Gene technologies part 2 Flashcards
1
Q
What are DNA probes?
A
A short, single stranded length of DNA with a label attached to make it easily identifiable
2
Q
What are the 2 most commonly used probes?
A
- Radioactively labelled probes - made of nucleotides with the isotope 32P, and identified using an X-ray film exposed to radioactivity.
- Fluorescently labelled probes - emit light under certain conditions, e.g. when the probe has bound to the DNA sequence.
3
Q
How do DNA probes identify particular alleles of genes?
A
- A DNA probe is made that has complementary base sequences to the DNA of the allele.
- The double stranded DNA being tested is treated to separate its strands.
- The strands are mixed with the probe, which binds to the complementary base sequence of one of the strands - DNA hybridisation.
- The site it binds to can be identified by the probes
4
Q
What is DNA hybridisation?
A
- Used to sequence the exact order of bases in a length of DNA of an allele trying to be located, so a specific probe can be made.
- It takes place when a section of DNA or RNA is combined with a single-stranded section of DNA which has complementary bases.
5
Q
What happens before hybridisation?
A
- The two strands of DNA must be separated.
- This is done by heating DNA until its double strand separates into complementary single strands (denaturation).
- When cooled, the complementary bases recombine (anneal) with each other to reform the original double strand.
6
Q
What are the implications of hybridisation?
A
- Given sufficient time, all strands in a mixture of DNA will pair up with their partners.
- If however, other complementary sections of DNA are present in the mixture as the DNA cools, these are just as likely to anneal with one of the separated DNA strands as the two strands are with one another.
7
Q
What is the summary of the process of DNA hybridisation?
A
- One strand is labelled and mixed with an unlabelled comparison strand
- the more similar the strands, the more strongly they will bind, and more energy will be required to break the strands apart
8
Q
What is location of alleles?
A
- Using DNA probes and DNA hybridisation, it is possible to locate a specific allele of a gene.
- This could be used to determine whether someone possesses a mutant allele that causes a particular genetic disorder.
9
Q
What is the process of locating specific alleles of genes? -first half
A
- The sequence of nucleotide bases of the mutant allele is determined, using DNA sequencing techniques, or by referring to the genetic library of bases sequences of genetic diseases.
- A fragment of DNA is produced with complementary base sequences to the mutant allele.
- Multiple copies of the DNA probe are formed using PCR.
- A DNA probe is made by attaching a marker to the DNA fragment.
10
Q
What is the process of locating specific alleles of genes - second half ?
A
- DNA from the suspected person is heated to separate the strands.
- The strands are cooled in a mixture containing many DNA probes.
- If the DNA contains the mutant allele, one of the probes is likely to bind, because the base sequences are complementary.
- The DNA is washed clean of any unattached probes.
- The remaining hybridised DNA is fluorescently labelled with the dye attached to the probe.
- The dye is detected by shining light onto the fragments causing the dye to fluoresce, detected by a microscope.
11
Q
What are the origins of genetic disorders?
A
- Many genetic disorders are the result of gene mutations.
- If the mutations results in a dominant allele, all individuals will have the disorder, if recessive, then only homozygous individuals will.
- Heterozygous individuals will be carriers of the disease, and will not display symptoms, but are capable of passing it on to offspring.
12
Q
Why is it important to genetic screen?
A
- Individuals who may carry a mutant allele often have a family history of a disease.
- Screening can determine the probabilities of a couple having offspring with a genetic disorder.
- So potential parents at risk can obtain advice from a genetic counsellor about the implications of having children
13
Q
What is the process of genetic screening?
A
- It is possible to fix hundreds of different DNA probes in a pattern on a glass slide.
- By adding a DNA sample to this array, any complementary DNA sequences in the donor will bind to on or more probes.
- It is therefore possible to test simultaneously many genetic disorders by detecting fluorescence that occurs where binding has happened.
14
Q
What is another use of genetic screening?
A
- The detection of oncogenes, which cause cancer.
