Techniques Flashcards
(130 cards)
1
Q
Define fluorescence
A
- emission of light by a substance that has absorbed light or other electromagnetic radiation
2
Q
How does the energy level of photons change in relation to wavelength of light?
A
- shorter wavelength/larger amplitude = higher energy photons
- longer wavelength/smaller amplitude = lower energy photons
3
Q
What is a fluorophore and how do they emit fluorescence?
A
- specialised molecules able to absorb specific wavelengths of light and release this energy at a different wavelength
- absorption of these photons causes fluorophore atoms to gain energy and enter higher energy state or orbit
- to return to ground state must release energy as another proton
- net result is fluorescence, where photon emitted exists at lower energy level and longer wavelength than that which excited fluorophore
4
Q
What is Stoke’s shift?
A
- loss of energy from a photon within a fluorophore
5
Q
What are dichroic mirrors?
A
- specialised mirrors able to selectively reflect/block passage of different
wavelengths of light - allow quick switching between filters when using multiple fluorophores
6
Q
What is a chemical fluorophore?
A
- fused to purified proteins, eg. Abs, FISH probes and DNA oligonucleotide primers
- eg. DAPI
7
Q
What are biological fluorophores?
A
- chromophores
- eg. GFP
- can be added to any protein in genome and visualised in live cells
8
Q
Why is signal bleed through an issue?
A
- if using multiple fluorophores essential they have distinct excitation values
- can cause false signals, making interpretation and analysis difficult
9
Q
What is photo-bleaching?
A
- reduction in fluorescence due to prolonged exposure to high intensity light, which damages their structure
10
Q
How can photo-bleaching be reduced?
A
- specialised mountants = compounds that polymerise into semi-solid matrix between sample and coverslip
- eg. Prolong Gold
11
Q
Why is it important for exposure/gain conditions to be constant between slides?
A
- to account for background fluorescence
12
Q
How is DNA purity measured w/ a Nanodrop?
A
- A260/A280 ratio (DNA/protein)
- approx 1.8 is pure
13
Q
What is copy number?
A
- no. of plasmids present w/in 1 cell
14
Q
How are diff plasmid subtypes observed on a gel?
A
- circular = larger than predicted size
- linear = predicted
- supercoiled = smaller than predicted size
15
Q
What is star activity of enzymes?
A
- restriction site indep enzymatic cleavage
16
Q
What factors can cause star activity?
A
- too much enzyme
- too little template
- non-optimal reaction buffer
- incorrect incubation time
- reaction vol too small/glycerol content too high
17
Q
Why are controls important?
A
- prove reaction components functional
- confirm reaction prepared correctly
18
Q
Which way does electrophoresis gel run?
A
- ‘run to red’ ie. +ve
19
Q
What are isoschizomers?
A
- REs which target diff restriction sites, but gen the same overhangs
20
Q
Why is phosphatase treatment of double digested vectors necessary?
A
- removes 5’ phosphate group from cleaved DNA
- prevents re-circularisation of single digested DNA (present in double digested vector)
- significantly reduces background in subsequent transformations
21
Q
What is the role of DNA ligase?
A
- catalyses formation of phosphodiester bond between 5’ PO4 and 3’OH on DNA strands
22
Q
How do you ensure the vector:insert ratio is correct for ligation?
A
- to achieve efficient ligation
- measure DNA conc of both
- calc DNA masses for appropriate ratio
- test a range of ratios
- in reality aim for highest ratio possible and have a ratio 50% smaller than this
23
Q
What is bacterial transformation?
A
- forcing competent E. coli to take plasmid into cell
24
Q
What are competent bacterial cells?
A
- chemically treated w/ Rb/Ca/Mn chloride
- facilitates attachment of DNA to cell membrane
25
What is the purpose of heat shocking cells in bacterial transformation?
- opens membrane pores to allow plasmid to enter cell
26
Why do cell need time to recover after heat shock in bacterial transformation?
- allow exp of antibiotic resistance genes successfully transformed w/ plasmids
27
How is selection performed after bacterial transformation?
