genetics S2 Y1 Flashcards
What is cytogenetics?
Study of chromosomes in health and disease
What is chromatin?
DNA compacted by forming complexes with histones
- What is euchromatin?
- How is it compacted?
- Loosely compacted but dynamic chromatin
- H1 proteins - can dissociate to loosen so RNA pol. can bind
- What is heterochromatin?
- What compacts it?
- Permanently tightly compacted chromatin
- Condenser proteins (recruited in cascade-like mechanism)
What separates euchromatin and heterochromatin?
Barrier proteins - stop condenser proteins compacting euchromatin
Why can barrier proteins sometimes be lost/moved?
Translocation of barrier element that codes for it can be moved
- What does structural heterochromatin contain?
- Where is it?
- Satellite DNA (repetitive DNA sequences)
- Centromeres and telomeres
What are telomeres and what are they for?
Short tandem repeats with a G-rich (overhanging) strand and a C-rich strand that protect ends of chromosomes fusing by capping the ends
- Role of G-rich strand?
- What promotes this?
- Acts as a longer G tail that loops over, displaces some the double stranded parts to create a T-loop
- Telosome-shelterin complex
- 6 steps of cytogenetics?
- Why fixed in metaphase?
- Chromosomes treated with colcemid (arrests cells in metaphase - prevents spindle formation)
- Harvested
- Hypotonic treatment (moves chromosomes to periphery of cell)
- Fixation
- Metaphase spreading
- DNA staining
- Chromosomes treated with colcemid (arrests cells in metaphase - prevents spindle formation)
- So the chromosomes are visible
Why do chromosomes in prometaphase provide more detail than those in metaphase?
Less compact
How are positions on chromosomes defined?
Coordinate system:
- Short p arm, long q arm
- Sub regions via numbers e.g. p22.1
Molecular cytogenetics:
- What is it for?
- Mechanism?
- Higher resolution analysis
- Specific, chemically-synthesised (hybridised) oligonucleotide probe for DNA sequence is labelled with a fluorophore and binds to heat treated chromosome
7 steps of fluorescence in situ hybridisation?
- Colcemid applied (chromosomes fixed in metaphase)
- Cells transferred into hypotonic solution
- Resuspended in methanol/glacial acetic acid fixative
- Cells placed on slide and fixed using formaldehyde
- Heat denatures chromosomal DNA
- Fluorescently labelled probes are hybridised to complimentary sequence
- Counterstain with DNA-binding dye (DAPI - blue)
How does chromosome painting work?
Many probes used to bind along length of chromosome
- What is spectral karotyping?
- How can it identify cancer?
- Entire set of chromosomes analysed by different coloured probes binding to different chromosomes
- Colours of cancerous chromosomes will be different
Sanger sequencing:
- How are DNA clones generated?
- What then happens to clones?
- How is the nucleotide sequence determined?
- Standard PCR reaction
- Polymerase-mediated synthesis step
- Random termination at each nucleotide using dideoxynucleotides/ddNTPs (OH on deoxyribonucleotide replaced with H) that stop any binding to create hundreds of fragments of varying sizes - pieced together to form sequence
3 limitations of Sanger sequencing?
- Necessity of having a clone of the DNA template (to create adequate levels of fluorescence for detection)
- Must have at least some sequence information beforehand
- Short sequencing read length
2 steps of Sanger sequencing?
- Constructing initial framework (contig)
- Sequencing and final assembly of genome
Step 1 of Sanger sequencing:
- What happens to chromosomal DNA?
- How are clones mapped?
- Fragmented into large fragments that are cloned into vectors called YACs
- In terms of original chromosomal location using FISH-type experiments and PCR-based screening for STS (sequence tagged sites)
Step 1 of Sanger sequencing:
- How does a FISH (fluorescence in situ hybridisation) -type experiment work?
- What are STSs and why are they useful?
- What is formed?
- DNA used as a fluorescent probe and matched to region on chromosome
- Sites that have already been sequenced previously - can have primers designed, if one clone gives +PCR result then it will be matched to chromosomal region
- A clone contig - series of overlapping clones that have been mapped for chromosomal location
Step 2 of sanger sequencing:
- Steps? (4)
- Why must the fragments be reordered?
