Lab tech Flashcards

Question

what info is required about the gene for a uv investigation?

Answer 1

- check pop freq (beware of later onset & lower penetrance)- in silico (splice, conservation, AA substitution), - lit/database search - previously reported variants, functional data, hotspots, - segregation data (watch out for LD, phenocopies & penetrance, non-paternity)- de novo - test parents, literature- allelic data - in trans with dominant pathogenic or in cis with recessive = benign / in trans with pathogenic for recessive = pathogenic- phenotype- MDT (assess variant in context of phenotype data)/referral info is specific- ACMG/ACGS guidelines - evolving through clingen & monthly webezespathogenic = >99% disease-causinglikely path = >90%class 3 (subdivided)likely benign < 10%benign <0.1%- 4 & 5s clinically actionable

Answer 2

- looks at splicing effects by studying cDNA generated from mRNA considerations: - normal isoforms may complicate results- sample type - is the RNA expressed in the blood?- quality - RNA degrades quickly- PTC can cause NMD of mRNA so product may not be present or be very low to sequence. biallelic expression of the variant rules out NMDmini-gene assays can overcome some issues

Answer 3

normal allele lost in tumour tissue usually due to large deletion suggests hemi variant is pathogenic- if variant allele is lost this suggests it is benign- be aware of variant in cis (ie. variant studied is not the causal variant) - presence of normal tissue may skew results

Answer 4

copy number changes, for single nucleotide polymorphism (SNP) genotyping, but can also be used to study DNA methylation, alternative splicing miRNAs and protein-DNA interactionstechnique for genome-wide profiling of DNA-binding proteins, histone modifications or nucleosomes used for studying transcriptional regulation and epigenetic mechanisms. It is an in-vivo protein-DNA binding assay where antibodies are used to select specific proteins or nucleosomes which enriches for DNA fragments that bind. These DNA fragments are sequenced directly.

Answer 5

the complete set of transcripts in a cell and their quantity at a specific developmental stage or physiological condition

Answer 6

cataloguing all species of RNA transcript, including mRNAs, non-coding RNAs and small RNAs to determine the transcriptional structure of genes, start sites, 5' and 3' ends, splicing patterns and other post-transcriptional modifications and to quantify changing expression levels of each transcript during development and under different conditions. This may be done by hybridisation techniques (incubating fluorescently labelled cDNA with microarrays or sequencing cDNA libraries

Answer 7

RNA > cDNA with adaptors at one or both ends. Each molecule is sequenced and reads are aligned to reference to produce transcription map that includes transcriptional structure and/ore level of expression of each gene

Answer 8

- larger RNA molecules must be fragmented to <500bp to be compatible with deep-sequencing technologies which may introduce bias- may need strand-specific libraries which yield information about the orientation of transcripts valuable for transcriptome annotation especially for regions with overlapping transcription from opposite directions

Answer 9

restriction enzyme digest, sonication, transposases (Transposases fragment DNA by cleaving and inserting a short double-stranded oligonucleotide to the ends of the newly cleaved molecule. The inserted oligonucleotide must contain a sequence that is specific to the particular transposase being used. While this method is fast and has low input requirements, the known sequence bias associated with transposases make them incompatible with some applications. )

Answer 10

- DNA denatured and ssDNA migrates through charged capillary containing polyacrylamide gel- rate of migration is dependent on the size of the fragment and requires an internal size standard to be run for each sample- ‚Ä¢ Applications for this include MLPA, genotyping such as QF-PCR for the analysis of aneuploidies and microsatellite analysis of tumour samples (HNPCC / Lynch syndrome), and Sanger sequencing

Answer 11

- ‚Ä¢ PCR with one primer with a fluorescent tag- ‚Ä¢ Products are analysed by capillary electrophoresis‚Ä¢ able to resolve products 1 bp apart‚Ä¢ limited by size of fragment able to be amplified by PCR ~ 5kb‚Ä¢ Preferential amplification of smaller fragments means that large alleles may not be detected when present with smaller ones

Answer 12

- larger fragments >5kb- additives can be used to overcome high GC eg. DMSO which destabilises secondary structure and weakens base pairing so primers can bind- Traditionally, long range PCR has been performed using a blend of Taq DNA polymerase (for fast elongation) combined with a small amount of proofreading polymerase eg. Pfu (for accuracy). The proofreading enzyme repairs DNA mismatches incorporated at the 3‚Äô end of the growing strand, allowing Taq polymerase to continue to elongate the DNA much further than it would otherwise, resulting in longer DNA amplification.

Answer 13

-used to detect expansions that are too big to amplify with conventional pcr- three primers, one binds flanking the repeat, one binds to the repeat, and one is a reverse primer - amplifies from multiple priming sites within the repeat giving rise to mixture of products5' end of reverse primer is complementary to repeat primer- Large expansions (>100 repeats) are not accurately sized, however this method will still show if an allele is in the affected range

Answer 14

- large amounts of DNA are subjected to restriction digest to isolate framnets of interest (can be double digest with methylation-sensitive enzyme- digested DNA undergoes electrophoresis and then denatured- DNA transferred to a membrane (usually positively charged nylon) by blotting- labelled chemiluminescent probe hybridised to DNA- wash to remove unbound probe- radiolabelled blots visualised bu autoradiography

Answer 15

- DNA denatured- hybridised with probes which are ligated if adjascent. if there is a del or dup ligation doesn't happen- probes amplified and separated based on size of stuffer fragment- amount of ligated probe is proportional to copy nu,ber- compared to control probes to indicate copy number

Answer 16

- array with immobilised oligos that are allele-specific targeted to SNPs- fragmented target DNA - hybridisation signal detection system measures signal intensity of the probe following hybridisation to the target sequence- depends on amount of target (CNV) and affinity between DNA and probe (less affinity if SNPs present).‚Ä¢ The fluorescence emitted is dependent on which alleles are present in the patient at the SNP site targeted by the probe- In contrast to aCGH, in which CNV calling is done by direct comparison with a control sample, SNP arrays use in silico based methods for copy number calling. Copy number changes are calculated by comparison of signal intensity for a probe to that of a set of in silico reference samples within the analysis software

Answer 17

- B/B+A- AA homo 0/0+2 = 0- AB het 1/1+1 = 0.5- BB hom 2/2+0 = 1duplication of B = 2/2+1 = 0.66duplication of A = 1/1+2 = 0.33deletion of B = 0/0+1 = 0deletion of A = 1/1=0 = 1 mosaic cases have data values that lean more towards the middle 0.5 as there are more normal cells

Answer 18

ABB = 0.66 AAB = 0.33 AAA = 0 and BBB = 1

Answer 19

yes the logR ratios are skewed. diluting out of the AB 0.5 allele on frequency chart. As the proportion of maternal cells present increases, the greater the divergence away from the expected 1.0, 0.5 and 0.0 values on the chart will be for all chromosomes. MCC % can be calculated.

Answer 20

yes - seen as a large drop in the LogR ratio and so spots/features appear randomly between 0.0 and 1.0

Answer 21

isodisomy -both copies inherited from one parent so every SNP is homozygous. looks the same as a deletion in the B-allele frequency chart but no copy number change. heterodisomy - both homologoues from same parent. detected when you have a trio for comparison of mat and pat alleles. mixture of AA AB and BB alleles eg. dad has BB mum has AA and proband has AA but copy number is normal. also used to identify sample mix-up

Answer 22

- isodisomy UPD- consanguinity - identical by descent. some software calculates the % LOH across the genome giving an indication of familial relationships but this is usually switched off. It may be useful for malignancy arrays.

