MCBG Session 13 - Molecular Diagnosis Flashcards
What is a mutation?
Mutation: A heritable alteration in a gene or chromosome (a change in the sequence of nucleotides) And, the process that produces the alteration (the source of most alleles).
What are the two sources of Exogenous mutations?
- Ionising radiation/particles & free radicals
- Mutagenic chemicals & anti-cancer agents
Outline the sources of ionising radiation/particles & free radicals.
In the UK…
- 84% of the radiation dose is natural
I. 50% radon gas from the ground
II. 9,5% from food
III. 12% cosmic rays
IV. 13% gamma rays from the ground and buildings
- 16% of the radiation dose is Artificial
I. 15% medical
II. Remaining 1% - nuclear discharges, products, fallout, occupational
Outline the formation of free radicals.
- UV light
- Ionising radiation
- Smoking
- UV
- Air pollution
- Inflammation
- Metabolism
What are the two sources of Endogenous mutations?
- DNA replication errors
- Transposable elements
Identify some examples of DNA replication errors.
- DNA lesions
- Limiting nucleotides
- Ribonucleotide incoporation
- Fragile sites or oncogene-induced stress
- DNA secondary structure
- Repetitive DNA
- Transcription and/or RNA-DNA hybrids
Outline the transposable elements.
- Specific DNA sequences (>1 gene)
- Supernumerary (many copies)
- Always contained within other DNA molecule; never in a free form
- Move (transpose) as a discrete unit
- Move (transpose) to random sites
- Insertionally inactivate target gene
- Ubiquitous
Identify micro mutations (DNA).
- Deletion
- Insertion
- Substitution
Identify macro mutations (chromosomal).
- Deletion
- Duplication
- Inversion
- Substitution
- Translocation
Identify and elaborate upon single nucleotide changes.
- Mutations that change gene products (change in amino acid – missense mutations)
I. Alter promoter activity
II. Alter translation initiation at AUG
III. Prevent mRNA splicing
IV. Reduce mRNA stability
- Mutations that change the amount of gene product (mutations affecting transcription and mutations affecting translation)
- Mutations that change the polypeptide length (frameshift mutations, mutations of stop codon, nonsense mutations)
- Mutations that do not have an effect (silent/neutral mutations, synonymous mutations)
What are the types of substitutions?
- Transition: Change to same type of base: purine to purine (A ßà G) pyrimidine to pyrimidine (T ßà C)
- Transversion: Change to different type of base purine to pyrimidine or vice versa (A/G ßà C/T)
Identify some haemoglobinopathies.
Outline DNA hybridisation.
- When a double-stranded molecule of DNA (dsDNA) is heated (or treated with an alkaline solution) it is said to denature.
- That means the hydrogen bonds between the bases are broken and single-stranded DNA (ssDNA) is released.
- If we subsequently cool the mixture containing the ssDNA then these can come back together – we say renature or anneal – to form the dsDNA molecule because the hydrogen bonds reform.
- The molecule is able to reform as the 2 strands have complementarity.
Outline DNA hybridisation - using a DNA probe.
- Now lets denature the same piece of dsDNA again. Before allowing the mixture to cool we add another piece of ssDNA that has an identical sequence to one of the strands.
- The only difference is that we have labelled this molecule with a radioactive (or fluorescent) marker.
- When the ssDNA reanneals some of the molecules will do so with the labelled piece of DNA.
- It is now possible to identify this labelled DNA using photographic film.
- This forms the basis for molecular hybridisation which is used in many molecular techniques
Outline the different types of hybridisation techniques.
- Southern blotting – named after it’s inventor Prof Sir Ed Southern this uses DNA probes to identify complementary DNA sequences after gel electrophoresis.
- Northern blotting – a molecular biochemist’s joke here! Uses DNA to detect RNA species in a similar way to the above.
- Western blotting is NOT a DNA hybridisation technique – as we discussed this involves the detection of proteins by antibodies after protein gel electrophoresis
Outline the steps involved in Southern blotting.
- Digest (genomic) DNA with restriction enzymes
- Initial DNA gel electrophoresis step to separate fragments of DNA
- Followed by the subsequent transfer to a membrane (nylon)
- Hybridisation of a probe to detect a specific piece of DNA
- Detect hybridisation (and hence DNA of interest) by exposure of filter to X-ray film - Visualisation of the labelled probe.
