Manipulating genomes Flashcards
(120 cards)
1
Q
What is DNA profiling used for?
A
Identification of individuals or familial relationships.
2
Q
What is DNA profiling?
A
Producing an image of the patterns in the DNA of an individual.
3
Q
What are introns?
A
Non-coding regions of DNA.
4
Q
What is the genome of an organism?
A
All of the genetic material that it contains.
5
Q
What is satellite DNA?
A
Repeated short sequences of DNA within introns, telomeres ad centromeres.
6
Q
What is a microsatellite?
A
Smaller region of 2-4 bases repeated 5-15 times.
7
Q
What is a minisatellite?
A
Sequence of 20-50 base pairs repeated from 50 to several hundred times.
8
Q
What are microsatellites also known as?
A
STRs
9
Q
What do STR stand for?
A
Short tandem repeats
10
Q
What position do satellites appear on a chromosome?
A
Same position but the number of repeats vary.
11
Q
What are the 5 main stages of producing a DNA profiling?
A
1) Extracting the DNA
2) Digesting the sample
3) Separating DNA fragments
4) Hybridisation
5) Seeing the evidence
12
Q
DNA profiling- What does extracting the DNA involve?
A
PCR/ Polymerase Chain reaction
13
Q
How much tissue is needed to carry out PCR?
A
Tiniest amount is enough
14
Q
What are the stages of PCR?
A
1) Temperature is raised to 90-95 degrees for 30 seconds. This denatures the DNA by breaking H bonds so strands separate.
2) Temperature is decreased to 55-60 degrees and the primers bind to the ends of the DNA strands.
3) Temperature is increased to 70-72 degrees for at least one minute. DNA polymerase adds bases to the primer, building up double stranded DNA indentical to the orginal sequence. The enzyme Taq polymerase is used.
15
Q
How is the enzyme Taq polymerase obtained?
A
From thermophilic bacteria found in hot springs.
16
Q
Why in stage 3 of PCR is the temperature raised to 70-72 degrees?
A
Optimum temperature for DNA polymerase.
17
Q
Why are primers needed in PCR?
A
For the replication of strands to occur.
18
Q
DNA profiling- What is involved in digesting the sample?
A
Strands of DNA are cut into small fragments using restriction endonucleases. They make two cuts, oncr through each strand of the DNA double helix.
19
Q
Where does a restriction endonuclease cut the DNA?
A
Recognition site or restriction site.
20
Q
Why are restriction endonucleases vital for scientists?
A
Gives the ability to cut the DNA strands at defined points in the introns.
21
Q
DNA profiling- What is involved in the separation of the DNA fragments?
A
Electrophoresis
22
Q
What are the steps of electrophoresis?
A
1) DNA fragments are put into wells in agarose gel strips which also contain a buffering solution.
2) When an electric current is passed through the electrophoresis plate, the DNA fragments in the wells at the cathode end move through the gel towards the positive anode at the other end.
3) When the first fragments reaches the anode, the electric current switches off.
4) Gel is placed in the an alkaline buffer solution.
5) Strands are transferred to a nitrocellulose paper or nylon membrane, which is placed over the gel. The membrane is covered with several sheets of dry absorbent layer, drawing the alkaline solution containing the DNA through the membrane by capillary action.
6) The single stranded fragments of DNA are transferred to the membrane as they are unable to pass through it. They are transferred in precisely the same relative positions as they had on the gel.
7) They are then fixed in place using UV light or heated at 80 degrees.
23
Q
Why is the often DNA fragments of known length used in the first and last well?
A
To provide a reference for fragments sizing.
24
Q
What does the rate of movement of DNA fragments depend on?
A
Mass and length
25
Why can DNA fragments move from the cathode to the anode?
Phosphate groups are negatively charged.
26
What move faster, small or large fragments?
Smaller
27
When is the electric current switched off in gel electrophoresis?
When the faster smallest fragments reach the anode of the gel.
28
How are DNA fragments fixed in position at the end of gel electrophoresis?
Using UV light or heated at 80 degrees.
29
What technique is given to the process of placing strands onto a nylon membrane/nitrocellulose paper?
Southern blotting
30
In gel electrophoresis, why is the gel placed in an alkaline buffer solution after the current is switched off?
To denature the DNA fragments, the two strands separate, to expose the bases.
