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Biology: Module 6: Genetics, Evolution and Ecosystems > Manipulating Genomes > Flashcards

Manipulating Genomes Flashcards

1
Q

How DNA sequencing got started

A

Sanger first did it using radioactive labelling of bases and gel electrophoresis, done manually so took lots of time, radioactive bases swapped for fluorescent bases, led to automation and scaling up of process, led to capillary sequencing used in HGP

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2
Q

Principles of DNA sequencing

A

DNA mixed with different chemicals, PCR carried out in a thermal cycler, DNA polymerase builds up new strand with the nucleotides as part of PCR, addition of terminator base stops the replication, results in many different lengths of DNA, separated by length in capillary sequencing, fluorescent mark on terminator base used to identify the final base using ‘lasers’, order of bases shows sequence for complementary strand, used to find sequence of original strand

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3
Q

Things that DNA is mixed with for sequencing

A

Primers, Taq polymerase, normal nucleotide bases, terminator bases

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4
Q

Capillary sequencing

A

Gel electrophoresis in capillary tubes

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5
Q

Current developments in DNA sequencing techniques

A

High throughput sequencing

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6
Q

How does high throughput sequencing work?

A

Done on a flow cell, fragments of DNA attached slide, replicated in situ by PCR, clusters of identical DNA strands form, addition of fluorescent terminator bases can stop the reaction to take an image, clusters all sequenced at the same time

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7
Q

Other terms for high-throughput sequencing

A

Massively parallel, next-generation

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8
Q

Bioinformatics

A

Development of the software and computing tools needed to analyse biological data. The best type of science.

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9
Q

Computational biology

A

Studying biology using computational techniques. The best type of science

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10
Q

Applications of DNA sequencing

A

GWAs between individuals and species, determining the sequences of amino acids in polypeptides, synthetic biology

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11
Q

Further detail on how DNA sequencing has allowed for genome wide comparison between individuals and species

A

Show patterns of inherited DNA, show diseases that we are vulnerable to, affects epidemiology

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12
Q

How has DNA sequencing allowed for the study of evolutionary relationships?

A

Compare the sequences from different organisms, rate of mutation used to figure out when the organisms had a common ancestor

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13
Q

Questions that DNA sequencing can help with

A

Studying genotype-phenotype relationship, epidemiology, evolutionary relationships

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14
Q

Fields that contribute to research into genotype-phenotype relationships

A

Bioinformatics, computational biology, proteomics

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15
Q

Reasons why the DNA sequence doesn’t completely determine the amino acid sequence in a polypeptide

A

Spliceosomes, protein modification

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16
Q

Spliceosomes

A

Enzyme complexes that join exons together in any order

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17
Q

Protein modification

A

Length might change to give other proteins

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18
Q

Things that epidemiology covers

A

Finding source of infection, identifying antibiotic resistant strains of bacteria, tracking the progress of an outbreak, identifying drug targets in a genome

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19
Q

Uses of synthetic biology

A

Genetic engineering, use of biological systems in industry, synthesis of new genes to replace faulty genes, synthesis of new organisms

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20
Q

Example of production of new genes to replace faulty genes

A

Replacing faulty genes in cystic fibrosis

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21
Q

Example of synthesis of new organisms

A

Genome of a bacterium made and put in a bacterium

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22
Q

Introns

A

Non-coding regions of DNA that are removed from mRNA before translation

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23
Q

Satellite DNA

A

Short sequences of DNA found in introns, centromeres and telomeres that are often repeated