- Random fragmentation - ligated into vectors - make clones of each fragment in bacteria - sanger sequence the clones
- Many overlapping fragments of a single clone are joined to vector molecules, these are then cloned in bacteria and sequenced, then re-constructed using homology
Whole genome shotgun sequencing:
- Who?
- Why was it better than Sanger?
- Celera genomics
- Rapidly reordered fragments (faster)
What is annotation?
Identification of genes present
- What indicated protein-coding genes initially?
- Issue with this?
- An open reading frame with no stop codon in the sequence
- Genes are made up of introns and exons –> introns have stop codons so there are only mini open reading frames
What can inter-species sequence comparison be used for?
If an ORF represents a true protein coding gene
What are mini ORFs associated with?
Evolutionary conserved regions
How are ORFs proven to be representative of protein coding genes?
Using expressed sequence tags (ESTs)
- reverse transcription allows cDNA formation for RNA –> cDNA library cloned –> put into cloning vector to form EST library
What are non-highly conserved regions?
Regions with no protein-coding unit but encode non-coding RNAs
Long non-coding RNAs:
- Main role?
- Conserved?
- Why do they have higher flexibility to sequence changes than protein coding regions?
- Regulating gene transcription by interacting with DNA
- Poorly
- Unlikely to have a negative effect on organism
MicroRNAs:
- How do they regulate translation?
- Conserved?
- Bind to 3’ UTR of mRNAs
- Highly
Transposable elements:
- What are they?
- What can they do?
- Main role?
- Highly repetitive transposon-based genes
- Change position and multiply
- Promote genome evolution by regulating genome complexity
What are DNA transposons?
Sequence is ‘cut and pasted’ from one region to another
What are retro-transposons?
RNA copy inserted (creates 2 copies in genome)
Long interspersed nuclear elements (LINEs):
- What are they?
- Why are they autonomous?
- Type of retro-transposon
- Mini-systems encode enzymes for insertion (e.g. LINE-1 has RNA copy encoding reverse transcriptase and endonuclease to aid with insertion)
How can the LINE-1 gene create a hybrid gene after insertion of the corresponding hybrid cDNA?
LINE-1 in introns has a polyA tail to signal end of transcription but if this messes up, transcription will occur up to the following polyA tail so there will be an exon fused after transcription
What are long terminal repeats (LTRs) associated with?
Endogenous retrovirus (ERV) sequences that derive from non-infectious retroviral sequences with maintained transposon activity
How do short interspersed nuclear elements (SINEs) reinsert themselves?
Hijack LINE-1 mechanisms
Mitochondrial genome:
- Shape?
-Number of genes?
- Circular
- 37
What are siRNAs for?
Regulate gene expression and have a viral defence mechanism
siRNA pathway?
Machinery generates small double-stranded RNAs from viral genomes and then uses them to destroy viral mRNAs
6 steps of detection and degradation of viral mRNA?
- Dicer enzymes produce siRNA from viral dsRNA
- siRNA taken uo by RISC protein complex
- One of the siRNA strands is degraded and the other becomes a guiding strand
- mRNA match found by base pairing
- RISC complex detects match
- RISC complex degrades viral mRNA
Why do small siRNAs need to be in a very precise molecular configuration?
RISC complex is very specific
Why is RNAase not perfectly aligned?
Cuts siRNA so there is an overhang of 2 nucleotides between the two strands - RISC complex only accepts this form of siRNA
Purpose of siRNA guiding strand?
Finds viral RNA
RISC complex:
- What is argonuate?
- Role of N-terminal?
- Role of PIWI protein?
- How is guiding strand locked into RISC complex?
- Major component of RISC complex
- Separates strands
- Breaks down viral mRNA
- 3’ bound to PA2 and 5’ bound to MID
What do microRNAs encode?
MicroRNA-precursor RNAs that are then processed to be shorter by cellular enzymes
Role of DROSHA complex?
Processes pri-miRNA to produce pre-miRNA with an overhang
What happens to pre-miRNA?
Undergoes dicer-mediated cleavage to produce mature miRNA to be recognised by RISC complex
What is the complimentary region between 3’ end of mRNA and 5’ end of miRNA called?
Seed
When is translation inhibited?
If miRNA is partially complimentary to the mRNA strand (if it is a perfect match mRNA degradation occurs)
PIWI-interacting RNAs (piRNAs):
- Role?