- transformation mixture plated on selective agar approp to plasmid inserted
- eg Ampicillin plates select for cells that have integrated plasmid containing AmpR gene
- transformed cells = survive
- non-transformed cells = die
28
How is transformation efficiency calc?
- CFU / ug pUC19 plasmid
| - higher then efficiency, the higher the quality of competent cells originally
29
When are higher efficiency cells required?
- to transform smaller quantities of DNA
30
What factors can effect transformation efficiency?
- temp
- DNA sample
- heat shock (temp and time)
- media used for recovery step
- plasticware used
- recovery time
- user handling/error
31
What is auxotrophy?
- the inability of an organism to synthesise a particular organic compound required for growth
32
What are the general steps of yeast transformation?
- prepare competent cells
- incubate competent cells with DNA
- heat shock
- selection using appropriate agar plates
33
How should cultures be grown for metabolic processes like transformation?
- cell cultures should be harvested when in the mid-log phase of growth (S.cerevisiae OD600 0.4-0.9)
- these cells are rapidly dividing and metabolically active
- harvesting in other growth stages significantly affects experimental outcome
34
How do you refresh a culture?
- measure OD after o/n culture
- dilute to OD600 0.1
- incubate for 4 hours, check after 2
35
What is the purpose of PEG 4000 in yeast transformations?
- thought to help bring DNA close to cell membrane
- stabilises cell membrane
- acts as molecular crowder, effectively increasing the DNA conc around cells
36
What is the role of salmon sperm DNA in yeast transformations?
- acts as carrier molecule, to aid uptake of plasmid DNA
| - target for nucleases, reduces chance of plasmid degradation
37
What are some common mistakes in yeast transformations?
- incorrect/old PEG solutions
- ssDNA not prepared correctly, must be boiled and quickly chilled to maintain its single-stranded DNA
- incorrect selection plates
38
What must be done before lysis of yeast, and how?
- degrading cell wall
- enzymatic or mechanical methods req, eg. zymolyase
- then chemical lysis of lipid bilayer can be performed
39
What are the diff components of digestion solution and their function?
- SCE buffer = buffering agent
- zymolyase = degrades cell wall
- beta-mercaptoethanol = protein denaturation/RNAse inhibition
40
What are the components of lysis solution and their function?
- SDS = disrupts cell membrane/denatures protein
- TrisHCl pH9 = buffering agent
- EDTA = nuclease inhibition by ion chelation
41
What are the apps of PCR?
- amplification of DNA --> for cloning, addition of restriction sites, qPCR to quantify amount of DNA in sample, measure gene exp, RT-PCR, sequencing
- diagnostic apps --> eg. prenatal screening and genetic testing for disease causing mutations
- forensic apps --> DNA fingerprinting, paternity testing
42
What are the components of a PCR reaction mix, and what is their role?
- enzyme buffer = to maintain an optimal pH
- dNTPs (dinucleotide triphosphates) = are incorporated in newly synthesised DNA
- DNA template = contains the specific sequence to be amplified
- forward and reverse primer
- polymerase = extends the DNA primers by catalysing the polymerisation of dNTPs
- high quality dH2O
- optional: DMSO to lower denaturation temp (high GC content?
43
What happens during 3 stages of PCR?
Denaturation
- DNA incubated at high temp (96-98°)
- disrupts H bonds holding 2 DNA strands together
- prod ssDNA
- essential for primer annealing in following stage
Annealing
- temp decreased to favour reformation of H bonding
- PCR req binding of primers to template DNA
- temp here is critical to success of PCR
Extension
- temp held at 72° to allow pol to extend primers using original DNA as a template
- time allowed here dep on size of product to be amp
44
How are annealing temps for PCR decided?
- should be based on Tm value of 2 primers
- Tm = primer melting temperature, ie. temp where 50% of the primer forms a duplex with the template DNA
- can be calc manually or w/ online tool
- pol will state e.g annealing temp 3°C above the Tm of the lowest primer
- temp grad may be useful when designing new PCR strategy, as highlights more optimal conditions
45
What happens if annealing temps are too high or too low?
