Topic 8.3 and 8.4 Flashcards
Gene technologies and Genome projects
1
Q
What is reverse transcriptase?
A
An enzyme that uses mRNA as a template to produce cDNA (a copy of DNA).
2
Q
Describe how reverse transcriptase can be used to produce a fragment of DNA:
A
- Take a sample of mRNA from a cell (that is expressing the protein you want)
- Use reverse transcriptase to make cDNA from mRNA template (cDNA is single stranded). Reverse transcriptase essentially joins them together via complementary base pairing.
- Heat separates these 2 strands of mRNA and cDNA.
- Use DNA polymerase to make the cDNA double stranded by joining free complementary DNA nucleotides
- You’ve now got a copy of your gene!
3
Q
What is a palindromic sequence of DNA?
A
- A sequence of DNA which is read the same in opposite directions.
4
Q
What is a restriction endonuclease?
A
An enzyme which cuts DNA into shorter sections at specific recognition sequences.
5
Q
What is the benefit of using mRNA in this process?
A
mRNA contains only exons , no introns.
Whereas DNA does, which would be nonsense and unhelpful.
6
Q
Why do restriction enzymes only cut at specific recognition sequences of DNA?
A
Because the active site of the enzyme is complementary to the shape of the specific sequence of DNA.
7
Q
Describe how restriction endonucleases are used to produce a fragment of DNA:
A
- Select a restriction endonuclease, which has a recognition site at either end of the gene you want.
- Use the restriction endonuclease enzyme to cut out the gene you want.
8
Q
Some restriction enzymes leave ‘sticky ends’ what is meant by this and why is it useful?
A
- ‘Sticky ends’ are sections of unpaired bases
- Producing DNA fragments with sticky ends is useful because it means you can also cut a plasmid with the same restriction endonuclease so that the sticky ends are complementary to each other and it is easier to join the fragments together.
9
Q
Explain briefly how a gene machine works:
A
- This is a machine that artificially produces the gene that we want.
- Select the protein of interest and find out its amino acid sequence
- Use this to work out the mRNA sequence
- Use this to work out the DNA sequence
- Put the sequence into a computer
- The computer will choose which oligonucleotides should be joined together to create the desired gene
10
Q
How could PCR be used to produce copies of the gene that you want?
A
- Put a section of DNA into a PCR machine
- Select two primers… one which is complementary to a sequence of DNA before (upstream) the gene you want and one which is complementary to a sequence of DNA after (downstream) of the gene you want
- Run the machine and you will produce lots of copies of the desired section of DNA
11
Q
What are the 3 methods that fragments of DNA can be produced?
A
- Using reverse transcriptase
- Using restriction endonuclease
- Creating the gene in a gene machine
12
Q
Now we’ve got the DNA fragment, how do we make lots of copies of it?
A
In vivo techniques - Transferring the fragments into a host cell using a vector, so they can make the copies of the genes we want.
In vitro techniques - Using the polymerase chain reaction
13
Q
If you are transferring a gene into an organism and you want that organism to produce a protein, what must you include with that gene?
A
A promotor region
A terminator region
14
Q
But how do you know which promoter and terminator region to use?
A
Use a promoter and terminator that are linked to a gene, which you already know is expressed in the cell you are transforming.
15
Q
EXAM DEFINITION:
What is a vector?
A
A vector transfers DNA from one organism into another.
16
Q
How do you insert your fragment of DNA into a plasmid?
A
- Cut the desired gene and the plasmid with the same restriction endonuclease, so that there are complementary sticky ends.
- Mix your fragment with the cut plasmid
- Use DNA ligase to join the sections of DNA together forming phosphodiester bonds.
- This forms a recombinant plasmid
17
Q
How do you insert your recombinant plasmid into a bacterial plasmid?
A
- Mix the plasmids with the bacterial cells in a solution of Ca2+ ions.
- Use heat stock to make the bacterial membrane permeable, so that the bacteria take up the plasmid.
18
Q
What are marker genes used for?
A
They are used to identify which bacterial cells have taken up the recombinant plasmid (i.e. the one with the desired gene).
19
Q
What is a marker gene?
A
A gene which codes for a protein which allow the cells (e.g. bacteria) that produce the desired protein to be identified.
Sometimes the plasmid won’t pick up the desired gene
Sometimes the bacteria won’t pick up the plasmid with the gene.
20
Q
Name 3 different marker genes:
A
- Enzymes (e.g. lactase)
- Green fluorescent protein (GFP)
- Antibiotic resistance
21
Q
What’s the problem with using genes which confer antibiotic resistance as marker genes?
A
The genes could end up in pathogenic bacteria
So if the pathogenic bacteria infect someone, they can no longer be treated by that antibiotic.