Answer 23

microarray. (aCGH and SNP) - Although the probes in these arrays are distributed across the genome, coverage is variable and highest in genes linked to developmental delay and known microdeletion/duplication regions.long stretches of homozygosity may be useful to unmask recessive disease but further testing is needed to confirm a homozygous mutation in the suspected gene.diagnostic yield is between 5-20%

Answer 24

in-house experience, minimum false positive calls, fast TAT, internal and external database access, extensive literature searching and communication with clinical geneticists. combined SNP/oligoarray platform provides extra reassurance and SNP arrays do not require sex matched controls and so the sex of the prenatal sample is not needed,

Answer 25

LOH - used to differentiate small cell lung cancer from non-small cell lung cancerCNV gain - 8q TPD52 prostate cancer overexpression androgen regulated genecombined LOH & CNV - AML 20% have UPDmethylation - restriction enzymes can be applied before hybridisation and compare DNA sequences between methylation sensitive enzyme samplesallele-specific gene expression - using cDNA instead of genomic DNA as starting material to look at allele specific gene expression which is implicated in tumourigenesis

Answer 26

- unable to detect balanced rearrangements/gene fusions/inversions- mosaicism not reliable <30% so not good for MRD detection or specific nucleotide mutations- coverage limited to where there is SNP variation

Answer 27

collection of probes attached to solid surface. each probe is a known sequence to which complementary DNA binds. patient and control DNA fluorescently labelled and compete to hybridise to array. Once bound, non-specific DNA is washed away. scanner measures fluorescence- this allows quantification of sequence within a sample. Differences in Cy3 and Cy5 fluorescence for each spot will indicate loss or gain of material in the respective chromosomal region. The log2 ratios of the test DNA (for example, Cy5) divided by the reference DNA (Cy3) are then plotted against the chromosome positioncan be used for DNA or RNA.

Answer 28

- multiple patients on one slide- ‚Ä¢ Genome-wide test - not targeted- reducing cost- high resolution (50 - 200kb),sensitivity, specificity - better reproducibility and batch-to-batch variation than BAC- Multiple consecutive probes indicating the same copy-number change are required to determine a gain or loss. This enhances the accuracy of the interpretation- Oligonucleotide based arrays include more flexibility in terms of probe selection, which facilitates higher probe density and customisation of array content- ‚Ä¢ Accuracy of copy number variant detection is higher than in Next Generation Sequencing-based assays, including Whole Genome Sequencing- can be custom designed or purchased from vendors with a pre-determined probe coverage eg. ISCA 8 x 60K array which has probes targeted in disease causing regions and the remaining probes spread across the genome to give an overall resolution of 70 kb.

Answer 29

- cannot detect balanced translocations or inversions - poor for mosaicism <30%- cannot provide structural or positional information - may need karyotype or FISH follow-up- small CNVs not detected- cannot detect sequence changes- large number of probes needed for accuracy- Poor signal to noise ratio due to small probe size, which can result in a significant number of false-positive outliers- Cannot detect UPD or LOH or triploidy- markers chromosomes may be missed depending on size, composition and array coverage for the region on the marker- variants of uncertain clinical significance difficult to interpret- ‚Ä¢ In prenatal diagnosis, more expensive and slower than QF-PCR

Answer 30

Chip - chromatin immunoprecipitation. allows researchers to examine the interactions between epigenetic regulators and DNA in their natural context. treat with formaldehyde to covalently link protein to DNA. cross-linked chromatin is isolated and fragmented and an antibody is used to precipitate the protein of interest (immunoprecipitation) with DNA. To identify attached DNA fragments, cross-links are reversed and DNA fragments are labelled fluorescently and hybridised to array. allows identification of protein binding sites that help identify functional elements in genome eg. transcription factorbisulphite modification - C> U except methylated cytosine. CpG islands in promoters often hypermethylated in cancer genomes. probes on microarray hybridise to specifically either converted or unconverted sequence to understand if promoter is hypermethylated.

Answer 31

- technique used to quantitate levels of DNA or RNA and Continually measures the amount of PCR product during a PCR by means of fluorescence (eg. CYBR green for ds-DNA or sequence-specific flourescent probe with attached quencher which is released - eg. taqman)Key point: During the exponential phase of the reaction, the amount of product is directly proportional to amount of template- The number of PCR cycles required to reach a set fluorescence threshold is proportional to the amount of starting materialCycle threshold (Ct) = this is the number of cycles required for the fluorescent signal to be detected above the background/baseline level, after which an exponential increase is seen, and quantification can occur- A lower number of cycles to reach the fluorescence threshold correlates to a higher amount of starting material- standards of known concentration are run on the plate with the unknowns in order to create a standard curve- a calibrator sample is run on each qPCR plate and expression levels are given proportional to the calibrator sample

Answer 32

Used in monitoring of minimal residual disease (MRD) with rt-cDNA, Counting bacterial, viral, or fungal loads, SNV detection with melt curve analysis and SNP genotyping with labelling two probes with different fluorophores. confirm copy number changes from microarray

Answer 33

- Probe is fluorescently labelled at 5' end and non-extendable at 3' end- Reported (5') emits wavelength absorbed by quencher 3' end- DNA polymerase extends primer moving towards the probe- probe is degraded, reported released and emits flourescence

Answer 34

the process that increases mutant allele concentration relative to wt alleles. may be for known or unknown mutants. known is easier to design for.

Answer 35

- destroying or blocking WT allele eg. restriction enzyme pcr cuts WT allele and amplified mutant allele, RFLPprimer binds to WT and introduces a RE site during PCR and so it is digested and smaller than mutant products when separated using electrophoresis- preferential amplification of KNOWN mutant allele eg. ARMS - primer has 3' end that matches mutant but not WT allele, taqman RT-PCR, probe binds specifically to mutant and quencher separated from reporter. - spacially separating variant from WT eg. digital PCR - DNA diluted into multi-well plates and flourescent pcr performed from single templates. individual wells are analysed for the presence of pcr products or mutant & WT sequences using fluorescent probes. can detect known with allele specific fluorescent probe and unknown mutations where NGS used to sequence products.

Answer 36

- cold PCR - full: induces formation of heteroduplexes after denaturation (mutant + WT bind) and by using a lower temperature, heteroduplexes denature first. amplification of homoduplexes is suppressed. fast: selectively denatures only variant sequences to be amplified, improved and complete: oligo complimentary to sense strand of WT has 3' non-extendable phosphate and so pcr of WT is inhibited and only the variant sequence is amplified- NGS but beware of preferential amplification, FFPE and sonication errors, polymerase mistakes such as FoSTeS, sequencing errors in platforms, need sufficient read depth. molecular idetifiers or barcodes can be used to trace back the strands of origin for variant detected.

Answer 37

used to enrich or purify a specific protein (or a group of proteins) from a complex sample using an antibody immobilized on a solid support (usually agarose resin beads).

Answer 38

Method for localising specific antigens (commonly proteins) in tissues based on antibody-antigen binding. This interaction is typically visualised using an antibody conjugated to an enzyme (e.g. peroxidase) that catalyses a colour-producing reaction (detectable via light microscopy), marking the sites of antibody binding. Provides information on the presence or absence and localisation of proteins and tissue structure/cellularity

Answer 39

Mass spectrometry (MS) measures the mass-to-charge ratio (m/z) of one or more molecules present in a sample (and calculates the exact molecular weight of sample components). It can be used to identify unknown compounds (via molecular weight determination), to quantify known compounds, and to determine structure and chemical properties of molecules.