31
DNA profiling- What is hybridisation?
Radioactive or fluorescent DNA probes are now added in excess to DNA fragments. They bind to the complementary strands of DNA under particular conditions of pH and temperature.
32
What are the role of primers in DNA hybridisation?
Identify the microsatellite regions that are more varied than the larger minisatellite regions.
33
What happens to the excess probes in DNA hybridisation?
They are washed off
34
What are DNA probes?
Short DNA or RNA sequences complementary to a known DNA sequence.
35
DNA profiling- How do you see the evidence if radioactive labels were added?
X ray images are taken of the paper/membrane.
36
DNA profiling- How do you see the evidence if florescent labels were added?
Paper/membrane is placed under UV light so the fluorescent tags glow.
37
Give a summary of DNA profiling.
1- DNA is extracted.
2- Restriction endonucleases cut the DNA into fragments.
3- Fragments of separated using gel electrophoresis.
4- DNA fragments are transferred from the gel to nylon membrane in a process known as Southern blotting.
5- DNA probes are added to label the fragments. The radioactive probes attach to specific fragments.
6- membrane with radioactively labelled DNA fragments is placed onto an X ray film.
7- Development of the X ray film reveals dark bands where the radioactive or florescent DNA probes have attached.
38
What can DNA traces be obtained from?
Blood, semen, saliva, hair roots, skin cells
39
Give 7 uses of DNA profiling.
- Giving evidence for guilt of a suspect
- Giving evidence for innocence of a suspect
- Prove paternity of a child
- Immigration cases to prove or disprove family relationships
- Identify the species an organism belongs to
- Demonstrate evolutionary relations between organism
- Identifying individuals who are at risk of developing particular diseases
40
How can DNA profiling be used to identify individuals that are at risk of developing diseases?
Certain non-coding microsatellites have been found to be associated with an increased risk of particular diseases such as heart disease. These specific gene markers can be identified and observed in DNA profiles.
41
What is used along with DNA profiling to make confident risk assessments for different diseases?
DNA sequencing
42
What is DNA sequencing?
Process of determining the precise order of nucleotides within a DNA molecule
43
What did the beginning of DNA sequencing involve?
Radioactive labelling of bases and gel electrophoresis on a single gel. Carried out manually and took a long time.
44
What the Sanger sequencing enable scientists to do in the 1970s?
Read sequences of 500-800 bases at a time.
45
Give a development of Sanger sequencing.
Swapping of radioactive labels for coloured fluorescent tags, which led to scaling up and automation of the process.
46
What is an alternate name for DNA sequencing?
Capillary method
47
What is the basic principles of DNA sequencing?
1) DNA is mixed with a primer, DNA polymerase, excess of nucleotides and terminator bases.
2) Mixture is placed in a thermal cycler which rapidly changes the temperature at programmed intervals. 96 degrees the double strand separates. At 50 degrees the primers anneal to the DNA strand. At 60 degrees DNA polymerase starts to build up new DNA strands.
3) Each time a terminator base is incorporated instead of a normal nucleotide, the synthesis of DNA is stopped as no more bases can be added. As the chain terminating bases are present in lower amounts and are added at random, this results in many DNA fragments of different lengths depending on where the chain terminating bases have been added during the process. The DNA fragments are separated according to their length by capillary sequencing.
4) The fluorescent markers on the terminator bases are used to identify the final base on each fragment. Laser detect different colours and thus order of sequence.
5) The order of bases shows the sequence of the new, complementary strand of DNA which has been made. This is used to build up the sequence of the original DNA strand.
6) Data is fed into a computer that compares fragments and finds areas of overlap.
7) Once a genome is assembled, you can identity the parts if the genome that code for specific characteristics.
48
What does capillary sequencing work like?
Gel electrophoresis
49
What is the colour of adenine?
Green
50
What is the colour of guanine?
Yellow
51
What is the colour of cytosine?
Blue
52
What is the colour of thymine?
Red
53
What can be used now instead of gel or capillaries for sequencing?
Reaction can take place on a plastic slide.
54
What is the plastic slide used in sequencing known as?
Flow cell
55
What are advantages of high-throughput methods of sequencing?
Efficient, fast, cheaper, more genomes can be sequenced.
56
How is bioinformatics different to computational biology?
