1
Q
Why is recombinant DNA technology possible with different organisms?
A
As the genetic code is universal- the same codons code for the same amino acids in all organisms
Transcription and translation mechanisms are also similar in different species
2
Q
Which type of enzyme can be used to produce cDNA?
A
Reverse transcriptase
3
Q
What is cDNA?
A
Complementary DNA
4
Q
Name 2 types of DNA probe
A
Fluorescent and radioactively labelled
5
Q
How is reverse transcriptase used to make DNA fragments?
A
- A cell that readily produced the protein is selected - such cells have large amounts of the target mRNA, which can easily be extracted.
- Reverse transcriptase is used to make a single-stranded complementary copy of DNA - cDNA, from the mRNA.
- cDNA is isolated by hydrolysing the mRNA using an enzyme.
- Double stranded DNA is formed on the template of cDNA using DNA polymerase.
- The copy of coding DNA in the gene is formed.
6
Q
What are restriction endonucleases?
A
Enzymes that can cut DNA at specific recognition sequences
7
Q
Uses of restriction endonucleases
A
Cutting out a gene for inserting it into a plasmid - can be done when the recognition sites are either side of the DNA fragment
8
Q
Where do different restriction endonucleases cut DNA and why?
A
At different recognition sequences, which have a shape complementary to the enzyme active site
9
Q
What are blunt ends? (Restriction endonucleases)
A
Where the restriction enzyme cuts both DNA strands at the same position- between 2 opposite/ complementary base pairs
10
Q
What are the 3 methods of producing DNA fragments?
A
Using reverse transcriptase, restriction endonucleases or the gene machine
11
Q
What are sticky ends?
A
Ends of DNA fragments when the restriction enzyme cuts in a staggered fashion- each fragment has some unpaired bases on 1 strand
12
Q
Are sticky or blunt ends more useful and why?
A
Sticky ends- the exposed bases mean that the fragment can be made to pair up with other bases in another DNA molecule.
13
Q
How can ‘the gene machine’ be used to produce DNA fragments?
A
- The amino acid sequence of the protein for which the gene codes is determined. From this, the mRNA codons and complementary DNA triplets are worked out.
- The desired nucleotide base sequence is fed into a computer.
- After safety checks, the computer designs a large number of small, overlapping single strands of nucleotides (oligonucleotides), which can be assembled to form the gene.
- The gene is replicated using the polymerase chain reaction. ‘Sticky ends’ are produced and the gene is inserted into a plasmid to be stored/ processed.
14
Q
What 4 things are needed for PCR?
A
DNA fragment, free nucleotides, primers, DNA polymerase
15
Q
Stages of the polymerase chain reaction
A
- DNA is heated to 95oC
- Hydrogen bonds between strands break, so strands separate
- Mixture cooled to 55oC, to allow primers to bind to DNA start sequence
- Temperature increases to 72oC
- Free nucleotides attach by complementary base pairing
- DNA polymerase joins nucleotides together
- Cycle repeats
16
Q
Role of primers in PCR (3-4)
A
- To bind to start sequence of DNA strands, allowing DNA polymerase to attach and start addition of nucleotides.
- The enzyme needs starting strand onto which to attach nucleotides.
- Also to stop strands from re-joining.
(and show enzyme where to start adding nucleotides)
17
Q
What is 1 common feature of a recognition sequence?
A
it is palindromic (same forwards of 1 strand as backwards on the other.)
18
Q
What are primers?
A
short nucleotide sequences that have a base sequence complementary to those at one end of each of the 2 DNA fragments.
19
Q
Stages of PCR
A
- Separation of the DNA strand - the reagents are placed in a vessel in the thermocycler which is heated up to 95℃. This causes hydrogen bonds between complementary base pairs to break, separating the 2 strands of DNA fragments.
