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Manipulating genomes Flashcards

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1
Q

What is DNA sequencing?

A

The process of working out the order of nucleotides within DNA.

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2
Q

What are the ingredients for DNA sequencing?

A

DNA for sequencing.
Primer
DNA polymerase
Excess of normal nucleotides
Terminator bases labelled with radioactive/fluorescent tags.

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3
Q

What do the terminator bases do?

A

They attach where a normal nucleotide would and stop DNA synthesis when they are included.

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4
Q

What do you need in each test tube for Sanger sequencing?

A

4 test tubes.
In each you need ;
The DNA being sequenced
A mixture of normal nucleotides in excess
One type of terminator nucleotide (ATC or G)
A primer
DNA polymerase

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4
Q

How can you then sequence the DNA in Sanger sequencing after the test tubes have produced lots of strands of DNA?

A

Eventually you will have built up all different lengths of DNA across the 4 test tubes.
If we visualise (gel electrophoresis) the fragments and line them up in order of length you can read the sequence from the terminator bases.

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5
Q

What is high-throughput sequencing?

A

A variety of approaches used to develop fast, cheap methods to sequence genomes.
Example is pyrosequencing.

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6
Q

What is PCR?

A

Polymerase Chain Reaction.
It can be described as the in vitro method of DNA sequencing.

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7
Q

What is PCR used for?

A

It is used to produce large quantities of specific fragments of DNA and RNA from very small quantities (even just one molecule of DNA or RNA).
Using PCR scientists can produce billions of identical copies of the DNA or RNA samples within a few hours, these can then be used for analysis.

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8
Q

How much DNA does PCR produce each cycle?

A

It doubles the DNA.

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9
Q

What is needed for PCR?

A

The target DNA being amplified.
Primers in excess.
DNA polymerase (Taq polymerase).
Free nucleotides.
Buffer solution.
Thermocycler to vary temperatures for the different stages.

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10
Q

What is the function of primers (forward and reverse)?

A

They are short sequences of single-stranded DNA that have base sequences complementary to the 3’ end of the DNA or RNA being copied.
They define the region that is to be amplified by identifying to the DNA polymerase where to begin building the new strands.
They also prevent the strands from joining up again.

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11
Q

Why is Taq polymerase used in PCR?

A

It is thermostable, doesn’t denature at high temperatures needed for the PCR to occur (95oC)

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12
Q

What are the 3 key stages of PCR?

A

Denaturation
Annealing
Extension/elongation

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13
Q

What happens at the denaturation stage of PCR?

A

Separates the DNA into 2 separate strands.
The hydrogen bonds between the 2 strands are broken.
(95oC)

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14
Q

What happens during the annealing stage of PCR?

A

Adding primers to the 2 separated DNA strands.
Temperature cooled to 55-60 oC to help the primers bind to the DNA.
The primers signal to a polymerase where to start synthesising new DNA.

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15
Q

What happens in the extension stage of PCR?

A

New DNA synthesised.
2 Taq polymerase molecule attached to the 2 primers on the 2 DNA strands and move along the strand.
As they move along they create new “complementary” DNA by adding nucelotides.
72oC

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16
Q

What does bioinformatics do?

A

It stores and organises large amounts of data.
Facilitates access to data on DNA and amino acid proteins and databases of metabolic pathways as well as fast retrieval and sharing of information as well as algorithms and stats tests.

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17
Q

What is the format of bioinformatics?

A

It is universal.

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18
Q

What does bioinformatics allow scientists to do in terms of disease and genetic disease? What does this then allow?

A

Identify the source of outbreaks and potentially vulnerable populations in outbreaks (can then be useful in developing vaccines).
Can be used after sequencing to identify alleles that cause genetic disease.
This then allows computer modelling of new protein structure from the base sequence.

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19
Q

What is the definition of computational biology?

A

The study of biology using computational techniques.

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20
Q

What is computational biology needed for?

A

Analysis of large amounts of data.

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21
Q

What does computational biology actually do?

A

It uses the data stored in bioinformatics to build theoretical models of biological systems which can be used to predict what will happen in different circumstances.

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22
Q

What does computational biology allow for?

A

Rapid processing of data.
Prediction of amino acid sequences.
Algorithms and stats tests.
EG analysing DNA sequencing to work out 3D structures of proteins and for understanding molecular pathways such as molecular pathways such as gene regulation.