- What are they complimentary to?
- Control/suppress bursts of retrotransposon activity by piRNA precursor being processed to form PIWI protein that fragments LINE-1 RNA to piRNA (silencing transposon)
- LINE-1 gene for transposon
How is a gene silenced?
Recognition of LINE-1 RNA by PIWI - DNMT enzyme recruited and methylates DNA (turns off gene transcription)
What biological function can non-coding DNA sequences have?
Transcriptional regulators (promoters, enhancers, silencers, insulators)
How does transcriptional control control lineage differentiation?
Ensures the correct expression of specific genes
Why are somatic cells different to one another in different areas of the body even though they have the same genome?
Specialised by regulated expression of genes
Genes transcribed by the 3 types of RNA polymerase?
- RNA pol. I - 5.8S, 18S, 28S, rRNA genes
- RNA pol. II - all protein-coding genes, plus snoRNA genes, miRNA genes, siRNA genes, lncRNA genes, most snRNA genes
- RNA pol. III - tRNA genes, 5S RNA genes, some snRNA genes, genes for other small RNAs
3 steps of transcription using RNA pol. II?
- Initiation - recruited to target genes along with general transcription factors and regulatory proteins in a complex called RNA pol. II holoenzyme
- Elongation - RNA pol. II moves stepwise along DNA, unwinds double helix at its active site, complimentary nucleotides are added in a sequential manner using anti-sense DNA strand as a template
- Termination - RNA pol. II stops at end of gene and is released from DNA strand
Promoters:
- What are they?
- Where are they?
- What do they contain?
- DNA sequences that define position where transcription of a gene by RNA pol. II begins
- Upstream of target gene
- DNA sequence motifs bound by GTFs and RNA pol. II in stepwise manner
What do GTFs position?
RNA pol. II to initiate transcription
What are enhancers and silencers needed for?
High levels of accurate transcription and modulation of the rate of promoter transcription
Where are enhancers and silencers?
On the same genes they regulate (cis-regulatory sequences) upstream or downstream of target genes and in introns or exons
- What do transcription factor proteins do?
- What do they also contact with?
- Read sequence of cis regulatory DNA to bind to specific motifs - DNA bound TFs interact with GTFs and RNA pol. II assembled at the promoter
- Intermediary proteins called transcriptional coactivators and corepressors
Role of enhancers?
Promote transcription from the gene promoter by speeding up the rate of RNA pol. II-GTF complex assembly
Role of silencers?
Slows transcription by blocking RNA pol. II-GTF assembly
Insulators:
- Role?
- Where?
- Specific type with essential role?
- Prevent innaporpriate regulation of adjacent genes and control genes/set of genes an enhancer can regulate
- Between enhancer and promoter (blocks enhancer action)
- Act as barrier elements
What does combined activity of multiple cis-regulatory elements generate?
The correct spatial and temporal gene expression patterns
What was the ENCODE project?
Mapped regulatory elements genome-wide using many experimental techniques
Two types of non-coding RNAs that are extensively transcribed but not into proteins?
Housekeeping ncRNAs
Regulatory ncRNAs
Long non-coding RNAs:
- Length?
- 2 ends?
- Difference from mRNA?
- Where?
- > 200 nucleotides
- PolyA tail, 5’ 7-methylguanosine cap
- Fewer exons, shorter transcript length, more tissue restricted expression
- Nucleus or cytoplasm
What are genes made up of?
Interlinked protein and non-coding transcripts
What happens when lncRNAs are coexpressed with protein coding genes?
Have a local transcriptional regulation role
What do most transcriptional enhancers generate?
A lncRNA
What have a new subset of lncRNAs formed?
New class of gene expression regulators that act either directly in the nucleus to regulate transcription OR indirectly in the cytoplasm to post-transcriptionally affect gene expression
When do genetic mutations arise?
During replication or cell division
What are heritable mutations also known as?
Germ-line mutations
Where do disease causing mutations mostly occur?
Protein-coding sequences
What is polyploidy?
Extra set of chromosomes
What is aneuploidy?
Extra or missing single chromosome
What can polyploidy and aneuploidy be caused by?
Nondisjunction (both chromosomes pulled to one end)
What are splice site mutations caused by?