Answer 40

fast and provides positional info

Answer 41

- cross-reactivity leading to false positive results- variability in fixation and processing- not quantitative- not high throughput (low level of automation possible)- need pathologist expertise- does not detect truncated or abnormal proteins with intact epitopes

Answer 42

- overexpression of HER2 protein in breast tumour predicts response to Herceptin- detection of DMD protein in dystrophinopathy- MMR protein detection in lynch syndrome and loss of MMR staining as evidence for mutation pathogenicity- EGFR detection in lung adenocarcinoma - good for low tumour cell content but false positives and negs

Answer 43

mRNA, tRNA and rRNA

Answer 44

1. isolate RNA2. gel electrophoresis 3. transfer to membrane4. detection with hybridised probe (often cDNA)

Answer 45

PROS: high throughput, low cost, reads gene expression compared to other samplesCONS: need prior knowledge of sequence, hybrid artefacts, very low or high gene expression genes are difficult to detect

Answer 46

PROS: can use mRNA or RNA (RNA best for relative quantification of targets). mRNA more sensitiveCONS: different yields for different mRNAs

Answer 47

PRO: can be performed on FFPE eg. detect ER and PR expression in breast cancer. fully automated, can view RNA expression in cells with cellular morphology and background intactCON: not quantitative

Answer 48

- uses ddNTPs which lack OH group on 3' C and so extension is inhibited. ddNTP is fluorescently labbelled and different fragments of varying length are produced. - chains are denatured and separated using electrophoresis

Answer 49

- lower sensitivity than NGS or qPCR- low level variants may be missed-

Answer 50

- confirm NGS- gap filling- founder mutations- familial mutationsPROs:- generates longer reads than NGS for repetitive regions for repeat expansions- less reliant on computational tools than NGS- easier to score indels or pseudogenes- less space to store data than NGSCONS: NAFNAP, poor sequence quality near primer binding site, NGS better at mosaicism - sanger = 15% threshold

Answer 51

- higher resolution 5mb vs <200kb- SNP arrays also detect UPD, LOH, mosaicism and parental origin- DNA from uncultured cells so processed quicker- custom arrays are targeted, limits IFs especialli in parental testing- array files are stored and can be reanalysed in future for newly identified conditions- enables identification of novel conditions- able to detect cryptic imbalances that may look balanced on karyotype

Answer 52

```Is the mutation known?Type of mutation.Tissue type testedCostHazardous materials needed?Specialist equipment needed?Turnaround time?Throughput?Polymorphic region?```

Answer 53

```Protein Truncation Test (PTT)Restriction Fragment Length Polymorphism (RFLP)SSCPCSGEdHLPCHigh Resolution Melt Curve Analysis (HRM)MALDI-TOFMutS```

Answer 54

High throughputRapidCan determine base composition of DNA

Answer 55

Expensive| Need a huge machine

Answer 56

```Cheap,Easy,High throughputCan use leftover PCR productsLow risk of contamination```

Answer 57

Doesn't detect actual mutation.Not 100% sensitiveNeed all DNA samples to be prepped in the same wayAll DNA samples must be run at the same concentrationNot suitable for highly polymorphic genesLots of optimisation needed

Answer 58

```FastCheapGood for genes with nonsense mutationsLarge coding regions covered in one fragmentCan detect mutations at 5-10%```

Answer 59

```Costly reagentsWon't detect missense variantsTime consumingRadiolabels requiredLarge deletions may be missed```

Answer 60

```ARMSAllele-specific PCROLAPyrosequencingMinisequencingRestriction Enzyme digest (needs mutation to create a new restriction site)```

Answer 61

```Long range PCRElectrophoresisSouthern blottingFluroescent PCRTP-PCRMolecular CombingNanochannel technology```

Answer 62

```CapiliaryAgarose GelPolyacrylamide gelNanowirePulse-phaseBioanalyzer```

Answer 63

Principle is the same as TP-PCR, but there is a single reverse primer. 5' binds to region outside of the repeat and the 3' binds to the repeat. 3' is more likely to bind at lower temperatures.

Answer 64

Inverse Shifting PCR (IS-PCR).This is used to detect in Haemophilia A inversion of intron 22.

Answer 65

```Restriction enzyme digest of gDNAGel electrophoresis to separateTransfer to membraneApply labelHybridiseWashDetect```

Answer 66

ms-MLPAms-PCRPyrosequencing (uses MIP primers)COBRA - introduces new restriction enzyme after bisulphite modification of the methylated DNA.ms-HRM (the conversion of C -> U in the methylated strand causes a reduction in the GC content and melting temperature of the strand).MethyLight and HeavyMethyl are both real-time quantitative techniques reliant on the binding of methylation specific taqman probes.

Answer 67

Restriction enzyme digest at unmethylated sitesSouthern blotting

Answer 68

Methylation-Indepedent PCR primer - these amplify methylated and unmethylated sequenceMethylation-Specific primers - these primers are specific to the methylated target DNA

Answer 69

5.2kb and 2.8kb.

Answer 70

Andrew: see revision notes :) 14m2.02 Methylation Detection page 5

Answer 71

164bp for the paternal allele131bp for the maternal allele.Exon 1 of SNRPN has been amplified.

Answer 72

```G-bandingaCGHFISHMLPAMAPHBAC arrayOligo arraySNP arrayNGSQF-PCRReal Time QPCR```

Answer 73

Slide labelled with allele-specific probesFragmented nucleic acid labelled with fluorescent dyeDetection system

Answer 74

The copy number of the target sequence.| The affinity between the DNA and the probe.

Answer 75

Illumina.| Affymetrix

Answer 76

Affymetrix arrays have oligos of 25bp in length; the oligos contain the SNP site. DNA with any SNP at a single position can bind that oligo. Presence of a SNP reduces the affinity of binding between the labelled DNA and the oligo so fluorescent signal is reduced.Illumina arrays have 50bp oligos; these oligos are complementary to the sequence ADJACENT to the SNP. Single base-pair extension of the next nucleotide incorporates a fluorescently-labelled A/T/G/C. Fluorescence determines next base/SNP in sequence.

Answer 77

Loss of Heterozygosity (LOH) and copy number changes.

Answer 78

LOH detection in imprinted regions (e.g. Prader Willi)DNA copy number changesMethylation analysis - DNA is fragmented using methylation sensitive or methylation specific enzymes.Gene expression analysis - use cDNA

Answer 79

A sex-matched control isn't required.

Answer 80

Can't detect balanced rearrangements.| Not highly sensitive.

Answer 81

BACOligoSNP

Answer 82

Low res, so fewer variants of uncertain significanceHigh signal to noise ratio due to long fragment lengthCheapDye-swap to check resultsFollow-up FISH is easy and FISH probes are also BACs

Answer 83

```Low res - may miss smaller abnormalitiesFiddlyExpensive as only 1 patient/slideArray design limited by BAC availabilityDetermining precise breakpoints is not possibleLots of batch-batch variation.```

Answer 84

Higher res than BAC arraysCheaper to run - multiple samples per slideCalls are dependent on multiple consecutive probes - more acurate

Answer 85

No UPD/LOHNeed sex-matched controlincreased number of variants of uncertain significancePoor signal to noise ratio.Some abnormalities are too small to be verified by FISH.

Answer 86

CNV detectionLOH detectionGene expression - compare tissues or tumour/normalEpigenetic expression

Answer 87

Mammaprint, breast tumours.

Answer 88

CHiP-chip (any proteins currently interacting with the DNA are covalently bound to it, these fragments are selected by immunoprecipitation. Unbound DNA then put on array.DamIDBisulphite modification and appropriate oligos on the array

Answer 89

```Gene contentAny known microdeletions/duplications in the regionSizePresence in a control populationInheritancePresence in a disease populationZygosityCNV location - imprinting regions etc.```

Answer 90

Only report de novo, fully penetrant CNVs, or those which correspond to a significant imbalance.Avoid reporting VUSs.

Answer 91

Prenatal phenotypes of disease may be different or poorly characterised compared with postnatal phenotypesMosaicism could be maternal cell contamination or confined placental mocaisism.

Answer 92

Non-specific fluorescent dyes (SYBR green)| Specific fluorescent dyes (TaqMan, FRET, Hairpin, Scorpion).

Answer 93

Small dye that binds to the dsDNA groove. Fluorescence emitted when bound to dsDNA.

Answer 94

Accumulation of dsDNA during PCR cycles.

Answer 95

Specific probes labelled with a fluorophore and quencher bind target DNA. Once the polymerase extends the sequence to the probe the fluorophore is removed from the quencher dye and light is emitted.

Answer 96

Two probes are needed.They bind adjacent to each other on the target sequence.In close proximity resonance is transferred from the donor probe to the acceptor probe and light is emitted.Probes are removed once the DNA sequence extends up to them.

Answer 97

As signal reaches threshold (Ct) the signal is above the baseline measurement and can be quantified.Product in measured during the exponential phase.

Answer 98

Absolute - test sample measured against set standards.| Relative - test sample measured against internal control.

