Human Genetics Gene therapy & DNA fingerprinting Flashcards
what is gene therapy
The treatment of genetic diseases by giving patients healthy/normal functioning copies of (mutated) defective alleles.
what are the three ways of altering the genotype?
- Repair of the gene
- Replacement of a defective gene with a normal one
- Addition of a normal gene leaving the defective one in place
what are the two types of gene therapy?
- somatic gene therapy
- germline gene therapy
what are the somatic cells?
body cells
how does somatic cell gene therapy work?
Copies of a functional gene are inserted directly into the body cells of sufferers.
If the normal gene has been successfully inserted/ taken up by the affected cells and the patient later has a child, since the gene is NOT in the germ-line, the children will NOT inherit the added functioning gene. Can be short-lived and repeated treatments required
what is a germline cell?
sperm/egg/zygote
what does totipotent mean, and give an example?
They can divide & specialise to become any cell within the body
example: egg/sperm/zygote
what is the germ-line?
The germ-line is the genetic information which is passed onto the next generation i.e. the DNA in the egg or sperm / the gametes
how does germline therapy work?
A corrected / functional, gene is inserted into an egg cell that has been fertilised in vitro (IVF).
Embryo then develops, then is re-implanted into mother’s uterus.
If gene transfer is successful, mitosis ensures that every cell in the embryo contains a copy of the corrected gene.
If there are any changes in these gametes then the offspring will inherit them
it crosses generations
what does germ-line therapy provide?
a treatment for an inherited condition that lasts a lifetime.
is germline therapy legal in humans?
NO
what are the problems related to gene therapy?
The problems are how to get the genes to the EXACT cells that need them, and then to make sure they are expressed – transcribed/translated into a protein.
what are vectors and what do they do?
Vectors are carrier molecules
Need vectors to get the normal functional gene into a target cell
what are examples of vectors?
Viruses
Plasmids
liposomes
how do you use adenovirus vectors for gene therapy?
A functional gene is injected into an adenovirus vector, which is used to introduce the modified DNA into a human cell. If the treatment is successful, the new allele will make a functional protein
what are advantages of viral vectors?
- Good at targeting and entering cells
- can target specific cell types - can be modified so they cant replicate and destroy cells
what are disadvantages of viral vectors?
- Can only carry limited size of gene
- May lead to immune response in patients which may cause side effects or block the virus getting to patient’s cells or kill cells once infected
what are the advantages of liposome vectors?
Can carry normal/functional alleles as a plasmid inside the bilayer
Lipid bilayer will fuse with cell surface membrane to deliver contents directly into the cell
Can carry large genes and don’t cause immune response in patients
why are liposomes used as gene therapy?
- Liposomes are spheres made of lipid bilayer
- Can carry normal/functional alleles inside the bilayer
- Lipid bilayer will fuse with cell surface
membrane to deliver contents directly into the
cell
what are vector delivery methods?
- in vivo
- ex vivo
what is ex vivo delivery?
Body cells are removed, copies of functional genes inside vectors are inserted into them and then the transfected cells are put back into the patient’s body.
what does ex vivo delivery work best with?
leukemia,
patient bone marrow cells are extracted, transfected, then replaced
less likely to trigger an immune response
what is in vivo delivery methods?
If a large number of body cells cannot be removed safely, functional genes inside a vector are inserted directly into the affected tissues.
what is in vivo delivery methods used for and how?
cystic fibrosis treatments,
Functional CFTR gene is inserted into a liposome and inhaled to get it to the target cells which are epithelial cells of the lung
what are the challenges in gene therapy?
- Gene Delivery and activation (produce functional protein)
- Reduce immune response
- Prevent disruption to normal host cell gene function
- Commercial viability – estimated cost of developing a new pharmaceutical drug is over $500,000 – how do companies make a profit if there is a small number of patients and only 1 treatment is required?
what vectors were used in 1993, against cf and what problems were encountered?
- Used adenovirus vector
- Gene didn’t enter cells
- Dose increased = immune response
what vectors were used in 1995, against cf and what problems were encountered?
- Used liposome vector
- Low levels of gene delivery into cells and DNA didn’t integrate so gene activity decreased over time
- Side effects in patients (fever/inflammation)
what genetic disorders make the best candidates for treatment using gene therapy?
genetic disorders caused by mutations in single genes
what vectors were used in 1998, against cf and what problems were encountered?
- Used adeno-associated virus vector
- No immune response/side efects
- Did not enter cells effectively or integrate into genoma as expected
What four things must a vector be able to do in order to be successful?
- TARGET the right cells.
- INTEGRATE the gene in the cells.
- ACTIVATE the gene.
- AVOID harmful side effects.
where has gene therapy shown some level of success?
- immune deficiencies
- hereditary blindness
- hemophilia
- blood disease
- fat metabolism disorder
- cancer
- parkinson’s disease
what is electrophoresis used for?
This technique is used to separate DNA fragments based on their SIZE so they can then be identified and analysed.
what is electrophoresis based on?
It is based on the fact that the rate at which a particular fragment of DNA moves is proportional to its mass (length).
The smaller the fragments will travel further than the larger ones.
how does gel electrophoresis work?
- DNA Samples are placed into the
wells at the negative end of the gel - Gel is immersed in a buffer solution and an electric current is passed through the solution for approx 2 hours. DNA is negatively charged, therefore fragments diffuse to the positive electrode.