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23
Q

How does computational biology and informatics link together?

A

Computational biology can use bioinformatics to make predictions about the structure and function of a synthetic protein using DNA sequences.

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24
What does whole genome sequencing determine? What does that mean for eukaryotes?
The complete DNA sequence of an organism's genome. For eukaryotes the is the genetic material of chromosomes, mitochondria and chloroplasts.
25
Where are genomes stored?
Gene banks.
26
What molecules are usually used to study evolutionary relationships?
Ribosomal RNA Cytochrome C Nuclear DNA Mitochondrial DNA
27
How is ribosomal RNA used to study evolutionary relationships?
It is integral to protein synthesis and so it changes slowly, making the base sequence useful for showing connections between species that diverged long ago.
28
How is cytochrome C used to study evolutionary relationships?
It is a highly conserved protein involved in cellular respiration, so slight changes changes in the amino acid sequence can help identify evolutionary links.
29
How is nuclear DNA used to study evolutionary relationships?
Species that are more closely related will have more similar DNA sequences.
30
How is mitochondrial DNA used to study evolutionary relationships?
It mutates faster than nuclear DNA, so differences in sequences show the origin of species and their subsequent migrations.
31
What does analysing the genomes of pathogens allow scientists to do?
Doctors to find the source of an infection, for example bird flu or MRSA in hospitals. Doctors to identify antibiotic-resistant strains of bacteria, ensuring bacteria are only used when effective. Scientists to track the progress of an outbreak of any potentially serious disease. Scientists to identify regions in the genome of pathogens that may be useful targets in the development of new drugs and to identify genetic marker for use in vaccine.
32
What is DNA barcoding?
Technique used to identify particular sections of the genome that are common to all species but vary between them, so comparisons can be made.
33
What is proteomics?
The study of amino acid sequencing of an organism's entire protein complement.
34
What is the definition of synthetic biology?
The synthesis of new genes/organisms.
35
What is synthetic biology used for?
Genetically modified organisms to produce drugs/enzymes/vaccines/useful molecules. Synthesis of whole new organisms.
36
How is artimsinin an example of synthetic biology?
It is an antimalarial drug. Until recently it was extracted from a plant. Using synthetic biology scientists have created all the genes responsible for producing the precursor to artimesin. These genes have been inserted into yeast cells and the yeast can be used to produce the drug.
37
Why might some people be concerned about the increase of synthetic biology?
Unknown consequences, Ethical concerns.
38
What is pharmacogenetics?
By testing people's genomes it is now possible to detect whether or not a drug will work. Doctors can prescribe a drug that will work rather than the one fits all system. Personalised medicine. Big advances for breast cancer patients.
39
Why are non-coding regions used in DNA profiling?
Coding regions are too similar in most people.
40
What is satellite DNA?
Short sequences of DNA that are repeated many times. They are found in introns (non-coding regions).
41
What is DNA profiling / fingerprinting?
Analysis of DNA from samples of body fluids or tissues, especially when conducted in order to identify individuals.
42
What are the most common applications of DNA profiling?
Forensics Analysis of disease risk Parentage testing
43
Why is coding DNA not used in DNA profiling?
Because in most people the genome is very similar so it wouldn't provide a unique profile.
44
What is satellite DNA?
Short sequences of DNA that are repeated many times.
45
Where is satellite DNA found?
Introns (non-coding region)
46
What is a VNTR?
In a region known as a minisatellite, a sequence of 20-50 base pairs will be repeated from 50 to several hundred times. These occur at more than 1000 locations in the human genome and are known as VNTRs. The number of tandem repeats shows a family resemblance.
47
What is a microsatellite?
A smaller region of just 2-4 base pairs repeated 5-15 times.
48
What are STRs?
Short tandem repeats. Microsatellites
49
Where do microsatellites appear?
They always appear in the same position on the chromosome but the number of repeats of each VNTR/STR varies between individuals.
50
Who has identical satellite patterns?
Identical twins only, although the more closely related you are to someone, the more likely you are to have similar patterns.
51
Why are STRs used instead of VNTRs?
STRs are similar to VNTRs but smaller. They stand up to the test of degradation over time so are useful in forensics.
52
How many STRs are in each person?
It varies from person to person.
53