Describe different aspects of the application of computer technology to biology.
57
What is bioinformatics?
Development of the software and computing tools needed to organise and analyse raw biological data including mathematical models.
58
What is computational biology?
Uses data from bioinformatics to build theoretical models of biological systems, which can be used to predict what will happen in different circumstances.
59
What does the quick sequencing of genomes of pathogens enable?
- Doctors find the source of infection
- Identify anti biotic resistant strains of bacteria
- Track progress of outbreak of a potentially serious disease
60
What is DNA barcoding?
Identify sections of the genome which are common to all species but vary between them
61
What is the region chosen for DNA barcoding in animals?
648 base pair section of mitochondrial DNA in the ogene cytochrome c oxidase, that codes for an enzyme in cellular respiration
62
Why is this 648 base pair section chosen?
It is small enough to be sequenced quickly and cheaply but varies enough to show differences between species.
63
Why is the 648 base pair sequence not used for plants?
DNA does not evolve quickly enough to show clear differences between species
64
What is used for DNA barcoding in plants?
Two regions in DNA of chloroplasts
65
How is the barcoding sequence not perfect?
Not yet found suitable regions for fungi and bacteria.
66
What is DNA barcoding used for?
Identifying species
67
How can genome sequencing be used to help find evolutionary relationships?
Build evolutionary trees
68
What is proteomics?
Amino acid sequencing of an organisms entires protein complement.
69
True or false,
| Genes can code for many different proteins.
True
70
What is the non-coding DNA?
Introns
71
Are introns or exons removed?
Always introns, sometimes exons.
72
After splicing of pre-mRNA, how are the exons joined together?
By spliceosomes.
73
What is pre-mRNA?
Exons and introns
74
How can a single gene produce several versions of functional mRNA?
Spliceosomes may join the exons in a variety of ways.
75
How can joining the exons in a variety of ways result in many different phenotypes?
Different mRNA code for different arrangements of amino acids which give different proteins, which give several phenotypes.
76
What is synthetic biology?
The design and construction of new artificial biological pathways, organisms or devices or the redesign of existing natural biological systems.
77
Give 4 techniques of synthetic biology.
1) Genetic engineering.
2) Synthesis of an entire new organism.
3) Synthesis of new genes to replace faulty genes.
4) Use of biological systems in industrial contexts.
78
What are the two ways restriction endonucleases are used to isolate the desired gene?
1) Straight cut to get blunt ends
| 2) Staggered cut to get sticky ends
79
What is the most common technique to isolate the desired gene?
Uses restriction endonucleases.
80
What is the advantage of sticky ends for isolating a gene?
Easier to insert the gene into the DNA of a different organism.
81
What is another technique to isolate a gene?
Isolate the mRNA for the desired gene and use reverse transcriptase to produce a single strand of complementary DNA.
82
What is the advantage of using the reverse transcriptase method to isolate a gene?
Easier to identify the required gene.
83
What is the most common vector in genetic engineering?
Bacterial plasmids
84
What are bacterial plasmids?
Small circular molecules of DNA
85
How are the plasmids used as vectors chosen?
Contain a marker gene
86
How is a DNA fragment put into a plasmid?
- Same RE is used to cut open the plasmid. So they have complementary sticky ends.
- Once the complementary bases are lined up, DNA ligase forms phosphodiester bonds, joining them together.
- Plasmid is the cut to insert the desired gene. If inserted correctly, marker gene will not function.
87
Why are the vectors usually given a second marker gene?
To show plasmid contains the recombinant gene.
88
Give two methods of transferring the vector to the host cell.
Electroporation.
Culture bacteria and plasmids in a calcium rich solution and increase temp, which causes bacterial membrane to become permeable and plasmids can enter.
89
What is the process of transferring the vector called?
Transformation
90
What is electroporation?
Small electric current is applied to bacteria which makes membrane porous and plasmids move into cells.
91
What precautions should be taken when doing electroporation?
Power of current should be monitored so no permanent damage is done to the membrane.
92
What is electrofusion?
Tiny electric currents are applied to membranes of two different cells. This fuses the cell and nuclear membranes of the two cells together to form a hybrid/ polyploid cell.
93
What is electrofusion used for?
Produce GM plants
| Producing monoclonal antibodies
94
Why is electrofusion different in animals?
