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Biology mod 6 Genetics, evolution and ecosystems > 6.1.3 Manipulating genomes > Flashcards

6.1.3 Manipulating genomes Flashcards

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1
Q

What does PCR stand for?

A

polymerase chain reaction

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2
Q

What is a pcr?

A

a method of DNA amplification used to produce large quantities of specific fragments of DNA or RNA from very small quantities which can then be used for analysis.

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3
Q

Outline some things every PCR reaction requires.

A
  • Target DNA or RNA to be amplified.
  • Primers
    -Dna polymerase (Taq polymerase)
    -Free nucleotides
    -Buffer soloution.
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4
Q

What is a primer?

A

A short sequence of single stranded DNA with base sequences complementary to the 3’ end of the DNA or RNA being copied.

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5
Q

Why are primers used in pcrs?

A

They define the region that is to be amplified by identifying to DNA polymerase where to begin building new strands.

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6
Q

What is the most common version of DNA polymerase used in pcr?

A

Taq polymerase

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7
Q

Why is taq polymerase used in pcr?

A

It does not denature at high temperatures.

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8
Q

How many key stages are there in a PCR cycle?

A

3

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9
Q

What are the three stages in a PCR cycle?

A

1) Denaturation
2) Annealing
3) Elongation

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10
Q

Outline the stages of a PCR cycle.

A

1) Denaturation- the double-stranded DNA is heated to 95º which breaks the hydrogen bonds that bond the two strands together.
2) Annealing- the temperature is decreased to between 50 and 60º so that primers can anneal to the ends of the single strands of DNA.
3) Elongation- the temperature is increased to 72ºc for at least a minute to build an optimum temperature for Taq polymerase to build the complementary strands of DNA using free nucleotides and each template strand to produce the new identical double-stranded DNA molecules.

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11
Q

What piece of equipment is used in a PCR reaction?

A

Thermal cycler

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12
Q

What temperature is DNA heated to in stage 1 of a PCR reaction?

A

95ºc

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13
Q

What temperature is DNA heated to in stage 2 of a PCR reaction?

A

Between 50º and 60º.

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14
Q

What temperature is DNA heated to in stage 3 of a PCR reaction?

A

72ºc

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15
Q

What is electrophoresis?

A

Using an electrical current to separate out fragments of DNA, RNA or proteins depending on their size/mass and their overall electrical charge.

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16
Q

What must you do before electrophoresis?

A

Amplify the DNA by PCR.
Restriction enzymes are then used to cut the DNA into fragments.

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17
Q

How is DNA cut into fragments?

A

Scientists use enzymes that will cut close to the variable number tandem repeat regions- which are regions found in the non-coding part of the DNA that contain variable numbers of repeated DNA sequences and are known to vary between different people (except for identical twins).

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18
Q

what are VNTR’s?

A

Variable number tandem repeat regions.

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19
Q

Outline the method of electrophoresis.

A

-Create an agarose gel plate in a tank. Wells (a series of groves) are cut into the gel at one end. The end of the gel tray with the wells should be closest to the negative electrode on the gel box.
- Submerge the gel in an electrolyte solution which conducts electricity in the tank.
-Load (insert) the fragments into the wells using a micropippete. With the same volume of loading dye Make sure the tip of the micropippete is in the buffer/electrolyte solution and just above the opening of the well.
-Repeat this process and add the same volume of each DNA sample to the other wells using a clean micropippete each time.
-Put the lid on the gel box and connect the leads from the gel box to a power supply. Turn this on and set it to the required voltage. This causes an electrical current to pass through the gel.
-Let the gel run for around 30 minutes and then turn off the power supply.
-Remove the gel tray from the gel box and tip off any excess buffer soloution.
-Wearing gloves, stain the DNA fragments by covering the surface of the gel with a staining soloution and then rinsing the gel with water. The bands of the different DNA fragments will now be visible.

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20
Q

Why do we use loading dye when adding the DNA fragments to the wells in electrophoresis.

A

This makes it easier to see the DNA samples sink to the bottom of the wells.