- too high = primers cannot anneal and failure to gen specific product
- too low = non-specific priming binding poss and failure to produce specific product
46
What cycle is specific PCR product gen?
- cycle 3
47
What PCR controls are generally used?
- compete reaction mix w/o pol --> shows amp products are a result of pols activity
- complete reaction mix w/o DNA template --> shows amp products are specific to DNA template
- control for each primer pair to confirm only specific amp and no contamination of reagents
48
What are important factors when designing PCR primers?
- primer length (18-24bp) --> longer primers more specific
- GC content (40-60%) --> higher gives a high annealing temp
- GC clamp at the 3’ end of your primer --> improves primer binding specificity
- melting temp between 50-60°C --> to limit annealing temperature
- primer pairs have Tm within 5°C of each other --> to anneal at similar temperature
- primer pairs should not have homology w/ each other --> to reduce primer dimers
49
What is a challenge in studying RNA?
- highly unstable and readily degraded by RNAse enzymes in env
50
What does the most common method of RT-PCR use?
- oligo dT primer to bind polyA tail of mature mRNAs
51
What are the first steps of RT-PCR?
- RNA extracted from cells and placed in a reaction mix with a reverse transcriptase enzyme, buffer, dNTPs and oligo (dT) primer
- heated to 37ºC, so reverse transcriptase enzyme produces cDNA based on the RNA template
52
What is the limitation of RT-PCR?
- reverse transcribes all mRNAs w/ polyA tail, resulting in heterogeneous cDNA pop containing thousands of diff DNA products originating from cellular RNA pool
53
How can cDNA yield or specificity of RT-PCR be improved?
- random priming approach uses series of random primers introduced to bind t/o RNA mol, increases cDNA yield and allows amp of mRNAs lacking polyA tail
- specific primers can increases specificity of reaction and amp only specific RNA req, but can cause poor yield and primers have to be synthesised for each specific RNA target
54
What is one-step RT-PCR?
- inc a DNA pol in same tube as RT reaction, so specific target is reverse transcribed and PCR amp w/in same reaction
- simple and rapid, and applicable when one/v few targets are to be amplified, whilst also reducing contamination risk t/ pipetting errors
55
What is two-step RT-PCR?
- amplify your cDNA products after the RT-PCR, w/ separate reaction such as PCR or qPCR
- allows greater range of targets to be amp from single RNA extract as cDNA sample produced can be divided between multiple diff secondary PCR reactions
- highly sensitive and also generates a higher cDNA yield as both random and oligo(dT) primers can be used in the initial RT-PCR, as sequence specificity not required
- but increased risk of contamination as requires extra open-tube step, more pipetting and greater
opportunity for errors
56
When and why would one and two-step PCR be used in mol cloning?
- one-step can be used to iso gene seqs from w/in cell for downstream cloning and analytical apps
- can't use normal PCR to amp complete genomic gene seq as will contain introns, and correct mRNA processing must occur to prod functional protein gene product, which is not always poss in downstream apps
- by selectively RT and PCR amp specific mRNA of gene of interest can prod dsDNA product that has already undergone intron spicing
- also can do this by two-step, offers much greater level of control to user over products formed, primers used for amp can be diff to those used in RT-PCR so can be mod to inc restriction sites etc. for downstream apps
57
What an qPCR be used for?
- copy number variation assessment of specific genomic sequences
- mutation detection
- SNP analysis
- gene expression analysis (mRNA)
- microarrays
58
How does qPCR differ from normal PCR?
- DNA product formation quantified by capturing fluorescence signal emitted from dyes or probes inc in reaction (rather than agarose gel or spec measurement)
59
What is a common fluorescent qPCR dye and how does it work?
- SYBR green
- intercalated into newly synthesised dsDNA during qPCR, causing conformational change in mol structure and 1000x increase in fluorescence
- fluorescent signal is proportional to amount of newly synthesised DNA, so quantifies relative DNA abundance
60
Benefits and limitations of using fluorescent dyes?