Reduced efficiency of spliceosome recognising the splice acceptor site due to sequence changes
- What do recessive mutations need for disease manifestations?
- Dominant?
- Both copies of a gene to be inactivated
- Only one copy inactivated
What is a dominant negative mutation?
Single mutated gene copy interferes with normal function of wild-type protein
What is mendelian (monogenic) inheritance?
Presence of mutation in a single gene is sufficient for disease manifestation
What is locus heterogenity?
Multiple genes can be mutated to cause the same disease
What is phenotypic rescue?
When the mutant phenotype is suppressed
What is incomplete penetrance?
Genetic mutations have varying degrees of penetrance - e.g. a dominant/disruptive mutation is very likely to cause disease, but environment and genetic background can influence phenotype
What is X-mosaicism?
One X chromosome inactivated in women during early embryo phase - can cause deviations from Mendelian predictions
What is a manifesting heterozygote?
If a zygote has one normal X and one mutant X, then some will be mutated and others not after inactivation - if more normal are inactivated then the zygote is a manifesting heterozygote
What is heteroplasmy and why does it complicate pattern of inheritance?
Mitochondrial inheritance is random so eggs will have varying numbers of mutant and non-mutant mitochondria
How does the number of mutant mitochondria affect disease manifestation?
Threshold will be present - worst affected areas will be where the body requires a lot of ATP such as the brain (will have lower threshold)
What was the main limitation of Mendel’s studies?
Only looked at binary traits - he believed one gene determined outcome of a trait (in reality, many will contribute)
What is the polygenic model?
Many genes contributing to end phenotypic outcome of a trait, also said that genes display ‘effect sizes’ rather than dominance and recessiveness
What is the polygenic threshold theory?
Specific number of disease risk alleles being present causes disease manifestation
What do many polygenic diseases display?
Reduced recurrence risks BUT also display a binary factor rather than continuous variables
What is the carter effect?
Some disease have sex dimorphism so disease threshold is different between sexes
- Why can monozygotic twin studies determine heritability of a disease?
- What are used as a control?
- Can determine extent disease is influenced by genetic and environmental factors
- Dizygotic twins?
Equation for phenotypic variance?
Phenotypic variance = heritable genetic variance + environmental variance + ‘personalised’ genetic variance
What is narrow sense heritability?
Genetic variants that contribute to phenotypic outcome are in a simple additive fashion
What is broad sense heritability?
Proportion of phenotypic variation due to genetic values that may include dominance and epistasis
Equation for broad sense heritability?
= 2 (CRmz - CRdz)
What may somatic mutations cause?
Non-cancerous diseases
What mutations have greatest impact?
Those that affect the neural lineage (higher up = whole nervous system affected)
Why can cancer cells evade apoptosis and replicate indefinitely?
Genetic defence mechanisms are inactivated by successive mutations
What mutations drive cancer the most?
Those that further accelerate the rate of mutation e.g. if they inactivate DNA repair pathways they increase cell division and DNA replication
Why is a stem cell mutating so dangerous?
Can become a cancer cell that can replicate indefinitely (self-renewing cancer cells)
What are targeted when degenerating a tumour?
Tumour stem cells
What are oncogenes?
Genes that drive cancerous transformation when activated (usually sporadically) - usually involves somatic ‘gain-of-function’ mutation
How is leukaemia caused?
ABL gene locked into ‘on’ position = uncontrolled haematopoietic cell division = leukaemia
How is a neuroblastoma formed?
MYC gene binds to promoter and acts as a transcription factor (interacts with another called MAX) - genes that drive proliferation of early neural cells are transcribed
Qualities of tumour suppressor genes?
Biallelic (have both copies of variant) and undergo tumorigenesis - inactivation by mutation)
What is Knudson’s two-hit hypothesis?
Tumour suppressor cases have one gene inherited and the other is somatically inactivated
2 breast cancer susceptibility genes?
BRCA1 and BRCA2
How is the bit of DNA that goes missing during a double-stranded break get fixed?
Via homologous recombination - sequence from homologous chromosome is used as a primer to form an extension and fix the missing information
Roles of BRCA1 and BRCA2 in homologous recombination?
BRCA1 senses the broken strand and recruits BRCA2 that recruits RADS1 for strand invasion and the strand is fixed
What mutation causes Fanconi’s anaemia and breast cancer?
