3.8.4.2 and 3 DNA fingerprinting and Genetic counselling (C21) Flashcards
What are the two methods that can be used in gene therapy?
germ line therapy and somatic cell therapy
How does germ line gene therapy work?
Replacing or supplementing the defective gene in the fertilised egg. This means all of the developing cells will be normal, as will the cells of the next generation. The solution is permanently but currently prohibited for ethical reasons
How does somatic cell gene therapy work?
Targets the affected tissues (such as the lungs). This does not pass to the next generation as it is not in the gametes. The treatment is also short lived for the patient as the lung cells are continually dying and being replaced. The treatment therefore needs to be repeated every few days in some cases.
Which two vectors can be used to get a healthy gene into somatic cell of a patient?
- Using a harmless virus
- Wrapping the gene in lipid molecules
Why might gene therapy be unsuccessful?
- Adenoviruses may cause infection.
- Patients may develop immunity to the adenoviruses.
- The liposome aerosol may not be fine enough to pass through the bronchioles in the lungs.
- The CFTR gene may not be expressed even when successfully delivered to the epithelial cells.
What is a DNA probe?
The DNA probe is a short, single stranded section of DNA that has an identifiable label attached to it.
What are the two types of DNA probes and how do they work?
- Radioactively labelled probes – made of nucleotides with the isotope 32P. A photographic plate exposed to -adioactivity is used to identify the probe.
- Fluorescently labelled probes – emit light under certain conditions.
How do DNA probes result in a scientist being able to identify a mutant allele in a patient?
A probe with bases complementary to the bases on the portion of the gene we want to find is made.
The DNA being tested is treated to separate the strands.
The separated strands are mixed with the probe which binds the complementary bases on the strands. This is DNA HYBRIDISATION.
The site at which the probe binds can be identified by radiation or fluorescence emitted.
What is the point of genetic screening?
Genetic screening is carried out on individuals who may carry the mutant gene, this is determined by looking at their family history.
Screening can allow a couple to find out the chances of having a child with a genetic disorder. Couple as risk can then be referred to a genetic Councillor for advice.
If a positive gene test result reveals that a seemingly healthy individual carries or has a genetic mutation associated with a specific disorder, what would a ‘negative gene test result’ be? How about a ‘false negative gene test result’?
A negative gene test result reveals that the faulty gene being looked for is not there. A false negative result means that further testing is probably needed as there was a problem with the procedure.
Why are introns (not exons) used in genetic fingerprinting?
Genetic fingerprinting is based on the fact that DNA the genome of any organism has many repetitive non-coding bases – INTRONS (e.g. 95% of human DNA does not code for any characteristic).
Introns contain repetitive sequences of DNA called core sequences which differ in everyone (except identical twins).
What are the stages of DNA fingerprinting?
Extraction, Digestion, Separation, Hybridisation, Development
In DNA fingerprinting what happens in Extraction?
extract DNA from the sample (however small) by separating it from the rest of the cell. PCR can be used to make more if necessary
In DNA fingerprinting what happens in Digestion?
the DNA is cut into fragments using restriction endonucleases (chosen as they cut close to but not inside core sequences).
In DNA fingerprinting what happens in Separation?
gel electrophoresis separates the DNA fragments according to size. The gel is then immersed in an alkali to separate the 2 DNA strands. The single strands are then transferred onto a nylon membrane by a technique called southern blotting
In DNA fingerprinting what happens in Hybridisation?
labelled DNA probes with complementary sequences are used to bind to the core sequences. The temp and pH must be correct for binding to occur. Different probes are used to bind different core sequences.
In DNA fingerprinting what happens in Development?
an X-ray film is put over the nylon membrane and the film is exposed by the radiation in the probes (fluorescence probes are seen visually). A series of bars are revealed in a pattern of bars unique to the individual.
How does southern blotting work in the separation stage of DNA fingerprinting?
- A nylon membrane is laid over the gel and several sheets of absorbent paper are placed on top which draws up the liquid containing the DNA via capillary action.
- The DNA fragments are transferred to they nylon in the same position.
- The fragments are fixed in place on the membrane using UV light.
How are genetic fingerprint results interpreted?
- A DNA fingerprint is compared for 2 or more samples of blood from the scene of a crime for example compared to the suspect(s).
- They are then visually checked.
- If there seems to be a match each fingerprint is passed through an automated scanning machine which calculates the length of the fragments from the bands (done via measuring the distance travelled by known lengths of DNA).
- Finally the odds are calculated of someone having an identical fingerprint .
- The closer the match between the two samples the more likely they came from the same person.
Give several uses of genetic fingerprints.
Forensic science.
Check immigration applications.
Confirm animal pedigrees / ownership.
Check evolutionary relationships.
To determine genetic variability within a population.
Paternity testing. Any bars which are not from mum will be from dad
What does VNTR stand for in genetic fingerprinting?
variable number tandem repeats
role of DNA polymerase
joins nucleotides together in DNA (complementary strand)
describe the role of restriction endonuclease when used to add a piece of DNA into a plasmid
cuts the plasmid
Describe 2 features of proteins what would allow different proteins to be separated by gel electrophoresis
- mass/length of AA
- charge
- R group differs
Describe Gel Electrophoresis
- DNA is cut at areas of tandem repeats using restriction endonucleases
- DNA fragments are placed in wells at the top of an agar gel.
- An electric current is applied over it
- DNA is negatively charged due to the phosphate group
- The DNA moves towards the positive electrode, but at different rates
- Small fragments move further through the gel
- A ladder/marker can be used to determine the size of the DNA fragments
Describe DNA fingerprinting
Extracted DNA is cut with a restriction endonuclease at sites of variable number tandem repeats/minisatellites
DNA is separated by gel electrophoresis, shorter fragments run further on the gel
Use Southern Blotting to transfer DNA to a nylon membrane
Use an alkaline solution to make DNA single stranded
Add a single stranded probe tagged with radioactive/fluorescent molecule
Visualise the DNA using and X-ray film or UV light
What is a DNA probe
A DNA probe is a short single stranded piece of DNA with bases that are complementary to the known genes or alleles.
It has a radioactive or fluorescent tag
What is a minisatellite?
Variable number tandem repeats (VNTRs) are areas of non-coding DNA between genes
Why are minisatellites useful?
These are repeated non-coding DNA, but the combinations and number of repeats and pattern produced are unique to individuals
How are VNTR used in in genetic fingerprinting?
Digest the DNA with a restriction endonuclease then run on a gel and southern blot. The VNTRs are unique to individuals, so DNA probes can be used to fins a specific banding pattern unique to individuals.
Genome
complete set of DNA in a cell/organism
Proteome
all the protein that can be coded for by the genome/DNA
What can genetic councillors give advice on?
Probability/Genetic risk of developing a disease based on DNA tests
Order genetic testing
Explain inheritance and how diseases are inherited
Options/strategies for managing a diagnosis
Pros/Cons of certain treatments
What can genetic councillors not give advice on?
Whether you will get a disease
What treatment/action you should take
How severe a disease will be
Can’t advise whether or not to have children
How to remember the order of genetic fingerprinting
Estaban Drove Sergio’s Hidious Digger
Extration Digestion Seperation Hybridisation Development