22
Q
Describe one way that antibiotic resistance genes can be used to identify which bacterial cells have picked up a recombinant plasmid?
A
- Use a plasmid which has two genes. One which codes for a protein that gives resistance to ampicillin and one that gives resistance to tetracycline.
- The plasmid is cut within the gene coding for tetracycline resistance and the desired gene should be inserted there
- The bacteria that have picked up no plasmids, will have no resistance to ampicillin and tetracycline
- The bacteria that pick up the plasmids without the desired gene will have resistance to both A and T
- The bacteria that pick up plasmids with the desired gene will only show resistance for A and not T (as the desired gene was inserted into the gene coding for resistance to T).
- Plate the bacteria onto a petri dish that contains ampicillin. This will kill type 1 bacteria.
- Transfer the bacteria from colonies which have not been killed by ampicillin to a new plate
- Make a replica of this plate (using a nylon membrane to transfer the bacterial colonies)
- Put tetracycline onto one of the identical plates. This will kill type 3 (i.e. the bacteria which have the desired gene).
- You can therefore identify which colonies contain the desired gene on your replica plate
23
Q
What is GFP?
A
Green fluorescent protein.
This glows green under UV light.
24
Q
Describe two different ways that GFP genes can be used to identify which bacterial cells have picked up a recombinant plasmid?
A
- The plasmid contains a gene which codes for a protein which glows in the dark (GFP gene)
- The desired gene is inserted into the GFP gene
- Therefore bacteria that do contain the desired gene will not glow in the dark
OR
- Insert the desired gene next to the GFP gene
- Therefore bacteria that do contain the desired gene will also have the GFP gene so it will glow in the dark.
25
Describe one way that genes which code for enzymes that causes colour changes can be used to identify which bacterial cells have picked up a recombinant plasmid?
- The plasmid contains a gene that codes for the enzyme lactase
- Lactase turns a colourless substrate blue
- Desired gene is inserted into the gene that codes for lactase
- So the bacteria that do contain the desired gene will not turn the substrate blue and it will remain colourless.
26
Describe how PCR can be used to clone genes:
First, mix together:
- The DNA you want to clone
- Thermostable DNA polymerase
- free nucleotides
- 2 primers (one that's complementary to the start and another that's complementary to the end of the section of DNA you want to clone).
- Heat up the DNA to 95 degrees so that the strands separate by breaking the hydrogen bonds.
- Lower the temperature to around 53 degrees so that the primers bind to the specific sequence by complementary base pairing
- The nucleotides attach by complementary base pairing
- Heat to 73 degrees so that DNA replication takes place, where the DNA polymerase joins the adjacent nucleotides together forming phosphodiester bonds
- Repeat the cycle many times
27
Why do we have to use primers?
- To mark the section of DNA that needs to be amplified
- To allow the DNA polymerase to attach
- To prevent the DNA strands from joining back together
28
What are primers?
- They are small sections of DNA that allow DNA synthesis to start
- DNA polymerase cannot catalyse the formation of a new DNA strand if they have nowhere to add the nucleotides to, obviously must be added to existing strands of DNA.
29
When you are genetically modifying an organism, as well as introducing the part of the gene which codes for the protein you must have the appropriate promoter attached to the front of the gene. Why is this important?
* This ensures that the gene is expressed (transcribed and translated) in the desired organ or part of the organism
30
What are some of the key concerns about genetically modifying organisms?
* Cross pollination with different varieties of the same species will cause the gene to spread to other plant species
* If antibiotic resistance markers are used during the production of the GM plant, then this raises concern about the spread of antibiotic resistances
* Impacts of food chains… (this will depend on the specific example)
31
Give a definition for gene therapy:
When a healthy gene is introduced to replace a defective gene
32
What sort of diseases are the best for gene therapy and why?
- Diseases caused by just1 or 2 genes
- Diseases which are caused by recessive alleles - so by introducing a dominant allele, you can mask the effects
33
What is somatic gene therapy? What are its limitations?
- The replacement of genes in body cells
- This is not a permanent solution because cells die so treatment must be repeated
34
What is germ line therapy?
What are the benefits and issues with this?
- The replacement of genes in fertilised egg cells.
- This means that all the cells in the organism will contain the healthy gene - so it's permanent
- It also means that the gametes produced will contain the modification, so all future generations will be affected.
- There are ethical issues with this because it changes the genetics for all future generations
35
Describe how a liposome can be used in gene therapy:
What are the benefits and limitations of this method?
- Package the desired gene into a liposome
- The liposome will be able to pass through the phospholipid bilayer so it will deliver the gene into the cells
A - this method does not trigger an immune response
A - There is no max length of DNA it can carry
D - there is a very low uptake of the gene
36
Describe how a viruses (e.g. adenoviruses) can be used in gene therapy:
What are the benefits and limitations of this method?