BRCA2
What causes Von Hippel-Lindau syndrome?
One mutated copy of VHL gene = manifestation, somatic inactivation of the second VHL copy = tumours in vasculature that supply various systems and organs
3 diseases that result from failure in brain communication and what type of neurones are affected?
- Alzheimer’s - cholinergic neurones in basal forebrain
- Parkinson’s - dopaminergic neurones in substantia nigra
- Huntington’s - medium spiny GABAergic neurones in striatum
What does Alzheimer’s do?
Destroys memory, ability to learn, reason and judge (leading cause of dementia)
3 pathological characteristics of alzheimer’s?
- Plaques of amyloid beta peptide
- Neurofibrillary tangles composed of Tau
- Inflammation composed of reactive glia (microglia)
Cause of Alzheimer’s:
- Sporadic or familial?
- Major risk factors?
- What allele increases risk?
- Mostly sporadic, some is familial due to autosomal mutations in amyloid precursor protein and presenilin 1 and 2
- Age and Down’s syndrome (higher APP gene dosage)
- ApoE4
What are the risks of Alzheimer’s onset associated with the Apolipoprotein E (ApoE) polymorphic variants?
ApoE2, ApoE3 and ApoE4 exist and most people have ApoE3, but individuals with ApoE4 are at a higher risk of getting the disease (but can never get it as well)
- 2 SNPs that increase the risk of Alzheimer’s?
- 2 that decrease?
- CR1 and TREM2
- Clusterin and PICALM
4 risk loci and associated pathways of Alzheimer’s?
Immunity, lipid metabolism, Tau binding, APP metabolism
What causes familial-inherited Alzheimer’s disease (FAD)?
Autosomal dominant mutations in APP and PS1 that are highly penetrant (PS2 less so)
What is APP and how does it relate to FAD?
A type 1 membrane glycoprotein that is on the long arm chromosome 21 – 50 mutations found and it accounts for 10% of FAD
What are PS1 and PS2?
Components of gamma secretase complex on chromosome 14 which cleave and process APP – >150 mutations on PS1, 13 on PS2 = 50% of FAD
Why can the amyloid-beta that sits in APP not be expressed and how is it released?
If expressed, Alzheimer’s manifests
Released via enzymatic cleavage
2 pathways of amyloid-beta cleavage from APP?
- Alpha-secretase = cuts the protein in half
- Beta-secretase cuts end of amyloid-beta protein (caused by APP swedish mutation) and then gamma-secretase cuts the protein so it is expressed and it can be DEGRADED or cause NEUROTOXICITY
Parkinson’s:
- Main cause?
- % inherited?
- Symptoms?
- Sporadic or idiopathic
- <5%
- Neuronal loss, loss of motor functions (loss of dopamine innervation in basal ganglia)
Pathology of Parkinson’s?
Lewy body observed - aggregates of alpha-synuclein
2 ways Parkinson’s is inherited?
- SNCA = chromosome 4, 18 mutations (mostly autosomal dom.), duplicates and triplicates of alpha-synuclein
What did GWAs for Pakinson’s indicate?
41 risk loci that indicate key role for autophagy and lysosomal biology in risk
What are repeat expansions associated with?
Neurodegenerative disease and developmental disorders (repeats can be in untranslated and coding regions)
Huntington’s:
- Symptoms?
- 2 qualities?
- Chance of offspring with one parent with it manifesting HD?
- Cause?
- Disturbs personality and jerky movement
- Autosomal dominant and highly penetrant
- 50/50
- Mutant HTT gene that codes for huntingtin protein
HTT gene:
- What is particularly high (>36) in HD patients?
- What is the correlation between polyQ number and age of onset of HD?
- When does age of onset decrease?
- PolyQ (trinucleotide repeat near N terminus codes for stretch of glutamine residues)
- Inverse (more repeats = lower age)
- In successive generation (especially male germline)
Why is HTT widely expressed?
Huntingtin required for early embryonic development and may have a role in nervous system development and neurogenesis
2 qualities of HTT?
- HEAT repeats (superhelix for protein interactions)
- N-terminal polyglutamine (polyQ) repeat
How does mutant HTT interfere with transcription?