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21
Q

During electrophoresis, the end of the gel tray with the wells should be closest to which electrode?

A

Negative

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22
Q

What does electrophoresis find?

A

DNA fragments are negatively charged so move towards the positive electrode at the far end of the gel. Small DNA fragments will move faster and travel further so the DNA fragments separate due to size.

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23
Q

What is the positively charged electrode called?

A

Cathode.

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24
Q

What is the negatively charged electrode called?

A

Anode

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25
What is DNA profiling?
Identifying the unique areas of a persons DNA in order to create a profile individual to them.
26
Why does everyone have a different genetic profile (usually)?
Every person has repeating short non-coding regions of DNA (20 to 50 bases) that are unique to them (VNTRs).
27
What determines the number of VNTR's we have?
The number of VNTRS are inherited from your parents so the more closely related you are to a person the more likely it is that the repeats have similar patterns.
28
Explain the process of DNA profiling.
1) Obtain the DNA. 2) Increase the quantity by using PCR. 3) Use restriction enzymes to cut the DNA molecule into fragments based on VNTR's 4) Separate the fragments using gel electrophoresis. 5) Add radioactive or floresecent probes that are complementary to specific VNTR regions. 6) X-ray images or UV light is used to produce images of the flouresecent labels glowing. 7) These images contain patterns of bars (the DNA profile) which are then analysed.
29
Give some uses of DNA profiling.
-To identify suspects of crimes and identify bodies that are unidentifiable. -Can be used to identify individuals which are at risk of developing particular diseases. -To determine familial relationships from paternity cases or immigration cases. -Species conservation to help captive breeding programmes.
30
What is a genome?
The complete set of genetic material present in a cell or organism.
31
What is a proteome?
The full range of proteins produced by a genome.
32
What is epitdemiology?
The study of the spread of infectious diseases within populations. The genomes of pathogens can be sequenced and analysed to aid research and disease control.
33
What is the human genome project?
An international, collaborative research programme where DNA samples were taken from multiple people around the world, sequenced and used to create a reference genome. The data is publicly available and in 2003 was sequenced to 99.9% accuracy and is over 3 billion base pairs long but only 25,000 genes.
34
Why does the human genome project involve the genomes of people all over the world and not just one person?
One person/organism may have anomalies/mutations within their DNA.
35
What is synthetic biology?
An area of research that aims to create new biological parts, devices and systems or to redesign systems that already exist in nature. It goes beyond genetic engineering and involves large alterations to an organisms genome.
36
What is bioinformatics?
A field of biology that involves the storage, retrieval and analysis of data from biological studies. These studies may generate data on DNA sequences, RNA sequences and protein sequences as well as the relationship between genotype and phenotype. Once a genome is sequenced, bioinformatics allows scientists to make comparisons with the genomes of other organisms to help find a degree of similarity between organisms which gives an indication of how closely related they are.
37
What does it mean if species are found to have a small number of differences between their genomes?
They are likely to share a more recent common ancestor.
38
How do we explore genotype-phenotype relationships?
By ‘knocking-out’ different genes (stopping their expression) and observing the effect it has on the phenotype of an organism.
39
Who developed the chain termination method of DNA sequencing?
Frederick Sanger (sanger sequencing).
40
What are developments of sanger sequencing called?
High-throughput sequencing.
41
What are the modified nucleotides called in sanger sequencing?
Dideoxynucleotides.
42
Explain sanger sequencing.