- Addition of primers - the mixture is cooled to 55℃, so the primers join to the complementary DNA base sequences at the end of the fragments. ⇒ start sequences for DNA polymerase to begin copying DNA, as it can only attach nucleotides to an already existing chain. Also - stops strands from re-joining.
- DNA synthesis - temperature is increases to 72℃, the optimum for DNA polymerase activity. New nucleotides are added from the end of the primer.
20
Q
How does the number of copies of the DNA fragment change with each PCR cycle?
A
doubles with each cycle - exponential increase. A graph may appear flat at first as there are very low numbers of DNA fragments.
21
Q
Purpose of heating the PCR mixture to 95oC
A
to break the H bonds between complementary bases and separate the 2 strands.
22
Q
What are DNA probes used for?
A
identifying where a particular DNA sequence is located, or identifying whether an individual has a certain/mutated allele of a gene which can lead to genetic disease.
23
Q
Process of using DNA probed to identify certain alleles
A
DNA probe with base sequence complementary to part of the target allele’s base sequence is made.
The double-stranded DNA being tested is heated/ treated so that strands separate.
Separated strands are mixed with the probe, which binds to its complementary base sequence on 1 strand. This is DNA hybridisation.
The site the probe has bound to can be identified with the radioactive/ fluorescent marker.
24
Q
Why does a DNA probe only detect a specific allele?
A
The probe has a base sequence complementary to the DNA of the allele, which it binds to. Only this DNA is labelled.
25
Why are promoter and terminator regions required when inserting DNA into a vector?
promoter region - required at start of gene for RNA polymerase and transcriptional factors to bind and stimulate transcription.
terminator region - required at end of DNA fragment so that transciption stops at the right point.
26
What is a vector?
a carrying unit used to transport the DNA into the host cell.
It is normally a small, circular section of DNA called a plasmid, into which the target gene can be inserted.
27
Which enzyme is able to join up the sugar-phosphate backbones of DNA fragments - in-vivo cloning
DNA ligase
28
Describe how restriction endonucleases and ligases can be used to insert DNA fragments into vectors.
- a restriction endonuclease is used to cut open the plasmid loop. and cut out the DNA fragment. This means the sticky ends of the plasmid and fragment are complementary and can pair together.
DNA fragments and open plasmids are mixed and may become incorporated. DNA ligase makes the join permanent by joining the sugar-phosphate backbones of the DNA strands.
29
What are restriction endonucleases and ligases used to do in in-vivo gene cloning?
insert DNA fragments into vectors.
30
How can host cells be transformed in in-vivo gene cloning?
by introducing the vectors - plasmids and bacterial cells are mixed in a medium containing Ca2+ and heated. This makes the bacterial membrane permeable, so allowing plasmids to pass into the cytoplasm.
31
What type of genes can be used to detect GM cells/ organisms in in-vivo cloning?
marker genes
32
Why will not all bacterial cells contain the plasmid with the desired gene in in-vivo cloning? (3)
- Only a small proportion of bacterial cells take up the plasmid upon mixing.
- Some plasmids close up again without incorporating the fragment.
- The fragment ends may join to form its own fragment.
33
give 3 different types of marker genes
Antibiotic resistant
Fluorescent
Enzyme
34
What are marker genes used to do?
identify whether cells have taken up plasmids with the target gene/ become genetically modified.
35
In general, how can marker genes work
the required gene can be inserted into the marker gene, which will therefore be non-functional if the required gene is present.
36
Advantages of in vivo gene cloning
- less risk of contamination - very specific. In in vitro cloning, contaminating DNA will also be copied.
- very accurate - few mutations
- cuts out specific genes rather than copying the whole DNA sample.
- transformed bacteria are able to produce the protein the gene codes for.
37
advantages of in vitro gene cloning
- rapid
- useful when initial amout of DNA is small
- no living cells/ culturing is required.
38
what is a DNA probe?
short, single-stranded sections of DNA which are complementary to the base sequence of the allele of DNA to be identified
39
what are the 2 types of DNA probe?
radioactively and fluorescently labelled.
