TD: Gene Therapy Flashcards
What are genes?
Where are they contained?
What happens to genes within DNA?
What is the central dogma?
- Building blocks of inheritance
- Genes contained within the DNA that make up the chromosomes which are present in every eukaryotic cells bar RBC.
- Sections of DNA contain specific and precise sequence to which specific proteins are translated
- DNA – mRNA - Protein
- •1in 300 nucleotides are polymorphic. Polymorphism are mutations in the DNA which occurs at specific frequency so are significant to the functioning of a particular gene
What is the most common polymoprhism?
How can these be detected?
What can RFLP be used for?
Describe analysis and how RFLA is carried out
Related this to the following example
Used to compare homologous DNA from two different samples:
Restricition endonucleases are used to detect base pair change (E.g. SNP) within DNA. These recognise specific sequences of DNA which are 6 bp long and termed restriction sites. RE cut DNA at specific sites. Therefore if there is a gene mutation and the bp sequence is affected then the RE will no longer recognise this and cut. Therefore SNP can be identified by loss or gain or new restriction sites.
The resolved DNA is put on gel. As the healthy DNA and mutant DNA will both produce fragments of different lengths these can be identified.
The fragments of varying length can be seperated by gel electrophoresis where the sample is loaded on the negative end of the agrose gel and a current applied. As DNA is negtaively charged, it will migrate towards the positve cathode.
As can be seen in the diagram the normal DNA is cut into 4 fragments. Due to the SNP present within the restriction site, the mutant DNA is only cut into 3 fragments
The larger fragments migrate less and smaller further. Some fragments are the same as bp has not been affected. - the difference allows for mutation to be identified and where.
In reality restriction digest will produce lots of fragments and not all will be of interest. Probe DNA with labelled nucleotide specific to gene of interest (hybridisation).
Screening - why is genotype information so desirable in terms of clinical data
Change in genes which encode for metabolising enzymes of receptor/transporter expression can affect safety and therapeutic effcet of a drug
It’s not enough just knowing a RFLP is there, you also need to look at how it has been passed down through generations.
why?
Describe RFLA in the passing of deleterious genes to future generations
In order to predict the likelihood of a deleterious allele appearing within a family, RFLP can allow you to screen for all the allelic variants of a particular gene
Would not expect the common allele to have a harmful effect, as natural selection would have deleted it.
Evolutionary processes are incomplete for rare alleles
What is gene therapy and what are the 3 main methods?
Experimental technique that uses genes to treat or prevent disease
- Replace a mutated disease causing gene with a healthy opy
- Inactivate or “knock out” a faulty gene that is not working properly (good for cancer)
- Introduce a new gene into the body to help fight a disease
Diseases with no known cure are prime candidates
What is the classification of gene therapy?
Somatic Cell therapy can be direct or indirect - describe both
in direct there is modification of the function of a gene involved in pathogenic process
What are the types of modification involved in gene therapy?
1. Gene augmentation
Supply functioning copy of the gene (where it is mutated or abscent):
- Cystic fibrosis (CF)
- Cystic fibrosis conductance transmembrane regulator (CFTR) gene mutation leads to production of more Cl- channels which are responsible for the movement of Cl- ions and fluid - leading to CF
- Cancer Therapy
- p53 (tumour suppressor gene) - enhance/augmentate P53 (tumour supressor gene)
2. Gene elimination
Restore function of mutated gene:
- Replace pathogenic mutation with healthy equivalent
3. Gene Inhibition
Target deleterious genes (aims to target abnormal/over expressed genes)
- Silence activated oncogenes
- Reduce effects of autoimmune disease
4. Gene targeted cell killing
- Specific
- Cancer treatment
- Cancer cells using genes encoding toxins
- Genes to provoke strong immune response
- Immunotherapy
- Cancer cells using genes encoding toxins
The required gene, nucleotide DNA can be inserted into the body in 2 ways: Ex vivo and In vivo.
Describe Ex vivo
The required gene, nucleotide DNA can be inserted into the body in 2 ways: Ex vivo and In vivo.
Describe in vivo