Benefits
- no sequence specific reagents req
- SYBR green master mix qPCR kits can be used to increase efficiency
- widely available and optimised for a range of qPCRs
- applicable for gene expression studies and DNA quantitation
- lower cost qPCR reactions
Limitations
- medium specificity and reproducibility
- higher nonspecific product and signal generation
- variable sensitivity
- only suitable for low level gene quantitation
- cannot be used for qPCR multiplexing (different colour signals in the same
reaction)
61
What are fluorescent qPCR probes, how do they work?
- eg. TaqMan probes
- designed to increase specificity
- uses probes designed to bind w/in DNA region to be amp and between forward and reverse primer binding regions
- plus quencher mols that restrict fluorescence emission
- Taq pol extends primers, when reaches probes the endogenous 5' activity cleaves probe and causes sep of fluorophore from quencher, allowing it to fluoresce
62
What are the benefits and limitations of fluorescent probes?
Benefits:
- high specificity and reproducibility
- high sensitivity and low background noise
- applicable for gene expression studies and DNA quantitation
- also for SNP genotyping, copy number variation and mutation
detection
- can be used in multiplexing qPCR
Limitations:
- specific probes must be synthesised for each qPCR reaction
- higher cost qPCR reactions
63
What pattern would be expected if track fluorescence emitted from single qPCR after every cycle?
- log pattern, unless reaction components depleted and plateau emerges
64
What is Ct or Cq value?
- critical threshold or cycle quantitation
- highlights which cycle no. begin to prod specific DNA product above background levels
- low value = higher abundance of DNA target initially
- high value = lower abundance of DNA target initially
65
What is an amplification plot in qPCR?
- when diff samples can prod plot w/ multiple trace patterns corresponding to indiv qPCR reactions
- indicates relative abundance of target between samples (more to left = higher abundance), but to fully quantify need DNA standards
66
How is amount of DNA in qPCR quanitfied?
- DNA standards = known amounts of total DNA template inc in qPCR protocol and their Ct values determined
- then used to compare experimental values w/ to more accurately quantify initial abundance of your target of interest
67
Why are reference genes needed in qPCR?
- check the amplification of specific product not a result of inconsistent template amounts in diff samples
- a set of qPCR reactions should be prepared
using the same DNA template conditions but targeting reference gene
- uses a set of primers to specifically amplify a housekeeping gene, eg. GAPDH and using abundance of reference product to normalise the values
68
What are primer melt curves, why are they needed?
- key requirement for producing reliable qPCR data is to check primers specificity and do not gen multiple random products or form primer dimers
- to do this, melt curve automatically performed at end of each qPCR run, where temp increased until dsDNA products formed in the reaction denature and ‘melt’ to ssDNA
- sudden decrease in fluorescence observed when the melting point is achieved
- plot this change against the change in temp
- 2 clear peaks present, highlighting presence of reference and specific product
- if multiple peaks observed, then need troubleshooting to optimise primer binding to the template
69
What considerations should be made when designing qPCR primers?
- amplicons should be 70-150bp (avoid fusion of primer dimers)
- 40-60% GC content
- melting temps should be 50-65°
- design primer to bind either side of exon-exon boundary (no introns to be amp), so specifically targets cDNA originated from mRNA target
- avoid seqs w/ single base repetition
- avoid seqs likely to cause 2° structures
- ensure no 3’ complementarity between forward and reverse primers (so no primer dimers)
70
Why are qPCR standard curves necessary and how to make?
- used to test primer efficiency
- shows specifically amp target, so know can trust data
- set up qPCR reactions using diff amounts of template DNA (ie. sequential dilutions)
- if primers specific should amp diff amounts of product and prod proportional dose-response curve
- so prod amp curve for each one
- plot log [DNA] against Ct and should be a line
- standard curve slope should be -3.32 for 100% efficiency (lower than this is less efficient)
- R^2 should be >0.98
71
What is the delta-delta Ct method?
