1
Q
What are some techniques used for studying genes and their function?
A
. The polymerase chain reaction (PCR)
. Cutting DNA using restriction enzymes
. Gel electrophoresis
2
Q
What can the pcr be used for?
A
To select a fragment of DNA and amplify it to produce millions of copies
3
Q
Describe the process of PCR
A
1) A mixture containing the DNA, free nucleotides, primers and DNA polymerase
2) denaturing: The mixture is heated to 95ºc to break hydrogen bonds.
3) Annealing: Mixture is cooled to 55ºc so the primers can bind (anneal) to the strands
3) Extension: Mixture heated to 72º so DNA polymerase can work. It joins onto the primer and joins complementary nucleotides together
4) Two new copies of the fragment are made. This cycle then starts over
4
Q
What are primers?
A
Short pieces of DNA that are complementary to the based at the start of the fragment you want
5
Q
What is DNA polymerase described as?
A
Thermostable because they dont denature under high temps. This means many cycles can be completed without them denaturing and having to be replaced
6
Q
What does anneal mean?
A
Recombining DNA into a double stranded form
7
Q
What is it called when you double the quantity each time?
A
Exponential growth
8
Q
How do you work out the number of DNA molecules after a given number of cycles?
A
2^n
n= number of cycles
9
Q
How do you work out molecules in log numbers?
A
Work out the molecules. Then do log(number) = x
Log10 value would then be 10^x.
To undo this you do e.g 10^7.53
10^7= 10000000
10^0.53 = 3.388…
Then you just x them together
10
Q
What is a thermal cycle machine?
A
A machine that goes through a series of set temp changes to allow cycles of replication to occur
11
Q
What is another way to get DNA fragments?
A
By using restriction enzymes.
12
Q
What do soke sections of DNA have?
A
Palindromic sequences of nucleotides. These consist of antiparalelle bas pairs (bases that read the same in opposite directions)
13
Q
What do restriction enzymes recognise?
A
Specific palindromic sequences known as recognition sequences
14
Q
When can you get a specific DNA fragment?
A
When recognition sequences are at either side of the fragment
15
Q
What are sticky ends?
A
Small tails of unpaired bases at each end of the fragment. Sticky ends can be used to bind (anneal) the DNA fragment to another peice of DNA that has sticky ends with complementary sequences
16
Q
What is a blunt end?
A
When a restriction enzyme cuts DNA and leaves no overhang.
17
Q
What enzyme joins ends together after they have been cut?
A
Ligase
18
Q
Are are restriction enzymes also known as?
A
Restriction endonucleases
19
Q
What is vitro cloning?
A
When PCR produces lots of identical copies of DNA so it can be used outside of a living organism
20
Q
What does the restriction enzyme EcoRI cut?
A
GAATTC
21
Q
What does the restriction enzyme Hindlll cut?
A
AAGCTT
22
Q
What is electrophoresis?
A
A procedure where an electrical current is used to seperate out DNA fragments, RNA fragments or proteins depending on their size.
23
Q
Describe how to carry out electrophoresis
A
1) This uses agarose gel that has been poured and left to solidify. A row of wells is made at one end of the gel. You put this in a tank, with the Wells near the negative electrode. You then add buffer to the reservoirs at the sides of the gel box so the surface of the gel is covered with the buffer
2) using a micropipette, add the same volume of loading dye to Wells. This helps the DNA sink and become more visible. Add a set vol of DNA samples into each well. Use a clean micropipette each time
3) Put a lid on the gel box and connect the leads to the power supply. DNA fragments move to the positive electrode because they are negative.
4) remove the gel tray when nearly at the end and tip of any excess buffer solution. Use gloves and stain the fragments by covering the surface with staining solution then rising with water.
24
Q
What molecules will move further in electrophoresis?
A
Smaller molecules
25
What needs to happen to proteins before they undergo electrophoresis?
They are mixed with a chemical that denatures proteins so they all have the same charge. This is because normally proteins can have different charges
26
What are the uses of electrophoresis of proteins?
Helps identify proteins in urine or blood to diagnose a disease
27
How is the size of DNA fragments measured?
In bases e.g 4 or 1kb = 1000 bases
28
What is the basis of DNA profiling?
Electrophoresis
29
What does agarose gel have?
Pores
30
How can the size of fragments be used after electrophoresis?
To map the location of restrictions. It can also be used to assess relatedness as an individual inherits the location of the cut sites from their parents
31
What must happen if you want to identify the exact identity of a fragment?