Cells don't fuse easily together as their membranes have different properties.
95
How can you genetically modify plants?
Use Agrobacterium tumefaciens, which causes tumours in healthy plants. Desired gene is placed in the Ti plasmid along with the marker gene. This is then carried directly into the plant cell DNA. The transgenic plants cells form a callus which can be grown into new transgenic plants.
96
What is a callus?
Mass of GM plants
97
How can transgenic plants be produced by electrofusion?
Cells produced have chromosomes form both original cells and so are polyploid. Plant cell walls are removed by cellulases. There is electrofusion to form a new polyploid cell and the use of hormones to stimulate the growth of a new cell wall. Followed by callus formation and the production of many cloned transgenic plants.
98
Why is it harder to engineer the DNA of eukaryotic animals then it is of bacteria and plants?
Cell membranes are harder to manipulate.
99
Why is it important to engineer animals?
So animals can produce medically important proteins and to cure human genetic disease.
100
What are the main techniques for genetic engineering?
Isolating the gene and then inserting into vectors, and transferring the vector.
Electrofusion
101
What are the stages of GE in plants?
1) Cut leaf
2) Expose to bacteria carrying a weedkiller resistant gene and an antibiotic resistant gene. Allow bacteria to deliver genes into leaf cells.
3) Expose leaf to antibiotic to kill cells which lack new gene. Wait for surviving cells to form a callus.
4) Allow callus to sprout shoots and roots.
5) Plants are transferred to soil where they develop into differentiated adult plants that are resistant.
102
What are the advantages of GM crops?
- Pest-resistant varieties reduce amount of pesticide spraying which protects the environment.
- Crops which are resistant to diease will have a greater yield.
- Herbidices can reduce competing weeds.
- Extended shelf life reduces wastage.
- Crops can grow in a more conditions.
- Nutritional value can be increased.
- Plants could prodce human medicines and vaccines.
103
What are the disadvantages of GM crops?
- Insect pests may become resistant to pesticides in GM crops.
- Transferred genes may spread to wild populations and cause problems.
- Biodiversity could be reduced if herbicides overused to destroy weeds.
- Fear of superweeds.
- Extended shelf life may reduce commercial value and demand for crop.
- People may be allgeric to different proteins made.
104
Why can patenting of GM crops affect those who need them most?
New inventions are patented, which means if someone wants to use it, they must pay. The people who need it most, cannot afford it.
105
What are microinjections?
Tiny particles of gold covered in DNA.
106
How can you produce GM vertebrates?
By use of microinjections and modified viruses to carry new genes into animal DNA.
107
Give an example of an GM animal.
Swine flu resistant pigs
108
What is pharming?
Use of GE to produce human medicines.
109
Give the two aspects to pharming.
Creating animal models- addition or removal of genes so that animals develop certain diseases, acting as models for the development of new therapies.
Creating human proteins- Introduction of a human gene coding for a medically required protein.
110
What are the ethical issues of pharming?
Should animals act as models?
Is it ok to put genes into animals?
Is it acceptable to put genes from another species into an animal without being certain it wont cause harm?
111
Give 2 diseases caused by a faulty/ mutant gene.
CF, Haemophilia
112
Give two methods of gene therapy in humans.
Somatic cell
| Germ line cell
113
What is somatic cell gene therapy?
Replaces mutant allele with healthy allele.
114
What is the positives of somatic cell gene therapy?
Successful treatments of diseases such as retinal disease, leukaemia.
115
What are the negatives of somatic cell gene therapy?
For some, it is only a temporary solution, somatic cells have a limited lifetime and are replaced from stem cells, which will have the faulty allele. This would be passed onto their offspring.
116
What is germ line cell gene therapy?
Insert healthy allele into germ cells, usually the egg or embryo. Individual would be born healthy and would pass normal allele to their offspring.
117
Why is germ line cell gene therapy illegal in most countries?
Medical and ethical concerns.
118
What are the medical concerns with germ line cell gene therapy?
Impact on individual of an intervention on the germ cells is unknown.
119
What are the ethical concerns with germ line cell gene therapy?
Done without consent of unborn individual.
May at some point be used to choose desirable characteristics of offspring.
120
Where has germ line cell gene therapy been a success?
On animal embryos.