- first normalise target Ct to ref Ct in each sample
- then normalise delta Ct of tumour to delta Ct of normal
- calc expression ratio
- so can compare quantitatively
72
How can qPCR results be quantified relatively?
- relative standard curve method
- compare signal to standard curve of known DNA amounts
- useful in techniques such as copy no. variant analysis
73
Eg. of qPCR use for bone marrow transplant patients?
- look for chimerism
- monitor engraftment to check if bone marrow survived and that host cells aren't reappearing
- sex-mismatched transplant can be monitored by XY-FISH
- in a sex matched transplant
- -> STR analysis used
- -> PCR based assay to look for diffs in these regions
- -> limited sensitivity (1-5%)
- -> but can be monitored by qPCR, diff SNPs identified in donor and host from panel, use ΔΔ Ct method and this has greater sensitivity
74
What is the pathway from patient sample to reporting findings to patient?
- collect approp sample
- assessment of test (clinical/diagnostic info)
- extraction of DNA/RNA/chrom preps
- direct mutation analysis (if know mutation) OR genetic mutation detection (if idea of condition so gene, but don’t know mutation) OR linkage
- results
- report
75
What is the difference between a mutation and polymorphism?
- mutation = disease causing alteration that can be inherited (if germline) or that has occurred after meiosis (somatic)
- polymorphism = alt which is w/o effect or advantageous and can be inherited
76
How can you tell whether a change is a mutation or a polymorphism?
- frequency vs incidence
- type of change, eg. if nonsense near end of ORF NMD less likely and get abnormal protein
- splicing variants --> some genes spliced diff dep on locations
- look at degree of conservation (MSA)
- structural info
- functional studies in vitro
- clinical info
77
What PCR based techs are there?
- amp of target DNA before sequencing and electrophoresis
| - but problems, need v good control, can get contam, pol can introd mutant, need to quantify amount of DNA
78
How is automated Sanger seq carried out in NHS?
- PCR w/ fluorescent chain-terminating ddNTPs, plus dNTPs
- incorp ddNTP terminates seq, get group products stopping at primer +1, 2 etc.
- ran in single capillary gel electrophoresis w/in sequencing machine, sep by size
- computer reads each band, using fluorescence to identify ddNTP, by laser exciting tag
- get trace differential (chromatogram), takes 1st trace away from 2nd, so see what diff is in peak height and area
- -> no diff = straight line, diff = bubble
- -> heterozygote has 2 peaks in same location at 2 height (as 2 seqs)
- -> sudden change after certain point likely fs (ie. all much lower and some changed)
79
How does NGS differ from Sanger seq?
- DNA seq libraries clonally amplified in vitro by PCR, rather than template prep by PCR
- DNA seq by synthesis w/ add of nts to complimentary strand, rather than ddNTPs
- products spatially segregated, rather than physically w/ gel tech
- much more data prod and much quicker
- can look at larger gene panels and whole genomes etc.
- could replace microarrays, chrom analysis, dosage, meth analysis, southern blotting
80
How does MLPA work?
- probes consist of probe, stuffer (to make all diff size), universal primer
- ligation only when 2 probes align and is quantifiable
- PCR from universal primers only when ligation completed
- can convert into bar chart to visualise
- problem if polymorphism under probe as prevents/reduces annealing, so may look like exonic del, but actually polymorphism
81
What risks may be an important consideration when deciding whether to have prenatal tests?
- posterior vs acquisory risks
| - if risk of intervention is greater than risk of having affected child then may decide against
82
Discuss PWS/AS locus
- close to centromere of c15
- related to imprinting disorders: Prader-Willi and Angelman
- in mat and pat diff patterns of methylation and repression
- in pat UBE3A meth and rep, not in mat
- can get dels (common), UPD, mutations in AS gene, imprinting defects
83
How can deletions be detected?
- chrom analysis
- FISH
- southern blotting/bisulphite mod PCR
- absence of PCR products
84
When is Sanger not useful?
- large exonic deletions or duplications
85
What can Southern blotting be used for detecting?
