Mutagenesis Flashcards
When does a mutation occur?
-When there is a change in the nucleotide sequence resulting in a triplet codon differing in the a’a it should represent, thus causing a different a’a to be translated
What is a silent substitution?
-A substitution which occurs at the third position of a codon which does not change the a’a produced
What are the three types of point mutations?
- Missense
- Silent
- Nonsense
What is a missense mutation?
-One a’a is substituted for another (may interrupt the tertiary structure by disrupting to bonding)
What is an example of a missense mutation?
-Sickle cell anaemia
When can silent mutations cause a problem?
- When the mutation disrupts RNA splicing
- The nucleotide sequence may change the splice sites in a way that the specific nuclease may no longer recognise the splice sequence or mat introduce a splice sequence
What is a nonsense mutation?
- When an a’a codon is mutated to produce a stop codon
- Produces a short non-functoning peptide
What is a frameshift mutation?
-Alterations in the reading frame of mRNA caused by insertions, deletions and splice site mutations
What is a common result of frame shift mutations?
-The introduction of stop codons causing the ribosome to terminate translation prematurely
Can a gain or loss of 3bp cause a frameshift?
-No the reading frame will be maintained
What happens to the mRNA when a gain/loss introduces a premature stop codon?
Why does this happen?
- mRNA is degraded by nonsense mediated decay and little or no protein is produced
- Happens as a protective mechanism
What happens when there is a mutation in an intron splice site?
-The exon is skipped immediately next to the mutation
In which two ways can sequence changes occur during DNA replication?
- Tautomeric Shift
- Slippage
What is tautomeric shift?
-Each of the 4 bases in DNA can undergo tautomeric shift where by a proton briefly changes position
How does tautomeric shift introduce a mutation?
-The rare tautomeric form of a nucleotide has altered base-pairing properties and behaves as an altered base template during replication
Which two bases undergo tautomeric shift and what does it cause them to behave like?
- C->A
- T->G
What is slippage during DNA relication?
-One of the nucleotides slips out of sequence above the rest of the mRNA molecule
How does slippage introduce a mutation?
- Slippage on the newly synthesised strand and the base is paired again introducing an extra nucleotide
- Slippage on the template strand and nucleotide is missed out on the new strand
How does nitrous acid cause mutations in DNA replication?
-Replaces amino groups with keto groups causing:
C->U
A->H
G->X
How does Ethyl Methane Sulphonate (EMS) cause mutations in DNA replication?
-Causes removal of purines and apurinic sites can be paired with any base during replication
What is a transition in DNA replication?
-Where the mutation occurs within the same group of purines or pyrimidines eg A->G or C->T
What is a transversion in DNA replication?
-When there is a mutation which changes the nucleotide group, ie purine pyrimidine
Name a base-stacking mutagen
- IQ is a food mutagen
- Ethidium Bromide
How do base-stacking mutagens lead to mutations?
-Disrupts the packing of DNA bases by intercalation and mostly causes single base deletions by forcing the bases further apart on one DNA strand so that DNA polymerase misreads leading to a deletion
What effect does ionising radiation have?
-Produces ions during interactions with cellular molecules
Name types of ionising radiation
- Solar radiation
- X-rays
- Nuclear power plant accident
- Environmental source such as radon gas
Exposure to which UV source is beneficial to a certain point?
-UVB
How do UVA,UVB, and UBC cause ageing?
-Damage collagen fibres in the skin
What vitamin do UVA and UVB destroy in the skin?
-Vitamin A
What effect does UV light have on thymine bases?
-UV photons cause adjacent T bases to form dimers
What is photo-reactivation?
-Spontaneous resolution of T dimers
What is the general rate of DNApol error rate?
-1 in 100,000 bases (120,000 mistakes every time DNA in a single cell replicates)
What is proof reading?
- Corrects most DNApol errors
- Detection of mispaired 3’ base in new;y synthesised strand and corrects
What is nucleotide mismatch repair?
- Multisubunit enzyme detects mismatched bases in newly synthesised strand and replaces them
- A ‘patch’ of DNA sequence is replaced by strand excision and re-synthesis
When does base-excision repair occur?
-When there is an accumulation of DNA damaged bases through oxidation, alkylation or deamination
What can be the result of failed DNA repair?
-Cancer
In hereditary non-polyposis colorectal cancer, what is often mutated?
-Mismatch repair enzymes
What often happens to the DNA in cancerous cells?
-Exhibits chromosomal and microsatellite instability
Early mutations affect functions increasing…
-The probability of successive mutations
What do BRCA1 and BRCA2 code for?
-Proteins involved in detecting DNA damage and signalling to cell cycle check points
What are proto-oncogenes?
-Genes which have the ability to become oncogenes through a’a substitutions (ie DNA damage)
What method are the majority of tests for detection of mutations based on?
-PCR
Why is DNA sequencing not used?
-Expensive
In recessive diseases, are the mutations in the alleles identical?
-They do not have to be
What is single strand conformation polymorphism (SSCP)?
-Single-stranded DNA has a defined conformation. Altered conformation due to a single base change in the sequence can cause single-stranded DNA to migrate differently during electrophoresis. Therefore wild-type and mutant DNA samples display different band patterns. 1) PCR 2)Denature 3)Snap Cool 4) electrophoresis
Where is it possible to isolate foetal DNA from?
- Amniotic fluid cells
- Chorion villus biopsy
- Foetal DNA in mothers blood
What is amniocentesis?
- Performed at 15-20 weeks of gestation by ultrasound guidance
- Injection through abdomen to recover cells (may need to be cultured)
- 0.5-1% miscarriage
What is a chorion villus biopsy?
- Performed at 10-13 weeks of gestation with ultrasound guidance
- Trans-cervical procedure recovering cells
- Foetal placental villi must be separated from maternal tissue
- 2% chance of miscarriage
Why is foetal DNA from the mothers blood not widely used?
-The DNA is very unstable
What is the principle of MLPA?
-To perform exon counts to identify whole exon duplications or deletions