What are the names of the 5 main stages of DNA profiling?
1- Extracting and purifying the DNA 2- Digesting the sample 3- Separating the DNA fragments 4- Hybridisation 5- Seeing the evidence
54
How do you do step 1 of DNA profiling, extracting and purifying the DNA.
Mouth swab / blood / hair / bone. Only tiny fragments are needed due to PCR. Purified with enzymes such as proteases to break down the proteins associated with DNA (such as histones)
55
How do you do step 2 of DNA profiling, digesting the sample.
DNA digested with specific restriction enzymes (restriction endonucleases) to cut DNA at particular recognition sites (cut out the STRs)
56
How do you do step 3 of DNA profiling, separating the DNA fragments.
The cut fragments need to be separated to form a clear and recognisable pattern. - 13 STRs are analysed simultaneously. - Fragments are separated by gel electrophoresis - The electrophoresis gel is immersed in alkali in order to separate the DNA double strands into single strands. - Single stranded DNA fragments are then transferred onto a membrane by a technique called Southern Blotting.
57
How do you do step 4 of DNA profiling, hybridisation.
Radioactive or fluorescent DNA probes are now added in excess to the DNA fragments on the membrane. They attach to specific fragments. The DNA probes identify microsatellite regions and bind to them. Excess probes are washed off.
58
How do you do step 5 of DNA profiling, seeing the evidence.
If radioactive labels were added to the probes, X-ray images are taken of the paper/membrane. If fluorescent labels were added to the DNA probes, the paper/membrane is placed under a UV light so the fluorescent tag glows. This is the most common method. The fragments show a pattern of bars - the DNA profile - which is unique to every individual except identical siblings.
59
How can DNA profiling be used for analysis of disease risk?
DNA profiling can show individuals that are at risk of developing particular illnesses. Research shows that specific VNTR sequences are associated with an increased incidence of particular diseases eg cancers and heart disease.
60
What is a DNA probe?
It is 50-80 nucleotides long made with complementary bases to the gene whose position you want to find.
61
How do you use DNA probes?
DNA strands are separated. Separate strands are mixed with the DNA probe. DNa probe binds to complementary bases. Visualised with fluorescence/radioactivity.
62
When are DNA probes used?
To identify the presence or absence of a specific allele for a particular genetic disease or that gives susceptibility to a particular condition. To identify the same gene in a variety of different genomes from different species when conducting genome comparison studies. To locate a specific gene needed for use in genetic engineering.
63
What is a DNA microarray and how is it used?
Scientists can place a number of different DNA probes on a fixed surface called a DNA microarray. A DNA microarray can be made with fixed probes, specific for certain sequences found in mutated alleles that cause genetic diseases, in the well. Reference and test DNA samples are labelled with fluorescent markers. Where a test subject and a reference marker both bind to a particular probe, the scan reveals fluorescence of both colours.
64
How can electrophoresis be used to separate whole proteins?
The same principle but it is carried out in the presence of a charged detergent such as SDS. SDS equalises the surface charge on the molecules - makes them all negative, and allows the proteins to separate as they move through the gel, according to their molecular mass. In some cases, the proteins can be separated according to their mass, and then, without SDS, according to their surface charge. This technique can be used to analyse the types of haemoglobin proteins for diagnosis of conditions such as sickle cell anaemia where the patient has haemoglobin S and not the normal haemoglobin A.
65
What is germ line gene therapy?
Inserting a healthy allele into the germ cells or into a very early embryo.
66
What is somatic cell gene therapy?
Replacing a faulty gene with a healthy allele in affected somatic cells.
67
How to perform germ line gene therapy?
The corrected gene is inserted into a fertilised egg produced via IVF. If successful, all cells of the embryo will contain the corrected gene when cell divides by mitosis. Germ cell therapy is permanent and also ensures offspring inherit the corrected gene. Currently illegal.
68
How do you perform somatic cell gene therapy?
Copies of the corrected gene are inserted directly into the somatic (body) cells of the sufferers. This type of gene therapy does not prevent the disease from occuring in the next generation because it does not affect the sperm and egg cells. Somatic cell gene therapy has to be repeated many times as the effects don't last long. Currently in clinical trials and in use for certain blood disorders such as sickle cell and haemophilia.

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