- UPD
| - imprinting defects
86
How does Illumina work?
- cut gDNA into 200-600bp fragments
- add adapters
- DNA fragments which bind adapters are made ss
- adapters bind oligos on flow cell surface
- unlabelled nt bases and DNA pol added to lengthen and join DNA seqs
- adapter seq at other end binds another type of oligo on surface and creates ‘bridges’ of ds DNA on flowcell surface (by seqs folding over and hybridising to oligos)
- in situ PCR = bridge amplification --> amplify original DNA to form small clusters of DNA w/ same seq
dsDNA bridges broken down to ssDNA w/ heat
- primers and fluorescently labelled bases added to flowcell
- primer binds DNA being seq and allows DNA pol to bind
- DNA pol adds bases to DNA
- lasers used to activate fluorescent label and camera detects this fluorescence
- each base gives off diff colour
87
What other Illumina advancements are there?
- flow cell clusters --> each cluster derived from single initial mol
- higher res cameras to allow clusters to be closer together
- patterned flow cells to increase cluster density --> amp in wells so cant overlap
- X ten --> targeted at seq human genomes
- 2 colour --> lowers costs as simpler optics
88
What is the main problem w/ Illumina?
- reads are too short, due to phasing
| - this means quality of reads reduces w/ length as chance of random base not being incorp and cause this to lag behind
89
How was NGS utilised pharmacogenomics?
- initial focus on cancer, can be affected by somatic and germline mutations
- can seq somatic genomes to target drive mutations, eg. BCR-ABL translocation in CML
- this allowed dev of imatinib, an inhibitor
90
How could NGS be useful for complex diseases?
- to identify disease sub-phenotypes
91
What is WGS particularly useful for and the problems w/ it?
- providing info on somatic mutations in non coding regions, esp in cancer
- need to analyse large amounts of data
92
Discuss an eg. of personalised medicine used now
- warfarin
- use dose prediction algorithms, accounting for age, weight, ethnicity, INR etc.
- further improved by considering genetic variants
93
What are some barriers to widespread use of personalised medicine?
- validity of data, esp w/ large vol prod, this is becoming a rate limiting factor, no single database of all genetic variants to help identify which are clinically relevant
- expense, consider value of info against cost of tech, if implemented large scale could provide savings by identifying non-responders
- ethical and social issues, genetic discrimination possible
- gen unwanted data, patients need to give informed consent but large amounts of data make this complex, may find info which affects relatives, who has the right to know, what should be disclosed to patients?
- impact of race, need further research to account for racial differences, as those of non-european ancestry v underrepresented and for warfarin have greater diversity in dosage reqs, certain variants may be common in these pops, studies must be on diverse pops
94
What type of FISH is used clinically?
- direct labelling of probes w/ fluorescent mols
| indirect has too high a failure rate
95
Role of DAPI?
- binds major groove of DNA so counter stain for nucleus
96
2 types of FISH?
- M-FISH = metaphase FISH
| - I-FISH = interphase FISH
97
Why is FISH useful?
- other techniques req DNA extraction and time to grow, but FISH much more rapid
- can double check abnormality found w/ other method
- M-FISH gives positional info, as can see which chromosome probe localised to
- gives copy no. info
- allows maintenance of tissue/cell morphology, good when investigating metastasis
98
Types of FISH probes?
- chromosomal enumeration probes --> for anueploidy
- locus specific probes
- whole chromosome paint
- break apart probes --> for gene fusions
- dual fusion probe
99
Why is FISH useful in cancer?
- preserve tissue architecture so can see how cancer progresses
- v rapid, good if aggressive cancer
- can confirm genetic abnormality at diagnosis and subsequently for monitoring
- eg. identify if of HER2 +ve
100
How are cells kept in metaphase for FISH?
- Colcemid inhibits spindle formation, and thus entry into anaphase
101
When is karyotyping recommended?
- recurrent miscarriages (3)
- of foetus
- if abnormality found, then parents cna also be tested
102
Why may chromosomal morphologies differ in karyotype?