40
Describe the process of using DNA probes to identify certain alleles.
1) DNA probe with base sequence complementary to part of the target allele’s base sequence is made.
2) The double-stranded DNA being tested is heated/ treated so that strands separate.
3) The mixture (of DNA and probe) is cooled and the probe binds to its complementary base sequence on a strand. This is DNA hybridisation.
4) The site the probe has bound to can be identified with the radioactive/ fluorescent marker.
41
Importance of genetic screening in identifying heritable conditions
- alleles for gene mutations which cause genetic disorders can be identified.
e. g. to determine the probability of a couple having offspring with a genetic disorder.
42
What are radioactively labelled DNA probes and how can they be identified?
Radioactively labelled probes - nucleotides have 32-P isotope of phosphorus. The probe can be identified using X-ray/ photographic film.
43
What are fluorescently labeled DNA probes ad how can they be identified?
emit light (fluoresce) under certain conditions, eg when the probe is paired with its target sequence.
44
What is required before a complementary DNA probe can be made?
The sequence of the sample DNA needs to be known
45
What is genetic screening? What can it be used for? (3)
Labelled DNA probes can be used to identify certain alleles and screen patients for heritable conditions, drug responses and health risks.
46
Why is it important to identify mutations related to cancer with genetic screening? (2) (health risks)
Individuals with these mutations are at higher risk of developing cancer.
If mutations are identified, individuals are able to mae informed decisions about their lifestyle and further treatment. e.g. giving up smoking, living more healthily, having regular checks for the early signs of cancer, surgery.
The type of mutations can be used to determine which type of cancer a patient has and therefore which drug will be most effective.
47
What kinds of genes relating to cancer can be identified with genetic screning?
oncogenes - mutations of proto-oncogenes
| mutated tumour suppressor genes
48
What is personalised medicine/ why is it important?
genes determine response to certain drugs, so different people respond differently. If your genotype is known, doctors can use this to predict how you will respond to different drugs and prescribe the most effective ones.
49
What is genetic counselling?
advising patients and their relatives about the risks of genetic disorders. e.g. explaining the results of screening and the treatment options available.
50
What are VNTRs? how do they differ between different people?
variable number tandem repeats - repeated base sequences which do not code for proteins and differ from person to person (apart from identical twins). The probability of 2 people having the same VNTRs is very low. The more closely related 2 individuals are, the more similar their VNTRs will be.
51
Technique of genetic fingerprinting
* **
1) Extraction: DNA is extracted from the sample and cloned by PCR.
2) Digestion: restriction endonucleases cut the DNA into fragments.
3) Separation: of fragments using gel electrophoresis.
4) Separation pt.2: DNA fragments are transferred from the gel to a nylon membrane.
5) Hybridisation: radioactive DNA probes are added to attach to and label specific fragments.
6) Development: the nylon membrane is placed on X-ray film. The film is developed and shows dark bands where the radioactive DNA probes have attached.
52
What happens in gel electrophoresis?
***
The DNA fragments are placed on an agar gel and a voltage is applied across it. The larger fragments move more slowly due to the gel’s resistance, so fragments of different lengths are separated. The final positions of DNA fragments can be determined if they are radioactively or fluorescently labelled.
53
What is the purpose of gel electrophoresis?
***
| to separate DNA fragments according to their size and produce a DNA fingerprint.
54
How can smaller DNA fragemnts be sequenced? what is required for larger fragments?
***
Smaller DNA fragments can be sequenced using gel electrophoresis, but larger fragments/ genes must be cut into smaller fragments by restriction endonucleases first.
55
Uses of genetic fingerprinting
forensic science,
| medical diagnosis, animal and plant breeding
56
Why do DNA fragmnets need to be cloned before genetic fingerprinting is done?
because the amount of DNA in the initial sample (e.g. from a crime scene) is often very small.