Answer 99

```Determining Viral/Bacterial/Fungal loadIdentifying and quantifying fusion genes in cancer, eg - CML (BCR-ABL1 transcript), AML (PML-RARA), ALL (ETV6-RUNX1)Single base mutation detectionSNP genotypingCopy number detectionNIPD```

Answer 100

NIPDDetection of ctDNAMRD monitoringheteroplasmy in mitochondrial disease.

Answer 101

Get rid of WT allele:AIRE-RFLP - artificially introduce a new restriction site into WT sequence and digest WT.REMS-PCR - digest WT sequence using naturally occurring restriction site.```Amplify mutant allele:Allele-specific PCRARMS PCRTaqman RQ-PCRMAMA```Spatially separate WT and mutation allele:ddPCR - very sensitive. Can be used to detect LDH1 mRNA in glioma patients' cerebrospinal fluid.

Answer 102

COLD-PCR - change the denaturing temperature to preferentially allow annealing of the primer to the mutant sequence (at lower temperatures the WT sequence will not denature so the primer cannot bind)NGS - need very deep sequencing to detect low level mutations - can be prohibitively expensive

Answer 103

Full - enriches all possible mutationsFast - enrichment of known mutationsICE

Answer 104

```Western blottingMALDI-TOFMass spectrometryImmunoprecipitation assaysIHC```

Answer 105

Lyse cellsRun components on a polyacrylamide gelStain protein to check it's workedTransfer to membraneBlock background using BSAHybridise antibodies specific to target antigen (direct or indirect)Induce reaction allowing detection - chemiluminescense, radiolabelling, peroxidase reaction.

Answer 106

By molecular weight

Answer 107

Run a 2D blotstep 1. run gel in a pH gradient to separate by charge/sizestep 2, rotate 90degrees in acidic buffer and separate by weight

Answer 108

run buffer type - some can change the protein structure and prevent antibodies binding their epitopes.

Answer 109

Use antibodies bound to beads to 'capture' proteins of interest.Beads are magnetic so can be pulled out of solution for analysisProtein eluted from antibodyReady for Western blot. ELISA etc.Uses: investigate protein half life in BRCA1/2 to determine affect of missense mutations

Answer 110

Antibodies to bind chromatin associated proteins, selecting them for analysis, then can study DNA to which they were bound.

Answer 111

No sequence-level informationNeed biopsy/sample on which to perform testNeed pathologist to prepare and interpret resultCross reactivity of antibodiesNot quantitativeLow throughputTruncated proteins or abnormal proteins with intact epitopes are not detected.

Answer 112

Tumour diagnosis and classification - expression of HER2 in breast tumoursDetection/absence of proteins in biopsies, e.g. dystrophinopathiesIdentify loss of MMR protein in Lynch syndrome - can direct gene sequencing.

Answer 113

Two MS machines, in tandem - only selected target material passes to second. It can be used to screen for metabolite markers in metabolic disease, e.g. MCAD, PKU.

Answer 114

Those that use protein capture agents, e.g. antibodies| Those that use protein-protein interactions to capture target sequence.

Answer 115

Provides comprehensive picture of the localisation, quantity, expression, modification of proteins in the patient.

Answer 116

Identification of new genes, biomarkers, signalling pathways, drug targets.

Answer 117

RT-PCR - fusion gene detection, monitoring MRDRNA-Seq - can detect novel transcripts, doesn't need known targets or specific probes. New transcript discovery.RNA expression arrays - tissue profiling. Need to know target sequences.Minigene assays (insert exon into a vector and culture in HEK293T cells) - e.g. insert an exon of PMP33 containing a missense variant into the vector, grow, harvest and purify DNA, run on GEL and see if there's a difference in size between WT and mutant.Northern blotting - gene expression in a small number of genes.

Answer 118

Allele specific PCR e.g. ARMs PCR for CFrestriction enzyme digest- when a mutation created or removes a restriction site e.g. F8 intron 22 inversion in maemophilia AFRET hybridisation- e.g. for JAK2 V617F mutation detection in myeloid diseaseDroplet PCR e.g. for BRAF V600E and EGFFR mutations testing in cancer (high sensitivity)

Answer 119

OLA PCRMini sequencingPyrosequencing

Answer 120

Benefits:- quick, sensitive, simple. - can be highly selective to targetLimitations:-cannot detect novel mutations

Answer 121

Allele specific PCR is a method for efficiently identifying specific SNvsAn allele specific forward primer (with different lenght stuffer sequences) and a common reverse primer amplify allele specific products which can be resolved by electrophoresis (usually capilliary). Based on the observation that under suitable conditions a primer cannot extend with a 3' mismatch. therefore only the primer that matches the allele(s) present will be amplified.

Answer 122

the CFeu2 kit is based on ARMs PCR it is multiplexed to be able to test for the 50 most common mutations in the EU Caucasian population. Mutation differs for other populations and this should be incorporated in risk calculations. - highly multiplexed assay. Uses A (mutant primers) and B (WT primers) tubes and also has primers to detect the polyT and TG tracts. - as well as the 50 targeted mutations, indels can cause shifts in the B tube peaks. If detected this can indicate which exons should be sequenced to ID the causative variant- products are distinguished by different fluorescent dyes and product lengths. - products are separated by capillary electrophoresis and viewed using genotyping software e.g. genemarkerif a 3' mismatch is werak a secondary mismatch can also be included to prevent non-specific amplification.

Answer 123

Benefits- quick, easy, simple- does not require specialist equipment- can be highthoughputLimitations- sensitivity of mutation detection is dependent on the population tested- PSPs can result in false results- may result in a failure to amplify the mutant allele- multiplexing can be difficult so commercial kits are often used- sensitive ti MCC compared to methods of MCC detection (QF-PCR can exclude significant MCC to ~10%)- need to confirm apparently homozygous results as it may in fact be due to a PSP or deletion. - need to confirm compound het muts are in trans for CF- if inheritance of a homozygous result can;t be confirmed consider: non-pat, deletion on other allele, PSP, UPD7, sample mix-up

Answer 124

Restriction enzymes digest the DNA forming DSBs at specific sites known as recognition sites- can be used when a mutation created or abolishes a specific restriction site.- for diagnostic testing the mutation should create a restrictions site 1) sequence containing potential restriction site amplified by PCR2) digested by restriction enzymes3) products resolved by electrophoresis (capillary electrophoresis used most commonly in diagnostic labs)it is good practice to include a second 'control' restriction site that will be digested in mutant and WT alleles to act as a digestion control. can also be used for the detection of changes in methylation using enzymes that will only cut methylated DNA

Answer 125

- genotyping for the hemachromatosis C282Y variantby RSa1| - F8 intron 22 inversion in hemophilia A by bcII

Answer 126

- cheap, simple, quick, no specialist equipment required- can only be used diagnostically if the sequence change created a restriction site. If the change abolishes a restriction site the exact sequence can't be determined as it can be any base but WT- polymorphisms can alter the restriction enzyme target sequence- only suitable if a restriction enzyme is available for the target sequence of interest- partial or overdigestion can affect interpretation- not quantitative as hetero-duplexes formed in a heterozygous sample will not cut

Answer 127

Flourescence resonance energy transfer hybridisationUses flourogenic minor groove binders (MGB) whihc bind stably to ss DNA. These are bound to the 3' end of a DNA probe and a flourescent quencher is bound to the other end.MGB probes are designed to be specific to be specific for a single base, hence are susefl for allele discrimination assays. Used for realt time PCR. 1) unbound probe free in solution, quenched by attached quencher2) probe and primer binds to target DNA. probe remain3 quenched3) DNA pol extends and from the primer. When it reaches the probe the 5'-3' exonuclease activity degrades the probe, releasing the the flourescent reported from the quencher4) the florescence emitted in measured in real-time e.g. Roche light cycler

Answer 128

Used for JAK2 V617F mutation testing in Myeloid disease (MF, PMF, ET)

Answer 129

methods fractionates sample into 1000;s of partitions (e.g. oil droplets in water-emulsion) so that each contains a single copy of the template DNAPCR is run in 1000's of parallel reactions using WT and mutant taqman probes (flourescent probe and quencher) and standard primers for region of interest. software reads +ve and negative reactions and can distinguish Wt from mutant

