Mechanisms and Effects of Mutations Flashcards
How do mutations arise?
- Random errors in DNA replication/repair
- Exposure to mutagenic agents
What are polymorphisms and what types are there?
- Non-harmful mutations
- Sequence variant in non-coding DNA
- Sequence variant in gene but doesn’t change amino acid
- Sequence variant changes amino acid but not protein function
What is a single nucleotide polymorphism?
One base in the sequence is changed (eg. C changed to T)
How can the bases of the genome be examined?
- Sequencing
- Microarray analysis
How can large blocks of DNA be examined?
- Microarray analysis
- Fluorescence in situ hybridisation (FISH)
How can chromosomes be examined?
Light microscopy
Method of DNA sequencing
Sanger sequencing:
- Amplify very small amounts of target DNA fragments (PCR)
- Fragments added to 4 mixtures, all containing all 4 deoxynucleotides (normal nucleotides), and each containing 1 of the dideoxynucleotides (OH group on 3’ carbon > H)
- No nucleotides can be added after a dideoxynucleotide as they can’t form phosphodiester bonds (no OH)
- This generates a set of fragments increasing by 1 base each time
- Fragments separated by size in their separate mixtures, so comparing them shows which the next biggest (and therefore which base next) in the sequence
- Base sequence put together and then inverted to show the original template fragment being tested
Why is DNA sequenced?
- Determine exact position of mutation
- Determine type of mutation
Endogenous mechanisms causing damage to DNA
- Depurination
- Deamination
- Reactive oxygen attacking purine/pyrimidine rings
- Methylation of cytosines
What is depurination?
- Spontaneous break of bond between purine base and the sugar
- Leads to deletion of base or incorrect nucleotide in new strand
What is deamination?
- Cytosine deaminates to uracil
- Causes substitution of A into new strand
What is methylation of cytosines and what can it cause?
- Cytosine at CpG dinucleotide is methylated
- Spontaneous deamination of 5-methyl-cytosine into thymine
Extracellular agents causing damage to DNA
- UV light
- Environmental chemicals
- Ionizing radiation
What does UV light do to DNA?
Cross-links adjacent thymines to form a dimer
What do environmental chemicals do to DNA?
Cause DNA breaks or chromosome aneuploidy
What does ionizing radiation do to DNA?
Cause breaks in DNA
2 types of mutation relating to location (and if they’re heritable or not)
- Germline mutation (heritable)
- Somatic mutation (non-heritable)
What are the stages of checking in order to remove mutations from replication?
- DNA polymerase adds a base, checks it and moves on
- The DNA mismatch repair system fixes most mutations missed by DNA polymerase
How does the mismatch repair system work?
- Mismatch is identified
- Newly synthesised strand is excised by a mismatch repair protein
- Gap left behind is refilled by DNA polymerase and ligase
How are DNA double stranded breaks fixed?
- Broken at same point on both strands = DNA on homologous chromosome used as template
- Broken at different points on strands = extra bits removed then ends joined
What is a splice-site mutation?
A single base substitution at the end of an exon where splicing was meant to take place
What is the normal method of splicing and how does a splice-site mutation affect this?
- Normally each exon has a splice donor site and splice acceptor site with the DNA in between being removed
- Due to mutation, one of these sites is removed
- Either an exon is removed or an intron is incorporated
How do copy number variants lead to large deletions or insertions?
- Areas of repeats at different loci on different chromosomes line up and crossing over happens here
- One chromosome then has more DNA and one has less than they usually have