- Modify a virus so that it is not harmful and select a virus that will target the cells you want
- Introduce the desired gene into the virus
- The virus will target specific cells and inject the DNA into the cells
A - There is a much higher uptake because viruses target cells
D - Viruses can cause an immune response
D -When the DNA is randomly inserted into host cell, this can disrupt existing genes. If a tumour suppressor gene is disrupted, it could lead to the cell dividing uncontrollably and therefore cancer.
37
What is a DNA probe?
Single stranded pieces of DNA
Complementary to a specific base sequence which it will bind to
38
What is DNA hybridsation?
When a single strand of nucleic acid anneals to complementary DNA or RNA to make a double strand.
So when the probe has bound to the sequence of interest, we say that it has been hybridised.
38
Describe how DNA probing is carried out:
1. Make a sequence of DNA that is specific and complementary to the DNA sequence of the gene of interest. This is the probe. The probe has a molecule attached to it, so that it is visible.
2. PCR the probe, to make lots of copies of it
3. Extract a sample of the person's DNA and PCR it
4. Use restriction endonucleases to cut the DNA into fragments
5. Use gel electrophoresis to separate the fragments of DNA
6. Denature the DNA and the DNA probe so that they both become single stranded (either using heat or a chemical solution to break the hydrogen bonds)
7. Add the probe to the DNA
8. If the target sequence is present, the probe will bind to the DNA, this is DNA hybridisation.
9. Wash with a buffer solution to remove any unattached probe
10. Screen it to see if it is radioactive OR fluorescent to identify if the target sequence of DNA is present.
38
List three things that DNA probing can be used for and for each one write a sentence explaining what it means:
- To screen for a heritable condition
i.e. check to see if someone is carrying a disease-causing allele such as the allele that causes cystic fibrosis
- To test for drug responses, i.e. the screen for genes which code for proteins that affect the way someone responds to a drug (e.g. it could be a gene which codes for a protein which is the target of the drug, or it could be a gene which codes for a protein which is an enzyme that could break down the drug)
- To screen for health risks, i.e. to test for the presence of alleles which are believed to make someone more likely to develop a particular disease
38
Explain why probing is sometimes carried out on chromosomes which are undergoing mitosis:
Because during mitosis the chromosomes are visible and this will allow you to find out which chromosome the gene of interest is on.
39
What is the aim of gel electrophoresis:
Separates DNA fragments based on their length and charge
40
How does gel electrophoresis work?
- The DNA is placed in a well at one end of a gel (near the negative electrode)
- An electric current is run across the gel
- The DNA migrates towards the positive end of the gel (because the DNA is negatively charged due to its negative phosphate head)
- The shorter fragments move furthest and fastest and the longer fragments move slow
41
What are VNTR's?
Small sequences of non-coding DNA which repeat several times
The number of repeats varies in different people
42
Describe how DNA fingerprinting is carried out:
1. DNA sample is obtained and it is run through PCR to obtain many copies
2. The DNA is cut using restriction endonucleases
3. The fragments of DNA are separated by electrophoresis according to size
4. An alkali solution is added to the gel to make the DNA single stranded
5. The DNA fragments are transferred to nylon membrane (nylon is pressed onto the DNa fragments so they are fixed in place and won't move around)
6. A DNA probe is applied which is complementary to specific VNTRs
7. The probe will hybridise with the complementary VNTR
8. Use a buffer to remove unbound probe
9. Use UV light or autoradiography to see where the probe has been bound
10, This will produce a pattern that will be different for every individual?
43
How can DNA fingerprinting be used for maternity/paternity tests?
All the child's bands should be present in either the mother or father.
44
How can DNA fingerprinting be used to investigate genetic variability?
- The more bands shard, the less the genetic diversity
- The more difference in the DNA fingerprint, the more genetically diverse and the more distantly related they are
45
How is DNA fingerprinting useful for captive breeding programmes for conservationists?
- Select the individuals whose DNA fingerprints are most different to each other and breed them
- This reduces the chances of inbreeding and creating offspring that have two copies of harmful recessive alleles
46
How would you work out the proteome of a prokaryote from the genome?
- 3 bases code for one amino acid
- Once you have sequenced the DNA you can work out the amino acid sequence and thus the primary structure of the proteins
47
How could knowing the genome of a pathogen be useful?
- You can work out the proteins the pathogen will express, including some of the surface antigens
- You can then produce those antigens and use them to create vaccines against the particular pathogen
48
Why is it challenging to work out the proteome of a eukaryote, even if you know its genome?
- There are introns within the genes
- There are regions of DNA which do not code for proteins but code for other things e.g. regulatory genes like siRNA or miRNA