- Cancers may develop from mutations that prevent tumour suppressor genes inhibiting cell division.
- Mutations on both alleles must be present to inactivate a tumour suppressor gene and to develop a tumour.
- Some people inherit a mutated tumour suppressor gene, and so are at greater risk of developing cancer.
15
Q
What is the result of genetic screening for cancer?
A
- Individuals more a risk of cancer can make informed decisions about their lifestyle and future treatment.
- They can choose to give up smoking, lose weight, eat healthier and avoid mutagens.
- They can check themselves more regularly for signs of cancer, which can lead to earlier diagnosis and better chance of successful treatment.
16
Q
What is the use of screening for personalised medicine?
A
- Genetic screening allows doctors to provide advice and health care based on an individual’s genotype.
- An individual’s genes can mean that a particular drug may be either more or less effective in treating a condition.
17
Q
What is personalised medicine?
A
- Genetic screening can determine more exactly the dose of a drug to produce the desired outcome.
- This can save money, that would otherwise be wasted on overprescribing drugs.
- It can also avoid medications that could cause harm or avoid raising false hopes.
18
Q
What is personalisation of prescribing painkillers?
A
- To function effectively, many pain medications need a specific enzyme to activate them.
- About half the population have this enzyme.
- Screening for the presence of these genes allow the dosage to be adjusted to compensate for how the genes affect the individual’s metabolism of the painkiller.
19
Q
What is the personalisation of vitamin E?
A
- for those who have diabetes, vitamin E can reduce the risk of cardiovascular disease for those with a specific genotype
- it can increase risk for those with a different genotype
- advantageous to screen someone who has diabetes before advising on whether they should take vitamin E supplements or not
20
Q
What is genetic counselling?
A
- expert advice provided by a counsellor to help individuals understand the results and implications of the screening and so make appropriate decisions
- important to research family history of an inherited disease and to advise parents on the likelihood of it arising in their offspring
21
Q
What is the use of genetic counselling?
A
- makes the parents aware of the chance of their children being affected with a disease and the chance of them being carriers
- can inform of the effects, so the parents can decide whether to have children or not
22
Q
What can genetic screening provide a genetic counsellor with?
A
- provides them with a basis for informed discussion
in cases of cancer it can help detect: - oncogene mutations, which can determine they type of cancer the patient has and the most effective drug or radiotherapy to use
- gene changes that predict which patients are more likely to benefit from certain treatments and have the best chance of survival
- a single cancer cell among millions of normal cells, identifying patients at risk of relapse from certain forms of leukaemia
23
Q
What are the benefits of genetic profiling?
A
- can identify disease very early, and therefore begin to treat them before symptoms develop, reducing impact on the individual
- treatment can also be personalised to make it more effective
24
Q
What is genetic fingerprinting?
A
- a diagnostic tool used widely in forensic science, plant and animal breeding and medical diagnosis
- based on the fact that the DNA of every individual, except identical twins is unique
25
What does genetic fingerprinting rely on?
- the fact that the genome of most eukaryotic organisms contains many repetitive, non-coding bases of DNA
- DNA bases which are non-coding are known as variable number tandem repeats (VNTR)
- for every individual the number and length of VNTRs has a unique pattern
- different in all individuals except identical twins and the probability of 2 individuals having identical sequences is small
- the more closely related 2 individuals are the more similar the VNTRs will be
26
What is gel electrophoresis?
- used to separate DNA fragments according to their size
- the DNA fragments are placed on an agar gel and a voltage is applied across it
- the resistance of the gel means that the larger the fragments, the more slowly they move
- therefore, over a fixed period, the smaller fragments move further than the larger ones
- so, DNA fragments of different lengths are separated
27
What is gel electrophoresis with labels?
- if the DNA fragments are labelled, e.g. with radioactive DNA probes, their final positions in the gel can be determined by placing a sheet of x-ray film over the agar gel for several hours
- the radioactivity from each DNA fragment exposes the film and shows where the fragment is situated on the gel
- only DNA fragments up to around 500 bases long can be sequenced in this way
- larger genes and whole genomes must therefore be cut into smaller fragments by restriction endonucleases
28
What are the stages of making a genetic fingerprint?