57
How can genetic fingerprinting be used in determining genetic relaionships?
paternity tests - individuals inherit half of theirgenetic information and VNTRs from each parent.
Each band on an individual’s DNA fingerprint should have a corresponding band on one of the parent’s fingerprints. This can be used to determine whether or not someone is a child’s genetic father.
58
How can genetic fingerprinting be used to determine genetic variablity withing a population?
the more closely related 2 individuals are, the more similar their genetic fingerprints. So if a population has similar genetic fingerprints, there is little genetic diversity.
59
How can genetic fingerprinting be used in forensic science?
***
matching DNA found at a crime scene with that of a suspect by comparing the bars on a genetic fingerprint.
If there is a close match, the suspect is likely to have been present at the crime scene.
The probability that someone else’s DNA matches that of the suspect is also calculated. This is based on the DNA that produces banding patterns being randomly distributed throughout the population.
60
How can genetic fingerprinting be used in medical diagnosis? (2)
- to diagnose genetic disease, by comparing fingerprints from the patient, people with different forms of the disease and people without the disease.
- Identifying the nature of microbial infection - comparing the fingerprint of the microbe found in patients with that of known pathogens.
61
How can genetic fingerprinting be used in animal and plant breeding? (4)
1) Preventing undesirable breeding in breeding programmes
2) Identifying plants or animals with certain alleles of a gene
3) Selective breeding - determining the likelihood of offspring having the characteristic coded for by a particular allele.
4) Paternity in animals, making family trees.
62
Issues associated with recombinant DNA technology in agriculture
- farmers might grow GM crop monocultures. here, crops may all be vulnerable to the same disease because plants are genetically identical. reduction in biodiversity.
- weeds resistant to herbicides could develop is transformed crops breed with wild plants. could lead to uncontrolled spread of recombinant DNA, consequences unknown.
63
Issues associated with recombinant DNA technology in medicine
- technology could be used unethically, e.g designer babies.
| - companies could limit the use of life-saving technologies.
64
Humanitarian benefits of recombinant DNA technology
- GM crops resistant to drought etc could be produced to reduce the risk of famine and malnutrition.
- transformed crops can be used to produce useful pharmaceuticals, helping make drugs available to more people.
- gene therapy to treat genetic disease in humans
65
what is gene therapy?
altering the defective genes inside cells to treat genetic disorders and cancers
66
What does whether or not an individual with a mutant allele develops a genetic disease depend on?
whether the mutation results in a dominant or recessive allele.
67
How can gene therapy be carried out?
a new allele can be inserted into a cell using vectors, which are produced as in recombinant DNA technology. The vector can be used to insert the DNA fragmnet into a person's DNA, e.g. using an altered virus.
68
Why is a base pair a suitable unit for measuring the length of a DNA fragment? (2)
DNA is formed of 2 chains of nucleotides with bases bonded in pairs.
Each base pair is a constant length
69
Why do all the plants produced when cells with recombinant DNA/ plasmid divide contain the target gene? (3)
- cells are produced by mitosis from original cells containing plasmid.
- all offspirng cells are genetically identical
- all new cells are produced from a cell with the target gene.
70
Why are there concenrs about GM crops with marker genes for antibiotic resistance being eaten by humans?
The antibiotic resistance gene could be spread to other bathogenic species of acterium.
These bacteria can no longer be killed with the antibiotic - so difficulties in treating disease.
71
explain how enzymes and vectors may be used to isolate genes and insert them into another organism
Restriction enzymes;
Cut DNA; at specific base sequences;
Same (restriction) enzyme also cuts DNA; into which gene is
inserted/plasmid/virus/Agrobacterium;
(DNA) ligase;
Joins two pieces of DNA together/forms recombinant DNA;
Vector needed to insert DNA into host/plasmid enters host/second
organism;
Correct ref. to sticky ends; Reverse transcriptase; mRNA DNA;
Biology A2
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