Answer 130

BRAF V600E and EGFR mutation testing in cancers

Answer 131

Benefits: - provides absolute quantification (assuming molecula distribution follows poisson distribution)- can detect low levels of mutant against a hgh level of background so ideal for detecting mosaicism of mutants in cancer samples. - in RT-PCR amount of target molecular give the threshold per cycle (Ct) (number of cycles before the flourescen can be detected) and the difference in Ct it used to calculate the amount of the target. this can be plotted onto a standard curve and compared to known standard to estimate the amount of target presentDisadvantages:- DNA qualtiy obtained from FFPE tumour block can often be poor resulting in poor amplification - fixation of tissues resulting in cross-linking of DNA and proteins, this damages DNA and can result in artefacts- accuracy of result depends on the quality of the DNA

Answer 132

single base extension of a single florescently labelled ddNTP at 3' end of a special oligonucleotide complimentary to a sequence 1 base upstream of the examined polymorphic site- ddNTP are flourescently to enable ID of the incorporated base and hence the complimentary dNTP in the target sequence

Answer 133

OligoNT ligation assay1) PCR primer hybridised to target (designed with WT or mutant 3' end and a different length stuffer seq for size fractionation)2) PCR performed3) ligation reaction- common primer with fluorescent FAM at 3' end meets the 1st primer over the mutation position in the altered mutant allele. If the 3' end of the primer matches perfectly with the target, both primers will be ligated. If there is a mismatch ligation wont occur

Answer 134

sequencing by synthesisfor sequencing a sinlge strand of DNA by synthesising the complimentary strand.- one base is added at a time. If incorporated the formation of the phosphodiester bond results inthe reslease of pyrophosphate. - this pyrophosphate is used by sulfarase to produce ATP which results in light being emitted by a luciferase catalysed reaction- light is detected by a camera and the amount is proportional to the number of bases added- best for short reads-unreliable for long mononucleotide repeats

Answer 135

Native DNA needs to be fragmented (restriction enzyme digest, sonication etc.) otherwise the DNA will be too large to pass through the gel or super-coiled and will pass through the gel at a rate disproportionate to its size. PCR products do not need to be fragmented but alleles dropout (due to PSPs or secondary structure), preferential amplification of the smaller allele, or lack of allele heterozygosity may hinder estimation of allele sizes

Answer 136

Agarose gele electrophoresisPAGE (polyacrylamide gel electrophoresis)Pulse field electrophoresiscapillary electrophoresis

Answer 137

- separates fragments of 500bp to 25kb- during gelation agarose polymers associate and form a network of bundles whose pore size determines a gels molecular sizing properties1) DNA is applied to Gel2) electric field is applied and negatively charged DNA migrates towards the positively charged anode3) speed of migration is inversely proportional to the log of the molecular weight (DNA has a constant mass/charge ratio)4) DNA can be visualized on the gel using an appropriate dye and size determined by comparison to a size ladder athe rate of the DNA migration is dependent on the size of the DNA fragment, secondary structure (needs to be denatured) charge applied and the size of the pores in the gel.

Answer 138

Most commonly used method in diagnostic labsUsed in combination with florescent labelled DNA can allow fragments to be sized to within 1bp of each other1) DNA denatured (heat and DMSO) and ssDNA is passed through a capillary containing polyacrylamide gel2) rate of migration depends on the size of the fragment and requires an internal size standard to be included with each sample- amplified fragments can be mixed providing they are of different sizes or have different fluorescent dyes]Used for MLPA, PCR genotyping, sanger seq etc

Answer 139

e. g. for FRAX, DM1, HD- can detect alleles up to ~5kb depending on template and PCR conditions. it is not suitable to large expansion alleles- there is preferential amplification of the smaller allele which may mask the presence of a second large expansion allele. - uses fluorescent PCR primers designed to flank the region of interest (1 of which is flourescently labelled)products are separated by gel electrophoresis

Answer 140

can be used to amplify fragments over 5kb by the use of PCR additives- betaine equalises AT and GC contributions to strand pairing- DSMSO weakens base pariing and destasbilises secondary structure (used for triplet repeats)use a mix of polymerases- taq pol is high processivity by low fidelity asd it lacks 3'-5' exonuclease activity so it can introduce errors- pfu is a proofreading enzyme that can correct taq errorsUses:provide a template for NGSnested PCR to overcome pseudogene interference e.g. PMS2-test for large common IKBkG deletion in incontentia pigmentia

Answer 141

Specialised type of PCR to enable to amplification of large triplet repeat expansion alleles- 3 primersA repeat primed primer primes from all the repeats to create products representing the full length of the repeat. It works with the forward primer to do this and is present in limiting amounts so that it is used up quickly, this prevent gradual shortening of the pool of PCR products. the repeat primer contains a unique sequence not present in the genome. a 3rd primer is complimentary to this and it works with the forward primer to amplify all the repeat primed productsAdvantages: periodicity of the peaks will indicate the number of repeats present-can identify AGG interruptions in fragile X, the presence on interruptions is associated with the risk of expansion on transmission. although this information is not currently reported on UK Fragile X reports. disadvantages: very large expansions will not be accurately sized but the presence of a peak for the full length expansion will identify samples in the pathogenic range. - PSPs can result in false results- interruptions can result in false -ves in DM1 so the reaction is run in both directions to overcome this

Answer 142

A type pf TP-PCR that has been used for HD but only uses 2 primers- forward primer- reverse primer is chimeric with 2 annealing sites. The 5' end is specific to the region of the HTT flanking the CAG repeat and the 3' end is complimentary to the CAG repeatthe 5' end will anneal at high temp- provind a full length product peakthe 3' end will anneal at low temperatures to generate the CAG stuttering pattern for allele sizing. this can detect all CAG alleles and expansions up to 40 repeatsPrimers avoid all SNPs and the assay can be used as a standalone test for HD. it overcomes the need to co-amplify both alleles using alternative primers in patients apparently homozygous for an allele in the normal size range (and exclude the risk of an undetected expansion allele)

Answer 143

Inverse PCR is used to detect distinct genomic rearrangements e.g. Hemophilia A intron 22 inversion:1) BcII digestion of DNA2) DNA ligated into circularized DNA3) primer added for PCR (primers would normally point away from each other in genomic DNA but will form a PCR product when the inversion is present)4) primers can be multiplexed so both fragments can be amplified in a single reaction

Answer 144

Can be used to resolve large fragments to large to be amplified by standard PCR- currently used for fragile X referrals where the methylation status is also required e.g. prenatals - new Asuragen TP-PCR with methylation status included. - testing methylation status requires a methylation specific restriction enzyme- also used for FSHD testing and c9orf72- labour intensive, not optimised for high throughput testing and requires a large amount of good quality DNA

Answer 145

1) genomic DNA digested by restriction enzyme (no need for PCR) to isolate region of interest. a double digest can be used to also test for methylation status2) DNA + controls and size ladder are applied to gel3) separate fragments using gel electrophoresis4) DNA is transferred to a negatively charge membrane by capilliary action and fix to the membrane5) a labeling probe is applied to label to label the DNA fragment. unused binding sites are blocked by BSA6) washed to remove unbound probe7) detect probe and visualize bands e.g. autoradiograph for radiolabeled blots

Answer 146

Most techniques require PCR amplification but methylation is lost during PCR so DNA need to be modified first so that methylated and unmethylated DNA can still be distinguished

Answer 147

bisulphite modification results in the conversion of unmethylated cytosine to uracil which is replicated as a T. methylated cytosines remain unchangedcan then either use PCR primers that are methylation independent for proportional amplification of methylated and unmtheylated DNA or methylation specific primers which are designed to amplify the methylated target only

Answer 148

1) bisulphite conversion2) methylation insensitive amplification if region of interest3) sequencing to report presence of T (unmethylated) or C (methylated) at each restriction sitesensitive to ~10% methyaltion but high cycle number required means it is prone to contamination