- Disrupts binding of CREB binding protein (transcriptional co-regulator) = CREB cannot allow neuronal transcription pathway to occur
- Could inhibit BDNF that is required for viability (control of transcription of neurotrophin genes)
- What is the toxic fragment hypothesis?
- 4 steps of toxic gain of function in HD?
- HTT has N-terminal cleavage sites, if cleavage occurs there is a build up of toxic cleavage fragments in the brain
- Proteosomal congestion
- Results in cytoplasmic aggregates of huntingin
- Causes mitochondrial dysfunction, nuclear shuttling and nuclear aggregates
- Leads to incorrect trafficking of BDNF
When does HD manifest?
40+ CAG repeats
Why is definitive diagnosis of HD possible?
Autosomal dominant nature
5 possible treatments of Huntington’s?
- Gene silencing
- Antisense oligonucleotides
- Viral vector delivery of RNAi-based therapies
- Tissue incorporation of grafts
- Blocking neuronal cell death/toxic gain of function
Motor neuron disease/amyotrophic lateral sclerosis (ALS):
- Cause?
- 2 symptoms?
- Familial or sporadic?
- Why does loss of motor function occur?
- What protein aggregates involved?
- Loss of motor neurones in spinal cord, brainstem and motor cortex
- Muscle atrophy and loss of motor function
- Mostly sporadic, 5-10% is familial
- Inability to innervate muscle due to loss of lower motor neurones
- Lewy body-like ubiquinated inclusion bodies in spinal motor neurones
4 autosomal mutations that are associated with ALS?
- C9ORF72 = 25-40% of FALS and associated with frontotemporal dementia
- SOD1 = 10-20% of FALS = an antioxidant enzyme
- TDP43 = RNA/DNA binding protein that is found in aggregates associated with sporadic ALS
- FUS = fused in sarcoma, RNA binding protein
What is SOD and how do mutations affect body?
Superoxide dismutase (Cu2+/Zn2+) converts superoxide to hydrogen peroxide
Mutations cause toxic gain of function
TDP43:
- Transcriptional functions?
- What does pathogenicity cause?
- What characteristic is seen in sporadic ALS?
- Repression, pre-mRNA splicing, translational regulation
- Involves perturbation of its trafficking between the nucleus and cytoplasm –> becomes sequestered as insoluble aggregates
- TDP43 ubiquitin inclusions in the brain
What are mutations in TARDBP gene associated with?
Familial forms of ALS and frontotemporal lobe degeneration
Frontotemporal dementia:
- Strongest component?
- 3 most commonly mutated genes?
- Genetics
- Microtubule-associated protein tau
- Progranulin
- Chromosome 9 open reading frame 72
4 steps of reverse transcription PCR to analyse mRNA?
- Primer anneals to mRNA poly(A) tail
- Reverse transcriptase then forms cDNA (copy of whole sequence)
- Alkali isolates cDNA
- PCR
Real-time PCR:
- What is it?
- What does it rely on?
- Semi-quantitative determination of the amount of a particular DNA/RNA species
- Specific enzymatic properties of DNA polymerase and real-time fluorescence detection
What happens if a DNA polymerase has 5’ to 3’ exonuclease activity?
Anything downstream on template is degraded
What is the probe like that is used in real-time PCR?
Sits between forward and reverse primers and has a reporter (dye) group and a quencher group (quenches fluorescence)
- Why does fluorescence occur during strand displacement?
- How can the completion of polymerisation be determined?
- Polymerase degrades probe and cleavage occurs – this means reporter is cleaved from quencher
- Fluorescence will stop increasing
What is digital PCR?
An expensive and time-consuming absolute quantification of DNA/RNA whereby the sample is partitioned into many reactions that show negative and positive PCR reactions and how much DNA is present
What is emulsion PCR (part of digital PCR)?
Microreactors (water droplets in oil) react with DNA and emit fluorescence
Why are there more microreactors than DNA molecules in digital PCR?
So there is only 1 DNA molecule per reaction
What can compartmentalised PCR be useful in?
Rare-allele detection
How does nucleic acid hybridisation work?
Synthesised oligonucleotide probes detect the presence of DNA/RNA species of interest
- Probe or DNA/RNA is fixed to a solid
support so binding/detection is visualised
spatially
- Once hybridised, they are washed and
only strongly bound probes remain
- What is in situ hybridisation?