You must use fluorescent dye or radioactivly labled DNA probe whose base sequence is complementary to the DNA fragment. The location of the DNA probe can then be identified using UV light or detecting radiation
32
What is some of a genome made from?
Repetitive non coding base sequences. The length of these varies. This can be analysed using electrophoresis and is called a DNA profile
33
What is a DNA profile not likely to be?
The same for any 2 people
34
How does forensic science use DNA profiles?
They use it to compare DNA collected from crime scenes. The DNA is isolated, microsatellites amplified using PCR, electrophoresis used, and DNA profiles produced
35
How are DNA profiles used in medical diagnosis?
DNA profiles can link to a unique pattern of several alleles and can assess the risk of genetic disorders.
36
Explain briefly the steps to identify genes
. Dna isolation
. Fragment separation
. Transfer to membrane
. Make a probe
. Hybridise to DNA
. Visualise
37
How can you make a probe?
Using DNA replication
38
What are the steps to do to hybrisise a probe and the target DNA.
. Nylon membrane
. Promote annealing
. Wash non specific binding of probe
. Autoradiography
39
What is the step "transfer to membrane"?
Place a nylon membrane on the agarose gel. You then place sheets of paper towel on top with a weight. This causes water to be dragged up bringing the DNA with it, causing it to be stuck kn the nylon membrane. The dna forms covalent bonds with the membrane causing it to become stuck. You then expose it to the probe
40
What is a microsatellite?
The non coding but repeating bases in the genome. These are polymorphic and vary in size due to mutations
41
What is Huntington disease and how can DNA profiles detect it?
It is a neurological degenerate disease which causes motor neurones to break down. Its caused by a long repeating base sequence. Everyone has the dominant alleles for it but it depends kn the base sequences after it. If its longer than 40 bases then you will get the disease. The longer it gets the earlier onset
42
What is cystic fibrosis caused by?
A recessive allele. Gene therapy can help this
43
What is the polymerase used in PCR?
Taq polymerase, found from extremophile bacteria
44
What is a bacteriophage?
A virus. REs naturally occurring cuts the dna of the virus at recognition sites
45
What is gene sequencing?
Finding the order of bases kn a gene. Genome sequencing is finding out the order in all of an organisms DNA
46
How can DNA be sequenced?
By using Sanger sequencing (also known as the chain termination method)
47
Describe Sanger sequencing
1. 4 tubes are set up. All have the template, DNA polymerase, primer, free nucleotides. A flourecently labled nucleotide (dideoxynuclrotide terminator). They are A, T, C and G, different ones in each tube.
2. PCR occurs. Strands are different lengths as each one terminates at a different points depending on where the terminator was added.
3. Fragments in each tube is separated by electrophoresis and visualised. This can be read to see the base sequences. The smallest nucleotide (one base) occurs at the bottom. So you can read the sequence from the bottom up.
48
What do dideoxynucleotides do?
Prevent attachment of another nucleotide
49
What gel is used for electrophoresis for gene sequencing?
Polyacrylamide gel
50
What can the Sanger sequence be used for?
Base pairs of around 750 pairs long
51
Explain how to sequence a genome
1. Genome is cut into smaller fragments us8ng restriction enzymes
2. Fragments are inserted into bacterial artifical chromosomes- man made plasmids. Each fragment is inserted into a different BAC.
3. The BACs are inserted into bacteria. Each bacteria contains a BAC with a different fragment
4. The bacteria divide and create colonies of cloned cells that contain the fragment. Together, the colonies make a complete genomic DNA library
5. DNA is extracted from each colony and cut up using restriction enzymes, producing overlapping peices of DNA
6. Each peice of DNA is sequenced
7. Prices are put back together in order to give the full sequence from that BAC
8. DNA fragments from all BACs are put back together in order by computers
52
What is high throughput sequencing?
A faster technique that sequences a lot faster than original methods
53
Describe high throughput sequencing
Dideoxy terminator nucleotides carry floursesnt tags to eliminate radioactive tags. Each terminator type floureces eith a different colour so they can be added to the same tube. The sample is electrophoresis through a capillary tube and a laser detector reads the signature of the fragments and builds up the sequence.
54
What is synthetic biology?
A large field that includes building biological systems from artificially made molecules emg proteins to see whether they work in the way we think they do, and redesigning biological systems to perform better and include new molecules. These molecules may not yet exist in the world
55
What is an example of synthetic biology?
Artemisinin is an antimalarial drug. Until recently we got this from a plant. Using synthetic biology we created the genes responsible for producing a precursor to it. They inserted these genes into yeast so we can use yeast to produce it.