Answer 149

The higher GC content of uncoverted (methylated) DNA makes it more resistent to melting. - methylation independent PCR performed in the presence of a dsDNA intercalating dye- results in peaks indicating melting temp. For a mix of methylated and unmethylated DNA 2 digest peaks are formed. results of a heterogenous mix can be complex and hard to interpret

Answer 150

methylight- methylation independent PCR and differentiates between methylated and unmethylated DNA using specific taqman probes (reported and quencher) with different flourophores

Answer 151

highly sensitive but they have a high false positive rate due to false priming events (mismatches between primer and template) and incomplete bisulphite conversion

Answer 152

NGS- of bisulphite converted DNA is possible for targeted regions or for the whole genome but is largely used int he research settingarray based methylation analysis- Human methylation 27 (illumina) offers coverage of over 450,000 methylation sites for GWAS studies

Answer 153

methylation sensitiev restriction digest and assay of the digestion1) southern blotting2) FMR1 mPCR3) MS-MLPA

Answer 154

e.g. used for fragiel X and can detect methylation status as well as the size of the expansion double digest using methylation sensitive restriction enzyme e.g. Eag1 to digest a larger fragment containing the expansion produced by a methylation insensitive EcR1 digest- normal male alleles will have 1 band from the Ecor1 digest- male expansion alleles will have a differnt sized band due to the methylation of the FM- may appear as a smear on the gel - all females will have tow alleles due the methylation of the inactve X- mosaicism is difficult to assess and incomplete digestion can be an issue - time consuming and labour intensive compared to PCR- BPG - do not use DNA from lith hep tubes as this can migrate anonymously in the gel- methylation pattern may not be reliably set in CVS samplers so AF are preferred for prenatals if southern blot is being performed. Usually only to confirm that a full expansion mutation is methylated following detection of an FM or large PM by TP-PCR in case of methylation mosaicism

Answer 155

amplidex TP-PCR contains a long range PCR for allele sizing and TP-PCR. In mPCR a methylation sensitive restriction enzyme is also used. Purified genomic DNAmixed with control DNA and reaction split into 2 tubes. - 1 tube has a control digestion that it not methylation specific (FAM labelled to detect repeat size)- 2nd tube has a methylation specific digestion (HEX labelled to detect methylation status)PCR is performed for both tubes using different coloured flourophoresamplicons are then pooled and seperated by Gel electrophoresis for data analysis- can determine the extent of methylation of each allele in male and female alleles eliminating the need for S. blot- methylation data is expressed as a percentage of the undigested DNA- faster and more accurate than S blot especially for low level methylation mosaics- if used with the asuragen FMR1 CCG kit can also assay the AGG interruption repeats to give full ascertainment of anindividuals FMR1 alleles.

Answer 156

BWS/SRS, PWS/AS and UPD 6, 7 and 14Same as MLPA except the hybridisationof MLPA probes reaction si split into two tubes. - methylation sensitive probes contain a HhaI site. - 1 tube is treated as standard MLPA for copy number determination- 2nd tube is incubated with a HhaI whilst probes are ligated- unmethylated probes and sample are digested and can't be amplified in subsequent PCR- digestion control is included to prevent false negative results

Answer 157

used to examine transcriptional inactivation if a TSG| e.g. MLH1 promoter methylation associated with sporadic CRC

Answer 158

- quantify gene expression (mRNA)- minimal residual disease monitoring as has high sensitivity (e.g. BCR-ABL)- detection of mutations against high background of WT - cancer - JAK2, V617F, NPM1, FLT3-ITD

Answer 159

- no positional information- targeted (not whole genome)- requires specialist equipment- unlikely to detect low level mosaicism- multiple reactions required to examine multiple loci (Not multiplexed)- non specific SYBR green intercalates with any ds DNA resulting in false results

Answer 160

- targeted- more labour intensive than PCR- cannot detect low level mosaicism- can be used for single cell screening e.g.PGD- no positional information- cannot detect balanced rearrangements- may not get the full breakpoints of a del or dup if it extends beyond the genomic region covered by the kit.- sensitive to contaminants- PSP can result in a false result - single exon deletions should be confirmed by another method

Answer 161

1. denature DNA2. hybridise probes3. ligate probes4. PCR amp pf probes (only probes that have the target region present will be amplified)5. separate probes by capillary electrophoresis6. analysiseach reaction needs a control to normalise against. Also need positive and negative controls to check for contamination and ensure that the reaction has worked. the controls also aid in interpretation, troubleshooting```expected ratio:1 - normal0 - hom del0.5 (0.3-0.7) - het del1.5 (1.3-1.7) - het dup2 - hom dup```

Answer 162

There are methylation probes included in the mix which contain a HhaI restriction site.- following probe hybridisation the reaction mix is split in 2- 1 tube has a standard MLPA reaction and is used to determine copy number- the second tube is treated with HhaI which will digest unmethylated. Methylation status is detemined semi-quantitatively by comparing the undigested tueb with the digested tube. a digestion control is required to confirm that full digestion has taken place

Answer 163

RNA is reversed transcribed to cDNA to allow RNA expression profiling. It is more efficient than northern blotting.

Answer 164

very high resolution- single base change and break points- can be genome wide or targeted- provides positional information-detects UPD and LOH balanced and unbalanced rearrangements can potentially be detected (pair-end and read-mate sequencing)- SNVs and CNVs can be analysed in a single assay- WES has lower const and higher coverage but less able to detect structural chages

Answer 165

1. read depth- duplication results in increased read depth and deletion results in a decrease. This technique requires high coverage to detect imbalances and to distinguish artefacts from real calls- can detect dels and dups but breakpints are unprecise and cant detect SVs2. read pairuses reads from paired end sequencing with an known distance between the two reads. If there is an increase in the distance between the reads this indicates and insertion and a decrease in the distance indicates a deletion- can detect most SVs but unable to detect the exact breakpoints3. split readuses reads from pair end sequencing where one of the pair perfectly matches and the other completely or partially fails to map to the genome. The unmapped reads are a potential breakpoint-can detect exact breakpints but is limited in the length of reads and NGS <1kb affects the accuracy and and precision4. de novo assembly and comparison to reference genome. less common as there is a huge bioinformatic demand. Performs badly on repetitive or complex regionsRP and SR are both poor in repetitive regions or regions with segmental dups as it is hard to accurately map reads to the reference

Answer 166

There are 3 mandatory components to an array:1) an array containing immobilized allele specific oligonucleotide probes 2) fragmented nucleic acid seq that has been flourescently labelled3) detection system to record and interpret hybridisation signalthe signal intensity depends on the amount of target DNA sequence is present in the sample and the affinity between the probe and the target

Answer 167

* CGH involves running the patient DNA against a sex-matched control * SNP: - a control DNA is not required as the data is normalised against a virtual cluster file which is composed of 100s of ‚Äúnormal‚Äù samples. * Knowing the sex before had is therefore not necessary for SNP array as it can be predicted using the SNP data. * For arrayCGH, the sex must be known beforehand to ensure it is run against the correct sex.* Both are limited in detecting mosaicism but SNP array is more sensitive due to additional data from B allele frequencies.* SNP array, but not aCGH, can detect copy neutral absence of heterozygosity (AOH). This includes uniparental isodisomy, where both copies of the region are inherited from one parental chromosome - uniparental isodisomy of imprinted regions is associated with imprinting syndromes. AOH, particularly mosaic AOH, may reflect acquired isodisomy, referred to as loss of heterozygosity in the context of neoplasia. SNP arrays therefore more useful in analysis of cancer samples.* SNP array requires lower input DNA* For prenatal samples, SNP array can reveal maternal cell contamination through the presence of additional genotypes in the B allele frequency plot. Maternal cell contamination is not detectable by aCGH.* SNP can detect triploidy (arrayCGH would appear normal as there would be an additional copy of every chromosome and when normalised against each other the log ratio would remain at 0)* SNP can predict origin of aneuploidies