-When DNA polymerase encounters a dideoxynucleotide on the developing strand it will stop replicating. -Once the incubation period has ended, the new DNA chains are separated from the template DNA and the resulting single-strand DNA chains are separated according to length by gel electrophoresis the four types of dideoxynucleotide either A*, C*,T* or G*. -The test tubes are incubated at a temperature that allows DNA polymerase to function. -The primer anneals to the start of the single-stranded template producing a short section of double stranded DNA at the start of the sequence. DNA polymerase attaches to this double stranded section and begins DNA replication using the free nucleotides in the test tube. -Hydrogen bonds form between the complementary bases on the nucleotides. -At any time, DNA polymerase can insert one of the dideoxynucleotides by chance which results in the termination of DNA replication. -Because the point at which the dideoxynucleotide is inserted varies with every strand, complementary DNA chains of varying lengths are produced. -Once the incubation period has ended, the new DNA chains are separated from the template DNA and the resulting single-strand DNA chains are separated according to length by gel electropheresis.
43
Why is high-throughput sequencing better than sanger sequencing?
The increase in speed enabled by high-throughput sequencing has allowed scientists to sequence and analyse the genomes of many organisms. Genes can be rewritten to alter their function and then inserted into cells using genetic engineering techniques so that scientists can potentially design new molecules with huge potential for drug production. Gene sequencing can also provide information about evolutionary relationships.
44
Define genetic engineering
The manipulation of the DNA sequence of an organism.
45
What feature of DNA means it is possible to take it from one organism and place it in another organism even if theyre from different kingdoms?
The DNA code is universal meaning every organism uses the same four bases.
46
What is altered DNA called?
Recombinant DNA.
47
Explain the very basics of genetic engineering.
Extracting a gene from one organism and then inserting it into another organism.
48
Explain the 5 stages of genetic engineering.
1) Isolation of the desired DNA fragment. 2) Insertion of the DNA fragment into a vector. 3) Transformation 4) Identification 5) Growth/cloning.
49
Give the two methods of isolation of a desired DNA fragment in genetic engineering.
Using restriction enzymes to cut DNA at specific recognition sites. Using reverse trancriptase to generate cDNA.
50
Some restriction enzymes cut straight across both chains forming ____ _____.
Blunt ends
51
How do most restriction enzymes cut DNA?
They make a staggered cut in both chains causing sticky ends.
52
What are sticky ends?
Overhanging regions of single-stranded DNA which expose complementary bases.
53
Sticky ends will only join with other sticky ends when what?
It has been cut with the same restriction enzyme.
54
Why would we use the reverse transcriptase method of DNA/gene isolation?
This method would be used as prokaryotes do not have non-coding regions (introns) as eukaryotes do so when human genes are transferred into bacteria, cDNA is used as this has been converted from mature mRNA (no introns).
55
Which enzyme turns single stranded cDNA into double stranded?
DNA polymerase.
56
What does the enzyme reverse transcriptase do?
Reverse transcription- turns mRNA back into (c)DNA.
57
How do we insert an isolated gene into a plasmid?
The plasmid is extracted from the bacteria and cut using the same restriction enzyme (at the marker gene) used to isolate the DNA fragment so the sticky ends are complementary to the sticky ends of the DNA fragment containing the gene. The vector and DNA fragment are joined together by DNA ligase which joins the sugar-phosphate backbone (ligation).
58
What are isolated genes usually inserted into?
A plasmid in a bacterial cell.
59
What is ligation?
When the enzyme ligase joins the sugar-phosphate backbone of the vector and the isolated gene together.
60
Difference between DNA polymerase and DNA ligase?
DNA polymerase joins individual DNA nucleotides together. DNA ligase joins DNA fragments at one specific point rather than joining each individual nucleotide.
61
What is the transformation step of genetic engineering.
The plasmids must be reintroduced into the host cell (e.g bacterial cell such as ecoli).
62
Name the three methods of putting a plasmid back into a host cell.
Heat shock Electroporation Electrofusion
63
What is electroporation?
Bacterial cells are mixed with the plasmid vector in a machine called an electroporator. The machine is switched on and an electric field is created which increases the permeability of the bacteria cells membranes allowing one to take up the plasmid.
64
What is heat shock?
Bacteria, plasmids and calcium ions are mixed together and by altering the temperature, the bacterias cell membrane becomes more permeable and the plasmid can pass through.
65
Why do we need the step of identification in genetic engineering?
Only a tiny number of bacterial cells take up a plasmid when the two are mixed together so we need to determine which bacteria did take up a plasmid.
66
What are the three possibilities that the bacterial cells will have undergone which we need to indentify?
-Some bacteria will have taken up no plasmid. -Some bacteria may have taken up the original plasmid. -Some bacteria will have taken up the hybrid genetically engineered plasmid.