- Shorter lengths of DNA move faster and further. The position of fragments is shown by a dye that stains DNA
- Gel electrophoresis can use either horizontal or vertical tanks to hold the gel
- After separation, the position of the DNA fragments in the gel can be shown by flooding it with a DNA-binding dye that stains & shows them up
What is the purpose of gel electrophoresis?
to analyse and identify nucleic acids, and to determine the purity, integrity, and fragmentation of samples.
What acts as a filter?
the gel
How is the agarose different to
jell-o?
argarose sets firmer at higher temperatures and is more brittle
What is the electric current for?
makes the DNA move
Why are DNA fragments drawn
towards the positive pole?
DNA molecules have a net negative charge due to their phosphate backbone, causing them to be attracted to the positively charged electrode (anode) when an electric current is applied
what is the purpose of the loading buffer?
to increase the density of a sample, allowing it to sink into the well of the gel, and to provide a visible dye that tracks the progress of the sample as it migrates through the gel during electrophoresis
What is the name of the holes left
in the gel as it cools in which the
DNA is loaded?
wells
What does the ‘DNA size standard’ contain? What is its purpose?
a mixture of DNA fragments with known, predetermined sizes, which is used as a reference point to estimate the size of unknown DNA fragments within a sample being analyzed on the same gel
How do you know when the current is running?
when you see bubbles forming around the electrodes in the gel chamber
Why do the longer DNA strands
migrate the least?
they encounter more resistance when trying to move through the gel’s pores
Why is ethidium bromide used to stain DNA? (2 reasons)
it intercalates between the base pairs of DNA, causing it to fluoresce brightly under UV light, making the DNA bands visible in the gel; and secondly, because this intercalation slightly alters the DNA’s mobility, allowing for size estimation of the DNA fragments based on how far they migrate through the gel.
Why is it necessary to avoid
contact with ethidium bromide?
it is a potent mutagen, meaning it can damage DNA and potentially cause mutations, posing a significant health risk
what is the difference between introns and exons?
exons are the coding regions of a gene that are used to produce proteins,
while introns are non-coding regions of DNA
What percentage of human DNA does not code for proteins?
95%
where are VNTRs located?
Regions of non-coding DNA between genes can consist of regions of variable number tandem repeats (VNTRs)
what are VNTRs?
These are repeating sequences of DNA bases between 10-80 bases long
different repeating sequences in each person are?
Different repeating sequences are found at various different positions on a DNA molecule. the number of times these sequences are repeated is different in different people
What experimental method could we use to determine how long these repeated sequences are from different DNA sources?
DNA sequencing using next-generation sequencing (NGS) technology
How could we cut out these VNTR sequences from samples of DNA?
restriction enzymes
The more closely related two individuals are, the VNTR?
the more similar the number of variable number tandem repeat (VNTR) sequences they share
How do we carry out a genetic fingerprinting experiment? (2)
- The gel is immersed in alkali in order to separate the double strands into single strands.
- The pattern of fragments are transferred to a nylon (or nitrocellulose) membrane by a process called Southern blotting.
- Radioactive probes are used to attach to the VNTR DNA sequences (hybridisation). The probes have base sequences which are complementary VNTR repeat sequence. Any probes not bound are washed off. Areas with the probe attached are detected by x ray film (will turn it dark)
what is southern blotting?
- A thin nylon membrane is laid over the gel.
- The membrane is covered with several sheets of absorbent paper, which draw up the liquid containing the DNA by capillary action.
- This transfers the DNA fragments to the nylon membrane in precisely the same relative positions they occupied on the gel.
- The DNA fragments are then fixed to the membrane using UV light.
what are the uses of genetic fingerprinting?
- solving crimes
- solving medical problems
what are the chances of two people having exactly the same DNA profile?
The chances of two people having exactly the same DNA profile is 30,000 million to 1 (except for identical twins).
how is genetic fingerprinting used to solve crimes?
- The pattern of the DNA profile is compared with those of the victim and the suspect.
- If the profile matches the suspect it provides strong evidence that the suspect was present at the crime scene (NB: it does not prove they committed the crime).
- If the profile doesn’t match the suspect then that suspect may be eliminated from the enquiry.
what can DNA profiles be used to determine?
DNA profiles can be used to determine whether a particular person is the parent of a child.
A child’s paternity (father) and maternity (mother) can be determined.
what can the DNA information be used in for medical problems?
- Paternity suits
- Inheritance cases
- Immigration cases
how does a paternity test work?
By comparing the DNA
profile of a mother and her
child it is possible to
identify DNA fragments in
the child which are absent
from the mother and must
therefore have been
inherited from the
biological father.
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.
how can Genetic Fingerprinting be used For Medical Diagnoses
Huntingtons disease diagnosis – due to CAG repeats at the end of a gene on chromosome 4
- Fewer than 30 repeats – unlikely to develop disease
- More than 38 repeats very high likelihood of developing disease
- Over 50 repeats – earlier onset of disease
how can genetic fingerprinting be used for animal/plant testing?
- Determine how closely related animals in captivity are to reduce inbreeding on farms or in zoos
- Establish pedigree(family tree) of an animal. (Allows animal breeders to sell animals with a good pedigree for more money)
what does inbreeding do?
Inbreeding reduces the gene pool and leads to increased risk of genetic disease/poor health/productivity
Which suspect’s DNA matches the crime scene DNA?
the suspect whose DNA profile matches the crime scene profile