- 4 steps of slide prep?
- Detection of presence/localisation of DNA/RNA in cells/tissues following fixation to a solid support (used fluorescently labelled probes)
- Tissue isolated –> embedded in paraffin –> thin sections cut –> mounted on glass slide
What do microarrays allow?
Transcriptome-wide analysis
4 steps of microarray?
- Oligonucleotides/DNA probes fixed at points on microarray grid
- Heterogeneous nucleic acids of test sample with fluorescent label
- Bind to oligonucleotide probes
- The more numerous the target sequence, the stronger the hybridisation signal
What did the human genome project and whole shotgun approach rely on?
Sanger sequencing
What did the first generation NGS technique remove from Sanger sequencing?
DNA size determination step (only relies on light detection)
2 key features of NGS methods?
- Clonal amplification of DNA fragments using ‘massively parallel’ PCR-based techniques
- Sequencing of all the produced DNA clones in parallel using a light detection-only method
What generates millions of clones in pyrosequencing?
‘Emulsion-PCR’ for massively parallel generation
6 steps of pyrosequencing?
- Genome fragmemented into tiny pieces
- Ligate bits of DNA (adaptors) of known sequence to the ends of each fragment
- Create DNA:waterfoil emulsion
- PCRs proceed from each DNA fragment in contained water droplet compartments
- Spread individual bead/droplet PCR reactions onto ‘picotiter plates’
- Perform pyrosequencing in wells, again using primers complementary to adaptors
How are clones sequenced in pyrosequencing?
One nucleotide at a time to work out sequence –> PPi is generated and converted to ATP by sulfurylase, then luciferase uses ATP to produce light (pyrase degrades nucleotides to stop signal confusion)
2 advantages and 2 disadvantages of pyrosequencing?
Advantage = cheaper, quicker
Disadvantage = not good for large genomes, high-error rate
What is illumina sequencing?
Localised ‘bridge-amplification’ on a glass slide to yield clonal clusters of DNA, DNA sequence of clusters is determined using reversible dye terminator
What is clonal bridge amplification?
Extension of human genome as it is fragmented, adaptors are added, bridges over, anneals to primers and then polymerase is added to extend
How is the sequence identified in illumina sequencing?
Reversible dye terminator (modified nucleotides with cleavable fluorescent tag) added and they also prevent addition of the next nucleotide
How is the sequence determined once the reversible dye terminators have been added?
Bind and terminate sequence –> fluoresce different colours
Once colour identified chemicals are added to deactivate it so next nucleotide can be added
What can newer NGS techniques now do?
Sequence DNA in real-time without PCR amplification and generate longer sequence reads
Single molecule real time sequencing:
- What is fixed to the bottom of the well?
- How is DNA polymerase exposed?
- How is sequence worked out?
- DNA polymerase
- Illumination of small part of the well
- DNA polymerase incorporates dntps into DNA whereby they are attached to phosphate group –> fluorescence emitted when excited and a flash is given off
Nanopore sequencing:
- Advantages?
- What does it rely on?
- How is sequence of DNA identified?
- High throughput and read length
- Cell membrane ion channels that are embedded in inert membranes
- Each nucleotide deflects the current flowing through the pores differently
When do polygenic diseases manifest?
When the additive factors reach the threshold
What are risk alleles?
SNPs found more frequently in disease cohort as identified by GWAs
How are GWAs performed with microarrays?
Oligonucleotide probes complimentary to all common SNP loci label the DNA fluorescently and hybridise to microarray and laser scans to find SNPs
What is missing heritability?
Genetic contribution to complex diseases that exist but aren’t identified by GWAs (could mean rare variants that are not identified are responsible) – NGS finds them
What can NGS find in relation to disease?
Find genotype-phenotype correlations in disease e.g. effects of genetic variations on drug responses and the possible progression paths of disease
Why is the whole genome of patients sequenced to find genotype-phenotype correlations?
So clinicians can use it to make informed decisions about tailored treatment
Why can nanopore sequencing be used to help treat an infection?
Identify microbial strain in a fraction of the time culturing would take
What is an advantage of nanopore sequencing?
Portable (can be used in the field) so it allows genome surveillance in acute viral outbreaks and determines evolutionary rate and confirms transmission paths
