Week 6 Flashcards
Types of variation in genome
-alterations in the sequence of bases in a specific section of DNA: single nucleotide polymorphisms (change in single base), small deletions or duplications (few bases)
-microsatellites (tandem repeats of 2-6 bp)- <100bp in total length
- minisatellites (variable number tandem repeats of 10-60bp)- can span several kb
-Larger deletions/duplications (copy number variation) of DNA segment, or segments of chromosomes
-Changes in number or structure of chromosomes
Variation in genome can lead to
Altered effects of a protein or control of genes leads to:
Normal human variation, differences in response to medication, influence the likelihood of disease, directly result in a genetic condition
How variation in genome affects health and disease depends on its type and where it is
How can a genome variant be classified
Size
Frequency -common or rare
Clinical effects- non-pathogenic (no change in phenotype) or pathogenic (disrupt gene function, clinical effect)
What is a mutation
An alteration or change in the genetic material
From exposure to mutagenic agents but more arise spontaneously through errors in DNA replication/repair
More likely to be recognised if effects are detrimental some are not recognised
DNA sequence variants
Mutation- harmful sequence variant alters gene function and phenotype
Polymorphisms- non harmful:
-sequence variant is in non functional DNA
-sequence variant is within gene but does not change amino acid
- sequence variant changes amino acid but does not alter protein function
SNP- single nucleotide polymorphism, commonest type of variant
What is a SNP
A change in a single base at a particular position
To be called an SNP, a base change has to have frequency of >1%
How can the genome be examined
Bases (small region)— sequencing or microarray analysis
Large blocks DNA— microarray analysis, fluorescence in situ hybridisation FISH
Chromosomal— light microscopy
DNA sequencing
Developed by Fred Sanger 1977
Use technology to read order of bases and compare to reference sequence
Amplify small amounts of target DNA usually by PCR
DNA is used as a template to generate a set of fragments that differ in length form each other by a single base
The fragments are then separated by size and the bases at end are identified, recreating original sequence of DNA
Why sequence DNA
Sequencing DNA determines exact position of mutation within gene
Determines the type of mutation (including single base changes)
Nucleic acid sequencing by chain termination is now automated
The use of fluorescently-labelelled ddNTP terminators has allowed automation and high-throughput sequencing
A different fluorescent labelling molecule (fluorophore) is used for each of the ddNTPs each with a different emission colour
All four sequencing reactions can be carried out simultaneously in a single tube
Next generation sequencing
Whole molecule sequencing in one reaction
Much faster and cheaper than Sanger sequencing
Patients can have genome sequenced- make predictions about health, make diagnosis, find out which medications they’re most likely to respond to
When do mutations occur
Cell division
From intrinsic and extrinsic attacks on DNA
How can errors in DNA replication and Meiotic division cause human disease
Mutation is in gametes so is passed onto offspring
How can errors in DNA replication and mitotic division cause human disease
Passed onto daughter cells
Mitosis is used for growth of embryo and for maintenance of tissues. Somatic changes
In body cells
Endogenous mechanisms causing DNA damage
Depurination: spontaneous fission between purine base and sugar, causes loss of adenine or guanine from helix-deletion of base or incorrect nucleotide in new strand)
Deamination: cytosine deaminates to uracil, causing substitution of A in new strand
Reactive oxygen: attack purine/pyrimidine rings
Methylation of cytosines: at CpG dinucleotides spontaneous deamination of 5-methyl-cytosine to thymine
A common mechanism of mutation: C to T at CpG
High frequency of C to T transitions in genome
Especially at CpG dinucleotides- can become methylated so C more likely to be spontaneously deaminated to a T
CpG to TpG mutations
Cytosines at CpG sequences are frequently methylated; 5-methyl-cytosine deaminated to thymine
Mutation rate at CpG 8.5x more likely than that of other dinucleotides
Frequent effect is production of a nonsense mutation: CGA—TGA arginine to stop codon
Extracellular agents causing DNA damage
-ultraviolet light
-environmental chemicals (interpolate into DNA or cause DNA breaks or chromosome aneuploidy)
-ionising radiation (causes breaks in DNA)
Ultraviolet light on dna damage
In presence of uv light two adjacent thymine bases covalently attach to each other forming a thymine dimer
Thymine dimers disrupt 3D structure and can stall DNA replication machinery
Bases damaged by sunlight are excised and the strand resynthesised
Where a mutation occurs has potential implications for offspring
Germline mutations- mutation in egg or sperm, meiosis, all cells affected in offspring, heritable
Somatic mutations- occur in non-germline tissues, specific tissues, mitosis, non heritable
Why are there checkpoints in cell division
Attempt to prevent mutations being passed on to daughter cells
G1- is environment favourable?
G2- is all DNA replicated?, is all DNA damage repaired?
Checkpoint in mitosis- are all chromosomes properly attached to mitotic spindle?
Correcting DNA replication errors
DNA replication machinery has proof reading
DNA polymerase adds a base, checks it, excises it if wrong, moves on
DNA mismatch repair system corrects 99% of residual errors from replication machinery
Replication copy errors leave mispaired nucleotides would cause permanent mutation when strand with error is copied in next round
Protein complex recognises DNA mismatch, excises newly synthesised mismatched strand and uses original template strand to re-synthesis new strand
Mismatch repair system mutations
As mismatch repair system is protein complex it’s coded for by genes
Mutations in mismatch repair genes themselves lead to accumulation of somatic mutations and so predispose to cancers
Damage to DNA due to ionising radiation and reactive oxygen species
Harmful, causes double stranded breaks- dont have a template strand
Repaired by either- the sequence from other homologous chromosome of pair used to synthesise missing DNA, accurate method (homologous recombination) or by end joining broken ends (Error prone), leads to deletion of nucleotides at repair site
Broken ends processed by nuclease, end joining by DNA ligation
Types of mutations affecting coding sequence
Missense
Nonsense
Frameshift
Duplication
Deletion
Insertion
Varying effects on health depending on where they occur and whether they alter the function of essential proteins
Pathological mutations associated with protein coding genes
Exons
Intragenic non-coding sequences necessary for correct gene expression
Can get variation in intronic regions but less likely to be harmful but can be pathogenic if in regulatory sequences
