L14 - Mechanisms and effects of mutations Flashcards
What are microsatellites?
Tandem repeats of 2-6 base pairs
- <100 bp in total length
What are minisatellites?
Variable number tandem repeats of 10-60 base pairs
- Can span several kb
How can variation in the genome occur?
- Alterations in the sequence of bases in a specific section of DNA
- Single nucleotide polymorphisms
- Small deletions or duplications (few bases)
What can variation in the genome lead to?
Altered effects of a protein or control of genes
- Normal human variation (i.e. eye colour)
- Differences in response to medication (effect of antidepressants)
- Influence likelihood of disease (diabetes)
- Directly result in a genetic condition (sickle cell disease)
How can a genome variant be classified?
Classified by size, frequency and clinical effects
- Large - small
- Common - rare
- Non-pathogenic - pathogenic
What is the definition of a mutation?
An alteration or change in the genetic material
- Harmful in clinical use
- From exposure to mutagenic agents but more arise spontaneously through errors in DNA replication/ repair
- More likely to be recognised if effects are detrimental
- Alters gene function and phenotype
What is polymorphism?
- Non-harmful
- Sequence variant is in non-functional DNA
- Sequence variant is within gene but does not change aa
- Sequence variant changes aa but does not alter protein function
What is a single nucleotide polymorphism?
- A change in a single base at a particular position
- Occur throughout the genome
- Most common type of genome variation
- The base change has to be a frequency of >1%
How can the genome be examined?
- Bases
- Sequencing
- Microarray analysis - Large blocks of DNA
- Microarray analysis
- Fluorescence in situ hybridisation (FISH) - Chromosomal
- Light microscopy
Describe the process of DNA sequencing
- Amplify v small amounts of target DNA (usually by PCR)
- DNA is used as a template to generate a set of fragments that differ in length from each other by a single base
- The fragments are then separated by size, and the bases at the end are identified, recreating the original sequence of the DNA
(Dideoxy or chain termination method)
Why sequence DNA?
- Sequencing determines the exact POSITION of the mutation within the gene
- Determines the TYPE of the mutation (including single base changes)
What is the Sanger sequencing method?
A method of DNA sequencing based on the selective incorporation of chain-terminating dideoxynucleotides by DNA polymerase during in vitro DNA replication `
When do mutations occur?
- Cell division
2. From intrinsic and extrinsic attacks on DNA
What are the different endogenous mechanisms that can cause DNA damage?
- Depurination
- Deamination
- Reactive oxygen
- Methylation of ctyosines
What is depurination?
Spontaneous fission of link between purine base and sugar
- Causes loss of adenine or guanine from helix
- Deletion of base or incorrect nucleotide in new strand
What is deamination?
Cytosine deaminates into uracil
- Causing substitution of an A in new strand
What is reactive oxygen endogenous mechanism?
Attack purine/ pyrimidine rings
What is methylation of cytosines?
Methylation of cytosines at CpG dinucleotides spontaneous deamination of 5-methyl-cytosine to thymine
What are the different exogenous (extracellular agents) mechanisms that damage DNA?
- UV light
- Environmental chemicals
- Ionising radiation
How dose UV light damage DNA?
Cross-linking of adjacent thymines on a DNA strand to form a stable dimer
How do environmental chemicals damage DNA?
Interpolate into DNA or cause DNA breaks or chromosome aneuploidy
What is aneuploidy?
The presence of an abnormal number of chromosomes in a cell
How does ionising radiation damage DNA?
Causes breaks in DNA
How can DNA replication errors be corrected?
- Proof reading from DNA rep machinery
- DNA polymerase adds base, checks it, excises it if wrong and move on - DNA mismatch repair
- Backup system which corrects 99% of residual errors from replication machinery
What happens if there is a mutation in the mismatch repair gene?
Mutations in the mismatch repair genes lead to accumulation of somatic mutations and so predispose to cancers
How can endogenous damage to DNA due to thymine dimers and chemical adducts be corrects?
- Mismatch identified
- Usually repaired by excision repair mechanisms (1300 genes involved)
How can damage to DNA due to ionising radiation and reactive oxygen species be corrected?
DNA double strand breaks repaired either by:
- The accurate method of using a sister DNA molc (homologous recomb)
- End-joining broken ends (error prone - resulting in deletion of nucleotides at repair site)
What are the different types of mutations possible?
- Inversion
- Insertion
- Deletion
- Substitution
- Translocation
- Non-disjunction
What are the different effects of mutations possible?
- Missense
- Nonsense
- Frameshift
- Silent
- Beneficial
- Neutral
What is a nonsense mutation?
The triplet for an aa is replaced with a triplet which is a STOP codon
What is a missense mutation?
The triplet for an aa is replaced with a triplet for a different aa –> ppc different 1o structure which will alter the 2o, 3o and 4o structures
- Changes to a codon for another aa
What is a silent mutation?
The triplet for an aa is replaced with a triplet for the same aa
What is a frameshift mutation?
A base within the triplet for an aa is deleted/ inserted
What is a splice-site mutation?
A change that results in altered RNA sequence
What can mutations at splice sites cause?
Mutations at splice sites can cause exon skipping (or incorporation of intron sequence into mRNA)
What are copy number variants?
[The number of copies of a particular gene varies between individuals]
Small arrays of triplet repeats in coding sequences of genes are prone to expand in number and disrupt the function of the gene
What could be the effects of copy number variants?
- Short tandem repeats can mispair and cause pathogenic deletions and insertions which cause a frameshift
- Expansion of the number of short tandem repeats within or in the vicinity of a gene can affect gene expression
- Repeats can predispose to large deletions and duplications
What is the repeat codon present in myotonic-dystrophy?
CTG
What is the repeat codon present in Huntingtons disease?
CAG (glutamine)
How do larger deletions and insertions of copy number variants occur?
- Usually caused by unequal crossing-over between repeat sequences
- May affect a gene, several genes or section of a chromosome
- Clinical effects depend on genes involved and gene dosage
1. Misalignment of large blocks of repeats with v similar sequence
2. Unequal crossing-over between chromatids
3. Results in deletion of segment of duplication of segment
What are the different possible variations/ mutations in genes?
- Mendelian inheritance
- Single gene - Copy number variation
- Chromosomal segment (or whole chromosome) and so affect thousands of genes - Multifactorial inheritance
- Several variants of genes acting with environmental influences