56
What is computational biology?
Using computers to study biology
57
What is bioinformatics?
Developing and using computer software that can analyse organise and store biological data
58
How can gene sequencing be used?
To compare gene and genome sequences between organisms. This is made easier with computational biology and bioinformatics.
59
What does synthetic biology use?
Oligonucleotide synthesiser
60
What can gene sequencing be used for?
.Studying genotype-phenotype
.Epidemiological studies
.Understanding evolutionary relationships
61
What are epidemiological studies?
The study of health and disease within a population. It considers the distribution of disease, its causes and effects. Computerised comparisons between genomes of people that have disease and those that dont an be used to detect particular mutations that may be responsible. This can also be done to pathogens to see different strains
62
How does sequencing help understand evolutionary relationships?
All organisms evolved from a common ancestor. Closely related organisms share more DNA. Whole genomes can be sequenced an analysed to tell us how closely related organisms are. Comparing genomes of members of the same species can also tell us about evolutionary relationships
63
Who acheived synthetic biology first?
Craig venter who worked on the human genome project.
64
What are genetically modified organisms also known as?
Genetically engineered
65
Describe how to genetically modify an organism (soybeans in this case) to make then resistant to something
Scientists have modified soybean plants to include a gene originally found in the bacteria bacillus thuringiensis (Bt). It codes for a protein that is toxic to insects.
. Isolated the gene from BT using a restriction enzyme and insert into a plasmids taken from the bacteria Argobacterium tumefaciens. The plasmid is engineered to remove its phagogenicity, where the target gene is replaced with that
. The plasmid is put back into the bacteria.
. Soybean plants are then deliberately infected with yhe transformed bacteria. The gene then gets inserted into thr soybean plant cells DNAcreating the genetically modified plant
66
What is genetic engineering?
The manipulation of an organisms DNA
67
What are organisms thag have had the DNA altered by genetic engineering called
Transformed organisms. These organisms have recomninant DNA
68
What is recombinant DNA?
DNA formed by joining together DNA from different sources
69
What does genetic engineering involve?
Extracting a gene from one organism and then inserting it into another organism. Genes can also be manufactured instead of extracted
70
What is a transgenic organism?
An organism that has genetically engineered to include a gene from a different species
71
Describe the first step of genetic engineering
The first step is obtaining DNA containing the desired gene. This requires the fragment being isolated using a restriction enzyme
72
Describe the 2nd part of genetic engineering
Making recombinant DNA.
. The vector DNA is isolated
. The vector DNA is cut open using the same restriction enzyme that was used to isolated the DNA fragment containing the desired gene. This means that the sticky ends of the vector DNA are complementary to the sticky ends of the DNA fragment
. The vector DNA and DNA fragment are mixed together with DNA ligase. DNA ligase joins the sugar phosphate backbone of the bits of DNA. This process is called ligation.
. The new combination of thr bases in the DNA is called recombinant DNA
Marker genes can be added to the plasmids so they can be identified
73
What is a vector DNA
Something that is used to transfer DNA into a cell
74
What is the 3rd part of genetic engineering?
Transforming cells.
The vector with the recpmbinant DNA is used to transfer the gene into the bacterial cells (host cells). If a plasmid vector is used, the host cells have to be persuaded to take in thr vector. With a bacteriophage vector, the bacteriophage will infect the host bacterium by injecting its DNA into it. The phage DNA (with the desired gene) then integrates it into the bacterial DNA. To do this the bacterial cells need to be exposed to calcium chloride to increase permeability
75
What is a bacteriophage
Viruses that infect bacteria
76
What is electroporation?
Where a suspension of bacterial cells is mixed with a plasmid vector and placed in a machine called an electoporator. This created an electrical field in the mixture which increases the bacterial cells permeability allowing them to take in the plasmids
77
What are the ethical issues with GM soybean plants
+
They reduce chemical pesticides needed
They can be designed to be more nutritious
-
It may encourage monoculture
Monoculture decreases biodiversity and could leave the whole crop vunruble to disease
Also a risk that GM soybean plants could intervreed with wild plants creating superweeds- resistant to herbicides
78
What is pharming
Producing genetically modified organisms such as animals
79
Give an example of pharming
Hereditary antithrombin deficiancy is a disorder that makes blood clots more likely. This can be reduced with infusions of the protein antithrombin. Scientists have developed a way to produce high yeilds of this protein in goats.