- chromosome is abnormal
- level of chromosome condensation
- tissue of origin
- chromosome overlaps others
- chromosome damaged during prep
- chromosome twisted or folded
103
How would you consider what an observed karyotypic abnormality means for the patient?
- is abnormality a common marker for certain disease?
- does the abnormality affect dosage of certain genes?
- does abnormality rearrange structure of chromosome and what
affect might that have?
- how often does this abnormality occur in
the patient, what % of cells
analysed had this abnormality?
- diagnosis?
104
What is NIPD and NIPT?
- NIPT = non-invasive prenatal testing, screen for aneuploidy w/o CVS
- NIPD = non-invasive prenatal diagnosis, used to detect non-maternally inherited mutations
105
What are the main methods used in NIPT?
- massively parallel shotgun sequencing = can detect trisomy, but can be costly
- targeted multiple parallel sequencing = similar principle to MLPA in that it amps probes, to quantify chromosome specific seqs
- SNP detection = differentiates maternal and fetal cfDNA, using kits w/ around 20,000 SNPs, and comparing profiles
106
How does NIPT work?
- look at total cfDNA in sample (inc maternal and fetal)
107
How accurate is NIPT?
- high sensitivity and specificity (>99%) for trisomy 21 detection (slightly lower for other trisomies and sex chrom imbalance detection)
- should be considered as advanced screening rather than diagnostic test (and positive predictive value of test is much lower than sensitivity)
- but negative predictive value is high, so a normal test result should be reassuring
- confined placental mosaicism is an issue to be considered
108
Discuss NIPD
- for fetal sex determination: can be undertaken from 7 weeks for pregnancies at high genetic risk (eg. in women who are carries for X-linked disorders, reduces testing rate by 50% as female fetuses don’t req further testing)
- for monogenic disorders: to exclude paternal or de novo alleles, can be used for dominant or recessive conditions where parents are carriers of diff mutations (eg. CF)
- current research focussing on extending use to detect mutation in fetal DNA against background of same mutation in mother
- dep on precise measurement of relative mutation dosage (RMD)
109
Discuss SNP arrays
- used to carry out SNP genotyping of millions of SNPs at once
- can be used in GWAS studies, to find if common SNPs assoc w/ disease (not necessarily causative, may just be a marker of disease)
- each indiv SNP carries a low individual risk, but can more signif risk in combo
- SNPs closer together are more likely to be inherited together, as less likely to be recombination events between them (this is linkage disequilibrium), so can use smaller no. SNPs to tag whole genome
110
How does linkage analysis work?
- aim is to track down genomic region containing mutation and look at candidate genes in region and seq them to try and identify which one causes mutation
- SNPs can be used as markers for this, or microsatellite markers (more expensive but more info, as SNPs binary)
- must identify markers present in unaffected indivs, but not unaffected
- eg. used in Huntington's, when patient doesn't want to know whether they’re affected but doesn't want child to be affected, so see which grandparents allele they have inherited
111
Discuss array CGH?
- microarray based
- used to detect chromosome imbalances by comparing patient/control DNA and comparing differences
- useful for detecting small chromosome deletions and duplications which would not have been detected with more traditional karyotyping techniques (by looking at copy no.)
112
What is a microarray?
- chip w/ small glass plate encased in plastic
- on surface thousands of short synthetic ssDNA seqs, which add up to normal gene being investigated and variants in pop
- eg. SNP arrays, array CGH
113
What is a gene panel?
- collection of genes to be sequenced together, which are usually linked by common biological pathways, or known disease associations
114
What kinda gene panels can be carried out?
- AML
- cancer
- CF
- mtDNA mutations
- custom panels
115
Discuss Southern blotting protocol
- DNA digestion at intended RE sites
- gel electrophoresis to sep by MW and charge
- DNA transferred to +ve nylon membrane
- probe w/ homologous seq to target seq is labelled w/ fluorescent (100-500bp)dye/enz/radioactivity
- hybridisation, then washing to remove unhybridised probe
- detection
116
Discuss STR-based analysis
- comparison of microsatellites at specific DNA loci
- then determine lengths from PCR products prod
- STR expansions are causal mutations in many Mendelian diseases
- used to monitor post-transplant donor engraftment
117
What is WGS used for in the NHS?