- extraction
- digestion
- separation
- hybridisation
- development
29
What is extraction?
- even the tiniest sample of animal tissue, such as a drop of blood or hair root, is enough to give a genetic fingerprint
- whatever the sample the first stage is to extract the DNA by separating it from the rest of the cell
- as the amount of DNA is usually small, its quantity can be increased by using the polymerase chain reaction
30
What is digestion?
- the DNA is then cut into fragments, using the same restriction endonucleases
- the endonucleases are chosen for their ability to cut close to, but not within, the target DNA
31
What is separation?
- the fragments of DNA are next separated according to size by gel electrophoresis under the influence of an electrical voltage
- the gel is then immersed in alkali in order to separate the double strands into single strands
32
What is hybridisation in fingerprinting?
- radioactive DNA probes are now used to bind with VNTRs
- the probes have base sequences which are complementary to the base sequences of the VNTRs and bind to them under specific conditions, such as temperature and pH
- the process is carried out with different probes, which bind to different target DNA sequences
33
What is development in fingerprinting?
- Finally, an x-ray film is put over the nylon membrane
- the film is exposed by the radiation from the radioactive probes
- because these points correspond to the position of the DNA fragments as separated during electrophoresis, a series of bars is revealed
- the pattern of the bands is unique to every individual except identical twins
34
What is the interpretation of genetic fingerprinting results?
- DNA fingerprints from 2 samples are visually checked
- is there appears to be a match, the pattern of bars of each fingerprint is passed through an automated scanning machine, which calculates the length of the DNA fragments from the bands
- it does this using data obtained by measuring the distances travelled during electrophoresis by known lengths of DNA
- the odds are calculated of someone else having an identical fingerprint
- the closer the match between the 2 patterns, the greater the probability that the 2 sets of DNA have come from the same person
35
What are the uses of DNA fingerprinting?
- genetic relationships and variability
- forensic science
- medical diagnosis
- plant and animal breeding
36
What is the use of DNA fingerprinting in genetic relationships?
- can help resolve questions of paternity
- each band of DNA fingerprint of an individual should have a corresponding band in one of the parents DNA fingerprints
- this can be used to establish whether someone is the genetic father of a child
37
What is the use of DNA fingerprinting in genetic variability?
- the more closely 2 individuals are related the closer the resemblance of their genetic fingerprints
- a population whose members have very similar genetic fingerprints has little genetic diversity
- a population whose members have a greater variety of genetic fingerprints has greater genetic diversity
38
What is the use of DNA fingerprinting in forensic science?
- DNA is often left at crime scenes
- Genetic fingerprinting can establish whether a person is likely to have been present at the crime scene, although this doesn't prove they actually did the crime
- even if there is a close match between a suspect's DNA and the DNA found at the crime scene, it doesn't follow that the suspect carried out the crime
39
How does DNA fingerprinting in forensic science not prove someone committed a crime?
- the DNA may have been left on some other, innocent occasion
- the DNA may belong to a very close relative
- the DNA sample may have been contaminated after the crime, either by the suspect's DNA or by chemicals that affected the action of the restriction endonucleases used in preparing the fingerprint
40
What is the use of probability in forensic science?
- calculation is based on the assumption that the DNA which produces the banding patterns is randomly distributed in the community
- this may not always be the case, e.g. may not apply to religious or ethnic groups who tend to have partners from within their own small community
41
What is the use of DNA fingerprints in plant and animal breeding?
- can be used to prevent undesirable inbreeding during breeding programmes on farms and zoos
- it can also identify plants or animals that have a particular allele or desirable gene
- individuals with this allele can be selected for breeding in order to increase the probability of their offspring having the characteristic that it produces
- another application is the determination of paternity in animals and thus establishing the pedigree (family tree) of an individual