Answer 168

* SNPs are not evenly distributed in the genome, being more common in non-coding regions, which are typically under less evolutionary constraint. Limited number of SNPs in the genome, which limits array design. Combined SNP/oligo arrays can improve coverage.* Areas of segmental duplication don‚Äôt contain SNPs so less accurate detection of CNVs* SNP density can be predicted by the presence of microsatellites: AT microsatellites are potent predictors of SNP density, with long (AT)(n) repeat tracts tending to be found in regions of significantly reduced SNP density and low GC content* When MCC detected cannot tell if the majority profile is fetal or maternal (unless abnormality present) i.e if 10% contamination detected cannot tell if it‚Äôs 10% fetus or 10% maternal* Artefacts due to misbehaving SNPs* Data is more complex to interpret due to additional data from B allele frequencies. Complex patterns observed when mosaicism is present. Therefore the training and interpretation requirements are more complex

Answer 169

‚Ä¢ Ethical issueso AOH ‚Äì AOH is relatively common throughout genome, when/what/how to report? Clinical significance of UPD for most genomic regions is uncertain, may choose not to investigate regions of AOH which do not involve well-establish imprinting syndrome regionso Safe guarding issues relating to high levels of AOHo Consanguinity?o Non paternity‚Ä¢ Difficulties with new tech (regulation, BPG, incomplete reference data for analysis, losing applicability of your local database of CNV)SNP array may detect CNVs in cancer-susceptibility genes that are not detectable using clinical aCGH platforms which have minimal coverage of these genes. The laboratory needs a policy for validation and reporting of such CNVs, including liaison with Clinical Genetics. Referring clinicians may not be aware of the risk of incidental findings and may not be counselling families appropriately.

Answer 170

method to separate proteins and can be used to determine the presence or absence of proteins, compare protein levels, assess purity of relative molecular

Answer 171

1. extract protein- can be from whole tissue or tissue cultures. cells are lysed to release the proteins of interest and protease inhibitors may be included2. separate proteins (SDS PAGE)3. blotting- protein are driven from the gel to a stable support membrane by exposure to an electrical field or a vacuum. - generally nitrocellulose4. block non specific ab binding with BSA5. labeling6. visualization and analysis

Answer 172

SDS PAGE is a mtheod to spearate proteins based on their moecular weight. Under denaturing conditions the -ve charged detergent SDS binds to hydrophobic regions of proteins causing them to unfold and dissociate from other proteins. SDS also confers a negative charge to the protein relative to its length, so when a charge is applied to the proteins in a gel they will migrate to the anode with the rate based on their molecular weight. - non denaturing conditions if protein-protein interactions or folding are being investigated, IN this proteins are separated based on net charge, size, shape as protein carry a net -ve charge in alkaline buffers

Answer 173

1- proteins are seperated by isolectric focusing and are run on a gel until they reach their isolecetric point. (pH at which the net charge on the protein in zero)2- proteins are separated by their molecular wieght.this allows separation of many more proteins (2000) that standard SDS PAGE (~50)

Answer 174

membrane is blocked to prevent non-specific ab binding using non-fat milk of BSA1. direct detection uses a labeled Ab targetd to the protein of interest2. indirect uses one Ab to bind to the Ag and a second labelled Ab which binds to the Ab

Answer 175

1) chromogenic methods (low sensitivity) use an enzyme e.g. horse radish peroxidase/alkaline phophatase plus the enzyme substrate. This results in the production of a colored precipitate which can be visualized on the membrane as a colored band2) chemiluminescence (high sensitvity)- uses a substrate that produces chemi-luminescence and can be visualised on x-ray film. 3) fluorescence using Ab coupled to a flourochrome. less sensitivity but allows multiple targets to be visualized at the same time.

Answer 176

used to enrich for a purify a protein from a complex sample. Purified Ags can then be quantified and anlaysed using ELISA or western blot and novel proteins can be identified by mass spectrometry

Answer 177

The Ab for selecting the Ag of interest can be pre-immobolised (most common) on a solid support e.g. agarose of magnetic beads. The Ag is incubated with cell lysate containing the Ag of interest. Unbound cell lysate is weashed away and the g can then be eluted from the bound AbFree Ab approach can also be used. In this case the Ab is free in the cell lysate solution and allowed to conjugate with the protein onf interest. The complexes are then retained by attachement to beads and the remaining cell lysate is washed away. This method is good for proteins present in low concentrations of when there is low Ab-Ag affinity

Answer 178

CIP is used to detect physiologically important protein-protein interactions by using protein specific Abs to target specific proteins and the bound partners. Protein complexes can then be analysed to ID binding partners and study the kinetics co binding interactions. pull down assays are similar to IP only the protein is attached to a non-Ab affinity system on solid support.

Answer 179

CHIP is an IP reactions where the targeted protein is isolated from cell lysate with the attached chromatin. This allows studies into gene regulation and sites of different protein intersctions e.g. transcription factors, silencers, promoters etc

Answer 180

Investigating protein stability, expression, activity, location and affinity can be useful for functional studies into the pathogenicity of a VUS, In practice this is by research as to specialist to perform as part of routine diagnostics. e.g. investiagtion of BRCA1/2 half life in proteins containing mismatch variants

Answer 181

IHC is a method to visualise the presence and location of proteins in a tissue but Ab-Ag binding and use of a labelled Ab e.g. conjugated to an enzyme such as horse radish peroxidase- non specific Ab binding should be blocked by BSA- detection methods can include enzymes in chromogenic reactions or directly labeled Abs

Answer 182

monoclonal Abs are targeted to a sinlge epitope of an Ag. they are less likely to cross-reactpolyclonal Abs wll recognise multiple epitopes of the same Ag. They are quicker and cheaper to make but more likely to cross react resulting in false binding.

Answer 183

Advantages:quick, cost effective and provide positional informationdisadvantages:Ab cross reactivity = false +vevariation in fixation and processing requires use of controlsrequires pathologist expertise for analysis and interpretationmostly qualitative (semi-quantitative)low throughputdoes not detect abn proteins that are still expressed e.g. missense mutationsrequires pathologist to accumulate mark slide and ID tumour material for study

Answer 184

Mass spectrometry measures the mass/charge ratio (m/z). It can be used to ID unknown compounds via determination of the molecular weight, to quantify unknown compounds and detemine the moelcular structure. It can be used to identify proteins in a mixture- determine a.a. frequencies of peptides, identify post--translational modifications, absolute and relative protein quntities in a mix, examine isoform expression , turnover rate and subcellular localisation

Answer 185

The samples is injected into the mass spectrometer and ionised and accelerated, the ions are separated by the m/z via electromagnentic deflectionthe mass spectrun is the m/z rations of the ions in a samples plotted against their intensities

Answer 186

uses 2 mass spectrometers in tandem. 1st is used to separate the mixture and the second to separate fragmented ions. this allows a greater of seperation and can be used to identify metabolites in numerous metabolic disorders e.g. newborn bloodspot screening e.g. MCADD and PRV- very specific method with a false positive rate of 0.05%

Answer 187

matrix associated laser desorption/tine of flight MSused for mutation detection- has been used to profile tissues in situ (tissue sections) for the classification of tumours e.g lung cancer with the distinction of primary metastasis and nodal involvement

Answer 188

- can comprehensively profile proteins without the need to separate them first- requires a small amount of protein- high sensitvity and specificity- high throughput = cost effective and suitable for screeningbut ..- preferential detection of proteins will lower molecular weight- proteins present in low amounts may be masked by the more common proteins

Answer 189

Type 1. use protein capure agents e.g. Abs on an array to capture proteins (Ags) to determine the abundance and type of proteins present in a mixType 2: arrays contain proteins or peptides and are sued to study protein-protein interactions, Used to detect disease, disease subtypes or response to treatment based on protein profiling of biological fluids

Answer 190

RNA based mutation detection has been used for:- the detection and charcterisation of gene fusions e.g. BCR-ABL1- detection of aberrant splicing, allowing the detection of deep intronic sequence changes that would be missed by exon sequencing

Answer 191

RNA needs to be converted to cDNA by reverse transcriptase for use in PCR. All RNA species in a cell can be reverse transcribed to cDNA1. RNA is RT into a hybrid of single stranded cDNA and RNA. the RNA strand can then be digested and DNA polymerase used to generate a second strand of complimentary DNA