- currently rare diseases and cancers --> where diagnosis will affect healthcare of patient or family members
- in the future, poss fetal anomalies
118
Discuss how WGS is carried out in NHS?
- diagnostic analysis focussed on gene panels
- samples from other family members may be req after results
- usually blood sample
- patient must be aware that may not be any signif findings, VUS, incidental findings, findings w/ connection to existing condition may affect future care
119
When might single gene seq be appropriate?
- CFTR gene for CF
| - BRCA1/2
120
What types of contamination are there in cell culture?
- chemical = incorrect reagent, excessive reagent conc, or accidentally introd chemicals
- human cell contam = indiv cell lines have unique characteristics, eg. specific mutations, so when culturing multiple lines can introduce one to another
- viral = need specific testing to confirm presence, often have little impact, but severe infections can change cell morphology/behaviour, when culturing human blood or tissue samples from patients risk of transmission of pathogenic viruses to user is higher, need good practise plus additional control measures, eg. PPE and specialised cell culture hoods
- bacterial = most common and easily identified as small dark cylindrical cells, causes physiological stress to cells, can inc antibiotics but not substitute for good practise
- ic bacteria, mycoplasma = no cell wall, can fuse membrane w/ host cell and compete for resources, v small so need PCR etc to detect
- fungal = yeast (small, uniform) or other fungi and moulds (large growths w/ hyphae)
121
How is aseptic technique achieved in cell culture?
- laminar flow hoods --> pull outside air down and away from work surfaces, then pass t/ HEPA filter to prevent contaminants reaching work area
- must spray and wipe everything that goes in w/ 70% ethanol (inc gloves)
122
What is req for maintaining cells in culture?
- media w/ electrolytes, AAs, vitamins, sugars and add supplements
- phenol red often in media, pH indicator to assess acidity of growing culture
- FBS, for prots, hormones, GFs to stim growth/prolif
- passage cells when become confluent, to provide excess of space and nutrients again
123
Role of trypsinisation in passaging?
- detach adherent cells from flask
124
Role of FBS in cell culture?
- stim growth and prolif
- quenches trypsin
- provide prots, hormones, GFs
125
How is cell conc and viability determined?
- haematocytometer to count cells
- conc = no. x 10^4 cells/ml
- dead cells take up Trypan Blue as membrane more permeable, so can calc viability as total live/total cells x100
126
What are primary cells? Adv and limitations?
- taken directly from an organism
- cannot divide indefinitely, ie. non-immortal
- adv = “normal”, non culture adapted, more physiologically comparable than established cell lines, easily obtained from tissue samples
- limitations = success rate of isolation is variable, variability between samples, access to samples, contamination, limited growth capacity, not suitable for LT experiments, non-immortal
127
What are immortalised cells, advs and limitations?
- derived from normal or cancer cells
- adv: divide indefinitely, allows LT/large scale experiments, diverse range available, maintains consistency between experiments
- limitations: have to immortalise, increased genomic instability, culture adaptation, often derived from tumours, not ‘normal’,
128
What is the Hayflick limit?
- no. of times a cell pop will divide before cell division stops
- 40-60 divisions
129
Steps of DNA extraction?
- lyse cells and add detergent to break down lipids
- separating DNA from proteins and other cellular debris, eg. by adding protease
- precipitating DNA w/ alcohol (eg. isopropanol)
- purification
- determine presence and quality of DNA, eg. OD, electrophoresis
130
Key NGS steps?
→ library prep, to fragment samples and mod w/ custom adapter sequence
→ capture step (optional), to pull out region of interest for sequencing
→ amp, to prod DNA clusters each originating from a single DNA fragment
→ sequencing, each seq is a read of 50-300bp
→ alignment, to ref seq
→ variant calling, identities diffs
→ variant annotations, ie. likely effect