67
How do we rule out the bacteria which have not taken up a plasmid at all?
Grow the bacteria on a medium e.g agar containing an antibiotic. Those which do not grow have not taken up a plasmid as they will have died.
68
If a bacteria has not taken up the genetically engineered plasmid and has taken up the original plasmid, what will their structure be?
They will have a fully intact non-interupted marker gene as well as the antibiotic resistance gene.
69
If a bacteria has taken up the genetically engineered plasmid, what will their structure be?
They will have an interrupted marker gene where the isolated gene has been added and will have the antibiotic resistance gene.
70
How do we determine between those bacteria which have taken up the original plasmid and those which have taken up the hybrid plasmid?
If the bacteria has the engineered gene, the marker gene (e.g blue) has been disrupted with the DNA fragment in the middle, so will not appear the colour of the marker gene (e.g blue) so it will appear colourless. All colourless colonies therefore contain the DNA fragment.
71
What is the process of growing bacterial colonies on a medium to see which have taken up a genetically modified gene called?
Replica plating.
72
What happens after we have identified which bacteria have taken up a genetically modified plasmid?
The bacteria which have taken up the modified gene are cloned.
73
If an organism contains nucleotide sequences from a different species it is called what?
A trasngenic organism.
74
whqt are some ethical issues of genetically engineering pathogens?
- could cause a mass outbreak among the scientists investigating the pathogen -genetically modified version of the pathogen could revert back to its original form triggering an outbreak of disease. -could get into the wrong hands and be used maliciously for biological warfare.
75
what are some positive ethical issues of genetically engineered organisms being owned by big companies?
It means the owner of the organisms will get money generated from selling the product.
76
what are some negative ethical issues of genetically engineered organisms being owned by big companies?
people in poorer countries may not be able to afford the genetically modified organisms such as seeds. Even if farmers for example can afford the seeds for one year, they cannot plant them past this time without paying again. This is unfair.
77
what are some negative ethical issues of genetically engineered organisms being owned by big companies?
people in poorer countries may not be able to afford the genetically modified organisms such as seeds. Even if farmers for example can afford the seeds for one year, they cannot plant them past this time without paying again. This is unfair.
78
What could gene therapy be used for?
To cure genetic disorders.
79
What could gene therapy be used for?
To cure genetic disorders.
80
If a genetic disorder is caused by two recessive alleles, how can we genetically engineer a cure for this?
Add a working dominant allele to make up for them.
81
If a genetic disorder is caused by two recessive alleles, how can we genetically engineer a cure for this?
Add a working dominant allele to make up for them.
82
If a genetic disorder is caused by a dominant allele, how can we using gene therapy cure this?
You can silence the dominant allele by inserting DNA in the middle of the allele so it does not work anymore
83
what are the two types of gene therapy?
Somatic therapy Germ line therapy
84
what are the two types of gene therapy?
Somatic therapy Germ line therapy
85
What is somatic gene therapy?
Involves altering the alleles in body cells. Does not involve sex cells so any offspring could inherit the disease.
86
What is somatic gene therapy?
Involves altering the alleles in body cells. Does not involve sex cells so any offspring could inherit the disease.
87
What is germ line therapy?
Involves altering the alleles in sex cells. Every cell of any offspring produced will be affected by the gene therapy too and won’t inherit the disease. This is currently illegal.
88
Give some positive ethical issues surrounding gene therapy.
It could prolong the lives of people with genetic disorders could give them better quality of life carriers of genetic disorders may be able to conceive a baby without the disorder (only in germ line)
89
Give some negative ethical issues of gene therapy.
Tech could be used in ways other than for medical treatment e.g cosmetic effects of ageing. Potential risk that you could do more harm than good. Expensive. Benefits may be short lived or have to undergo multiple treatments. Body could identity the vectors as foreign body and reject them and start an immune response against them. An allele could be inserted into the wrong place and cause problems e.g cancer.

Biology mod 6 Genetics, evolution and ecosystems (6 decks)

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