1. DNA fragments that code for the protein are extracted
2. DNA fragments injected into a goats embryo
3. Embryo is implanted into a female goats
4. When offspring is born it is tested to see if it can produce the protein
5. If it does selective breeding is used to produce a Gerd of goats that produce it in their milk
6. The protein is extracted from the milk and used to produce a drug (ATryn) that is given to the deficiency
80
What are the ethical issues with pharming?
+
Drugs can be made in large quantities to make them more available
-
Manipulating animal genes may cause harmful side effects
Enforces that animals are just assets
81
What other ways can a gene be isolated?
-restriction enzymes
-PCR with primers that will bind to either end of the gene
-isolated the gene from mRNA in cells actively expressing the gene converting it into cDNA using reverse transcritpionase
82
How else does transformation occur in genetic engineering?
Heat shock may be used whee they are mixed on ice the heat shocked to 40
83
What is the 4th stage of genetic engineering?
Selection and culture. The bacterial cells are put on an agar plate and allowed to grow. The bacteria that took up the plasmids are identified to be cultured. E.g using uv light
84
How else can transformed bacteria can be identified
If the bacteria contains antibiotic genes that where wanted, they are placed in antibiotics. The ones with the gene would survive. The gene is inserted in the plasmid into the middle of a.col9ur selection gene, so successful insertion inactivated the gene meaning the bacteria cannot convert substrate into colour. Plating the bacteria out on media that contains the substrate for the col9ur selection gene means coloured colonies dont have the gene
85
What else can gene modification be used for?
Genetic modification of pathogens for research
86
What are the ethical issues with GM for bacteria?
+
Improve patient outcomes
-
Potential for outbreaks
May be mutations making them pathogenic
May be used for bio warfare
87
What is the sharing on knowledge skills and technology called?
Technology transfer. Although this occurs, some people may choose to obtain legal protection for their GM products e.g by getting a patent
88
What are the ethical issues of ownership of GM organisms?
+
More money
Scientists compete so products are made faster
-
Farmers in poorer countries may not be able to afford patented GM seeds
89
What are genetic disorders?
Inherited diseases caused by abnormal genes or chromosomes
90
What can gene therapy be used for?
Curing inherited disorders
91
What does gene therapy involve?
Altering alleles inside cells. How you do this is dependant on whether the disease is caused by a dominant or recessive allele.
Recessive: can add a working dominant alleles
Dominant: you can silence the dominant alleles (by sticking a bit of DNA in the middle of the allele so it doesn't work anymore).
92
What are some vectors that may be used for gene therapy?
Altered viruses
Plasmids
Liposomes
93
What is somatic gene therapy?
This involves altering the alleles in body cells. This doesn't affect the individuals sex cells though so offspring mya still inherit the disease
94
What is germ line therapy?
This involves altering the sex cells meaning every cell of the offspring produced from the cells will be affected by the therapy. This is currently illegal
95
What are the positives ethical issues of gene therapy?
. Could increase life spans
. Increases quality of life
. Germ line would allow the carriers of genetic disorders to conceive a baby without the disorder
. Germ line may decrease the number of people that suffer from genetic disorders and cancer meaning fewer people will need treatment
96
What are the negatives ethical issues of gene therapy?
. Technology could be used in other ways apart from medical treatments
. Potential to do more harm than good
. Concern that gene therapy is expencive
97
What are the disadvantages of gene therapy?
. The body could identify vectors as friends bodies and start and immune response
. An allele could be inserted into the wrong place in the DNA
. Inserted allele could get over expressed producing too much of the missing protein
. The effects of treatments may be short lived in somatic therapy
. The patient may have to undergo multiple treatments with somatic therapy
. May be difficult to get the allele into specific body cells
Biology OCR
flashcards
Decks in class (25)
# Cards
-
Cell Structure And Microscopy51
-
Biological Molecules97
-
Nucleotides And Nuclaic Acids32
-
Enzymes35
-
Biological Membranes36
-
Cell Division And Organisation41
-
Exchange And Transport55
-
Transport In Animals66
-
Transport In Plants46
-
Disease And The Immune System71
-
Biodiversity59
-
Classification And Evolution77
-
Ecosystems80
-
Populations And Sustainability45
-
Neurology124
-
Homeostasis And Hormonal Control83
-
Plant Responses And Hormones79
-
Excretion133
-
Photosynthesis75
-
Respirstion56
-
Cellular Contril53
-
Patterns Of Inheritance58
-
Evolution ^31
-
Manipulating Genomes97
-
Cloning And Biotechnology57
