment, the further it moves
- this is how different lengths of VNTR's are separated
- The gel is then immersed into alkali/buffer liquid in order to separate the double strands into single strands
- The pattern of fragments are transferred to a nylon membrane by a process called Southern Blotting (Hybridisation)
126
Describe what happens during Southern Blotting?
- a thin Nylon membrane is placed over the gel
- the membrane is covered with several sheets of absorbent paper, which draws up the liquid containing the DNA by capillary action
- this transfers DNA fragments to the nylon membrane in the same relative positions they occupied on the gel.
- The DNA fragments are then bound/fixed to the membrane using UV light
127
Describe what happens in the Hybridisation process?
- Radioactive or Fluorescent DNA probes are now used to bind VNTR's .
- the probes have base sequences which are complimentary to the base sequences (single-stranded) of VNTR's, and bind to them under specific conditions, such as temperature and pH.
- The process is caried out using different probes, which bind to different DNA sequences
- Any probes not bound are washed off
- the membrane is dried
128
Describe what happens in the development stage?
- Finally, an X-ray film is put over the nylon membrane.
- The radioactive probes on the DNA fragments expose the film
- because these points correspond to the positions of the DNA fragments as separated during electrophoresis, a series of bars are revealed
- these pattern of bands are unique to each individual
129
Describe what happens in the Analysis stage?
- the results can then be analysed and interpreted in an automated scanning machine which calculates the length of DNA fragments from the bands
130
What are the uses of genetic fingerprinting?
- Testing genetic relationships and variability - paternity tests
- Forensic Science
- Medical diagnosis
- Animal and Plant breeding
131
Why can genetic fingerprinting be used to test paternity?
- Individuals inherit half their genetic material from their mother and half from their father
- Therefore each band on a DNA fingerprint of an individual should have a corresponding band in one of the parents DNA fingerprint
132
Describe how genetic fingerprinting can be used to determine genetic variability within a population?
- The more closely related two individuals, the more similar the genetic fingerprints
133
Describe how genetic fingerprint can be used in forensic science?
- can be used to determine whether a person is likely to have been present at the crime scene
- this does not prove the person committed the crime
- other explanations to why their DNA might be present
134
What problem occurs during the collection of a blood sample from a crime scene?
- problems with contamination
- forensic samples from rape victims are rarely pure
- DNA decays quickly so that restriction sites may be lost - may give too few or too many fragments
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Describe how genetic fingerprinting can aid a medical diagnosis?
Diagnosing sarcomas/diseases. The genetic
fingerprint of known sarcomas/diseases are
compared to a fingerprint of a patients
tumour/diseases
136
Describe how the genetic fingerprint can be used in plant and Animal Breeding?
- can identify animals with a desirable gene or particular allele
- individuals selected with this allele can be bred to increase possibility of their offspring having the same characteristics
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DNA probes, Locating genes, Genetic Screening and Counselling
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What are DNA probes?
DNA probes are short, single-stranded pieces of DNA that are labelled radioactively or fluorescently so they can be identified
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What are DNA probes used for?
This is used to locate specific alleles of genes and to screen patients for heritable conditions, drug responses or health risks
140
DNA probes are complementary to..?
the allele that is being screened for
141
Describe simply how DNA probes work?
- the patients sample is treated to make it single-stranded and is then mixed with DNA probes
- If the patient has the allele, then the DNA probe will Hybridise (cross breed) and the label (radioactive or fluorescent) indicates the presence
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What does this process involve?
DNA hybridisation
143
Describe DNA hybridisation
- The patients DNA sample is heated to make it single-stranded
- The heat causes the hydrogen bonds between the bases to break (denaturing)
- the patients single stranded DNA sample is mixed with the DNA probe, and cooled. Any complimentary sequences align and form hydrogen bonds (anneal)
- Some of the patient's DNA samples will anneal back together, but some will anneal with the DNA probe
144
Explain how would we locate a specific allele of a gene?
- to locate a specific allele, the base sequence must be known to then create the DNA probe
- This can be determined using DNA sequencing techniques (e.g Sanger method)
- the fragment of DNA can be produced using the gene machine
- The fragment can then be amplified using PCR
- The label is then added, either a radioactive nucleotide containing the Isotope 32P or a fluorescent label which emits light under UV exposure.
- After hybridisation, the DNA is washed so that any unbound DNA probes are washed away.