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24
Q

Other term for satellite DNA

A

Variable tandem number repeats

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25
Microsatellite
2 to 4 bases that are repeated 5 to 15 time
26
Other term for micro satellite
Short tandem repeats
27
How are satellites inherited?
Always in the same place on chromosomes, number of repeats varies between individuals, number of repeats inherited from parents
28
DNA profiling
Producing an image of the patterns in DNA
29
Stages of DNA profiling
Extract the sample, use PCR to get many copies, digest the sample with restriction endonucleases which cut at specific points in introns, fragments contain a mixture of mini and microsatellite regions, separate the fragments by electrophoresis, add radioactive or fluorescent probes, bind to complementary strands in hybridisation, take X-ray images or put under UV light to see the pattern
30
Process of electrophoresis
Agarose gel with wells in the bottom, fragments with known lengths in first and last wells, electric current put through the plate, DNA moves towards the positive electrode, rate of movement depends on size of DNA fragment, gel placed in alkaline buffer solution to denature the DNA fragments, bases exposed, transferred to nitrocellulose paper by Southern blotting
31
Process of Southern Blotting
Nitrocellulose paper placed on top of the cell, covered with sheets of absorbent paper, DNA drawn up in alkaline solution by capillary action, fixed in place by UV light
32
Chemicals that go in a PCR machine
DNA sample, excess of nucleotide bases, primers, Taq polymerase
33
Process of PCR
Temperature raises to 95 degrees celsius, denatures the DNA by breaking hydrogen bonds, decreases to 60 degrees celsius, primers anneal to the ends of the DNA, temperature raised to 72 degrees celsius, optimum temperature for Taq polymerase, adds bases to the primer to make complementary strands of DNA
34
Uses of DNA profiling
Forensics, paternity testing, immigration cases, identifying the species of an organism, showing evolutionary relationships, identifying individuals at risk of particular diseases
35
How does DNA profiling help in identifying individuals at risk of particular diseases?
Certain microsatellites and their patterns associated with cancers and heart disease
36
Uses of PCR
Forensics when tiny amounts of DNA available, lots of DNA made from small sample for DNA analysis
37
Methods of isolating a gene for genetic engineering
Restriction endonucleases, reverse transcriptase
38
How to use restriction endonucleases to isolate a gene for genetic engineering
Restriction endonucleases cut the DNA at a specific base sequence, leave sticky ends
39
How to use reverse transcriptase to isolate a gene for genetic engineering
Isolating the mRNA for the gene, reverse transcriptase makes the complementary DNA
40
How to get the isolated gene into a vector for genetic engineering
Use the same restriction endonuclease to cut open the plasmid, leaves sticky ends that are complementary to the original sticky ends, DNA ligase joints the two strands of the DNA
41
How to identify which vectors have the gene in them
Vectors have a marker gene in them for antibiotic resistance or fluorescence, often have two where one of them will have the desired gene put in the middle of it
42
Methods of transferring a vector into the host cell
Culture the bacterial cells and plasmids together and raise the temperature, electroporation, electrofusion
43
Why does the culturing together and raising the temperature technique work?
Raised temperature increases permeability of the bacterial membrane, plasmids enter
44
Stages of genetic engineering
Isolate desired gene, put in a vector, transformation
45
Transformation
Transferring the plasmid containing the recombinant DNA into the host cell
46
Electroporation
Applying an electrical current to the bacteria, membranes get very porous, plasmids enter
47
Electrofusion
Applying electrical currents to the membranes of two different cells to get them to fuse to form a polyploid cell
48
Disadvantage of electrofusion
Not used in animal cells as they don't survive it
49
Techniques for genetically modifying plants
Agrobacterium tumefaciens , electrofusion
50
How is Agrobacterium tumefaciens used?
Desired gene and marker gene put in Ti plasmid, carried directly into plant cell DNA, callus forms on plant which contains genetically modified plant cells
51
How is electrofusion done in plants?
Plant cell wall removed by cellulases, electrofusion done to make a polyploid cell, plant hormones used to stimulate growth of a new cell wall, callus formation
52
Word to describe the nature of plasmids used in genetic engineering
Recombinant
53
Examples of uses of genetic engineering
Insect resistance in genetically modified soya, genetically modified pathogens for research, genetically modified animals for pharming
54
How genetic engineering has been used to confer insect resistance in genetically modified soya
Gene for Bt protein inserted, Bt protein is toxic to lots of pest insects
55
How genetic engineering has been used to do research with genetically modified pathogens
Collections of sequenced DNA fragments stored in the pathogens, DNA can be used for further genetic engineering, used in development of novel medical treatments
56
How genetic engineering is used in pharming
To create animal models by adding or removing genes so the animals act as models for the development of new therapies, to create human proteins by introducing a gene for a therapeutic protein
57
How to ensure an introduced gene in pharming is only expressed in one place
Promoter sequence
58
Pros of using genetic engineering for pest resistance
Reduces pesticide spraying, protects environment, helps poor farmers, increases yield
59
Cons of using genetic engineering for pest resistance
Toxins in the plants may affect non-pest insects, pests may become resistant, decreased biodiversity, allergic reactions
60
Potential con of genetically modifying pathogens for research
Development of biological weapons
61
Types of gene therapy
Somatic cell, germline cell
62
Diseases that gene therapy could treat
Cystic fibrosis, haemophilia, severe combined immunodeficiency
63
Somatic cell gene therapy
Replacing the mutant allele with a healthy allele in the affected somatic cells
64
Why is somatic cell gene therapy hard?
Issues in getting the alleles into cells, getting the plasmids into the nucleus, maintaining expression of the allele
65
Type of vectors used in somatic cell gene therapy
Viruses
66
Diseases that have been treated by somatic cell gene therapy
Retinal diseases, immune diseases, leukaemia, myeloma, haemophilia
67
Disadvantage to somatic cell gene therapy
Only a temporary solution as somatic cells have limited lifespan, children still affected
68
Germline cell gene therapy
Inserting a healthy allele into germ cells or an embryo after fertilisation in IVF
69
Disadvantages of germline cell gene therapy
Potential impact on individual unknown, lack of consent, might lead to designer babies
70
Limitations of DNA profiling
Other evidence may be ignored in court as this is given too much weighting, mistakes and contamination can occur
71
Name of sites where restriction endonucleases cut
Recognition sites
72
How does DNA sequencing allow the origin of an infection site to be discovered?
Each strain has a different sequence and a strain may come from a place
73
Genetic engineering
Isolating genes for desirable characteristics in one organism and placing them into another organism using a suitable vector
74
How are animals genetically engineered for pharming?
Gene isolated, introduced into egg of the animal, promoter sequence added, fertilised and put back in mother
75
Ethical issues associated with pharming
Animal has to go through fertility treatment, low success rate so ova are destroyed, genes perpetually passed on
76
Role of PCR in DNA sequencing
To amplify DNA, to give a range of lengths
77
Role of restriction endonucleases in DNA sequencing
To cut the genome down to smaller fragments, to cut plasmids

Biology: Module 6: Genetics, Evolution and Ecosystems (4 decks)

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