- The presence of radioactive or fluorescent labels therefore indicate that the allele of interest is present in the patient's DNA
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What is Genetic Screening?
This method can be used to screen for potential genetic disorders or for the presence of Cancer causing oncogenes.
It is possible to screen for multiple diseases simultaneously using an array, where multiple DNA probes are attached
146
What is DNA microarray?
- a glass slide with microscopic spots of different DNA probes attached to it in rows.
- Human DNA is washed over the array
- Any of the human DNA that contains the DNA sequence which match to the DNA probe will ‘stick’ to the array.
- Array is washed, to remove any unbounded DNA
- therefore, you are able to test simultaneously for many different genetic disorders.
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What is the key reason having your DNA screened is advantageous?
is because of Personalised medicine
148
Give examples of personalised medicine?
- Certain drugs, such as some painkillers, are more or less effective depending on your genotype. It can also help determine the best dose, which increases the effectiveness, safety and can save money
- Vitamin E can be given to diabetics to reduce the risk of CVD for some, but for other with a different genotype, it can actually increase the risk
149
What is genetic Counselling?
Having your DNA screened, Allows genetic counsellors to give advice and information that enable people to make personal decisions about themselves or offspring
150
Why do people usually take genetic counselling?
- research family history of an inherited disease and to advice parents on the likelihood of it arising in their children
- so explains the potential risks to themselves or future offspring if they were carriers of a specific allele
151
Genome projects
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What is the genome?
The genome is the entire genetic material of an organism in the nucleus of the cell (in eukaryotes)
153
What is sequencing a genome?
Sequencing a genome means working out the DNA base sequence for all the DNA in a cell
154
What are genome projects?
In genome projects, scientists work to determine the complete genome sequence (the entire DNA nucleotide base sequence) of a wide range of organisms, including humans.
155
what does success of genome projects depend on?
Their success depends on the complexity of
the organism and the technology that is
available.
156
what was the aim of the genome projects?
To identify all the genes in the human genome.
To find the location of all the genes.
To determine the sequences of the base pairs that
make up the human DNA.
To find the functions of different genes.
To publish the results on a public database.
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How many base pairs does the human genome consist of and how many genes is this organised into?
consists of over 3 billion base pairs organised into around 20,000 genes
158
How long did it take to find out the Human genome?
Took 13 years
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What did they find?
Human genome consist of 3 billion base pairs organised into around 20,000 genes
160
What did they use to help them?
(do not necessarily need to know)
Bioinformatics - collecting + analysis complex biological data uses computers to read, store + organise biological data at a much faster rate than previously
161
What are the sequencing methods used for genome projects? (spec)
The methods used to sequence genomes are continuously being improved and updated and have now become automated
(do not need to know any methods because it is a fast growing area of science)
162
What are some sequencing methods?
Sanger method
163
What is the proteome?
The set of proteins produced by the genome
OR
The set of proteins in a given type of cell/ organism ( at a given time under given conditions)
164
What are examples of simpler organisms?
Simpler organism like prokaryotic cells (bacteria)
165
What was the first bacteria to have the genome fully sequenced?
Haemophillius bacterium - contains 1700 genes and 1.8 billion bases
166
What is the purpose of proteome projects?
- Hope that the information gained can be used to help cure diseases and
- provide knowledge of genes that can be usefully exploited
167
Explain Why is determining the proteome sequence in simpler organism e.g Prokaryotes easy?
- This is because vast majority of prokaryotes are just one circular piece of DNA and not associated with histones
- Prokaryotes do not contain introns - this means that the genome can be used directly to sequence the proteins that derive from the genetic code (the proteome) of the organism
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What is this useful for? - spec
This is useful for many reasons: IDENTIFYING POTENTENTIAL ANTIGENS TO USE IN VACCINE PRODUCTION
169
This means that..?
Antigen memory cells can be produced and trigger a a secondary response when encountered
170
What are examples of complex organisms?
Eukaryotes such as humans
171
What is a problem with studying the genome of more complex organisms?
- More complex organisms contain large sections of non-coding DNA.
- They also contain complex regulatory proteins which determine when the genes that code for particular proteins should be switched on and off
-
172
Why does the following make it more difficult to to translate the genome into the proteome?
This makes it more difficult to translate their genome into their proteome, because it's hard to find the bits that code for proteins among the non-coding and regulatory DNA.
173
DONE!!