Answer 192

RNA is converted to cDNA by RThas been used to detect fusion genes found in leukemia for prognosis, diagnosis and MRD. e.g. Used in CML to detect the t(9;22)(q34;q11) BCR-ABL1 fusion following complete cytogenetic response has been achieved with imatinib as the technique is more sensitive, It can also detect impending relapse before it becomes overt

Answer 193

Method to enable VUS and splicing pattersn to be investigated without needing a sample from the affected individual so can be useful for investigating mRNAs in tissues that cannot be easily sampled- variant of interest can be generated by site directed mutagenesis but the test may not replicate in vivo splicing patterns- can also be used to elucidate cis and trans regulatory elements and other regulators of pre-mRNA splicing in vivo-

Answer 194

RNA is separated by size by gele elecgtrophoresis- RNA is then bound to an solid support and exposed to labeled probes complimentary to the region of interest- can be used to investigate RNA exporession pattersn, splicing isoforms of genes of interest (based on size)- can identify tissue specific splicing and compare splicing between different tissues, developmental times, in disease and non-disease states

Answer 195

- able to detect small changes in gene expression- detects RNA szie- can observe all splice products- samples are degraded by RNAses- requires harmful chemicals- usually only suitable for investigating 1 or a couple of genes

Answer 196

Array with probes targeted to RNA and is used to measure RNA in order to ID changes in gene expression and commonly used for RNA profiling in humans- only known gene can be investigated so not suitable for identifying novel genes, fusion geneMicroarrays can also be sued for expression profiling of miRNAs-miRNAs are involved in regulation og mRNA expression, their expression is tissue specific and critical to the development of organisms and they have been implicated in oncogenesis. Therefore miRNA profiling has been used to ID biomarkers for diagnosis and prognosis in cancer

Answer 197

The main difference between RNA-Seq and microarrays is that the former allows for full sequencing of the whole transcriptome while the latter only profiles predefined transcripts/genes through hybridization.RNA microarray is cheaper, quicker and easier than RNA seq with reduced bioinformatics input- this results in lower costs, shorter TAT and it can also provide quantitative accuracyRNA seq- allows unbiased detection therefore it can be used to ID novel transcripts, gene fusions, SNVs and indels that the array can't - broader dynamic range- higher specificty and sensitivity- easier detection of low abundance transcripts by increasing the coverage)- However the bioinformatics requirements are much greater especially for de novo assembly of novel transcripts

Answer 198

RNAseq uses NGS sequencing technologies to analyse the transcriptome- RNA-Seq facilitates the ability to look at alternative gene spliced transcripts, post-transcriptional modifications, gene fusion, mutations/SNPs and changes in gene expression over time, or differences in gene expression in different groups or treatments.[4] In addition to mRNA transcripts, RNA-Seq can look at different populations of RNA to include total RNA, small RNA, such as miRNA, tRNA, and ribosomal profiling.[5] RNA-Seq can also be used to determine exon/intron boundaries and verify or amend previously annotated 5' and 3' gene boundaries. Recent advances in RNA-Seq include single cell sequencing and in situ sequencing of fixed tissuein library prep RNA is converted to cDNA as it is more stable that RNA and facilitates amplification using DNA polymerases- can also select for RNA of interest by looking for specific features e.g. mature processed mRNA can be isolated by virtue of the poly(a)tail using poly(T) oligomers covalently attached to a substrate, typically magnetic beads

Answer 199

method to sequence the full RNA in a single cell. Can look at RNA processing events e.g. recursive splicing by looking at read coverage patternscan compare RNA expression patterns between different cells (different tissue, developmental time point so disease vs non-disease)

Answer 200

Sanger sequencing is known as the chain termination or dideoxy method- it involes the use of ddNTPs whihc lack a 3'OH group and cant be extended from. These are flourescently labelled and are used to generate a series of fragments, each terminated at different positions whihc represent the full length of the sequence of interest. the fragments are seperated by gel ecletrophoresis and the sequence is determined by the fkrourescent signal as it pases through the detector

Answer 201

1. PCR amplify target DNA 2. run products on gel to check that the amplification has been successful and to rule out the presence of contamination (blank)3. clean up the sample to remove any unincorporated dNTPs4. sequencing the sample- each ddNTP is labelled with a different coluured flourophore. ddNTP present in limiting amounts - sequencing involves strand separation, primer annealing to ss template, polymerase extension (more dNTPs so these will be incorporated the majority of the time), random chain termination when a ddNTP is incorporated. this results in a collection of different lenght fragments

Answer 202

the fragments are separated by gel electrophoresis- as the fragments of different lengths pass the detector the flourophore is excited and the flourescence is detected by a detector to create an electropherogram

Answer 203

Becoming replaced by NGS for diagnostic sequencing as more genes and greater depth can be analysed with NGS- still considered the gold standard and is used to confirm NGS findings, for gap fill of regions poorly covered by NGS or to look for targeted mutations e.g. PND, PST and carrier testing

Answer 204

GC rich regions are present in the promoters, enhancers and regulatory regions of genes. They are more stable than regions that are AT rich and a higher melting temp is required to denature the DNAGC rich regions can form hairpin loops and other secondary structures- these can impede the progress of the DNA pol leading to truncated PCR products

Answer 205

- higher melting temp- DMSO and betaine additives facilitate strand separation- formamide is added to PCR products before being separated by capillary electrophoresis. It helps denature DNA by weakening H bonds between strands -

Answer 206

PCR uses thermocycling for repeated amplification of the target to produce 1000's of copies. At each round each of the new products can be used as a template resulting in exponenetial amp (until amount of substrate in the mixture and pol processivity becomes a limiting factor1. high temp to denature DNA2. temp lowered to enable primers to anneal to template3. temp raised to optimum temp for DNA pol and templates are copied* repeat*

Answer 207

PCR is very sensitive to contamination-need to keep pre and post PCR (very high conc so only a small amount could contaminate other samples) separate- can reduce the conc of the starting material to reduce the affect of contamination- run a blank to check for contamination- positive control can detect whether contamination has inhibited the reaction and helps calibrate the analysis e.g. if it has a known fragment size for sizing PCR

Answer 208

Anti-sense oligonucleotides- ss RNA or DNA ~20bp- can bind to DNA and block access to the translational machinery- used to block production of an aberrant protein- correct splicing mutations- correct aberrations by exon skipping- chemically modified to prevent degredation by nucleases- large molecules so enter the cell via endocytosis and need to be stable enough to survive in the endosome

Answer 209

delivery to target tissue- sustained effect- require readministration fo most applications- difficult to achieve complete inhibition due to the relatively large amount of target mRNA in the cell compared to the conc of the ASO

Answer 210

e. g. to convert a DMD phenotype to BMD by inducing exon skipping to restore the ORF so a full length protein is still produced but with reduced functionality- mutations specific but presence of mutation hotspots in DMD mean same treatment can be applied to multiple patients exon 51 skipping is applicable to most patients (14%)- Eleplirsen approved by FDA in 2018Golodirsen approved 2019 for exon 53

Answer 211

C>T transition in SMN2 results in aberrant splicing and transcripts lacking exon 7Blocking or enhancing the binding of proteins to an ESS or ISS can increase the amount of full length protein from SMN2Nusernisen has FDA approval- block the ISS in intron 7, blocking access of the spliceosome and promotes inclusion of exon 7 in the SMN2 mature transcript- this can make up for the lack of SMN1

Answer 212

IONIS-HTT-rx (roche) allele specific ASO designed to target HTT specific SNPs- 2017 1st clinical trial demonstrated that the treatment was safe for the duration of the study and follow-up, and the treatment produces a reduction of mHTT in the cerebrospinal fluid- further studies required to determine the best timing fro treatment and amount of mutHTT lowering required

Answer 213

siRNAs act by RNAi interference to induce gene inhibition- targeted degredation of RNA - formed by DICER which chops dsRNA into 20-25nt fragmentsdsRNA can be endogenous or exogenous fro a virus or other source- extensivley used in KO experiments- intrinsic cellular mechanism and sequence specificity can be used to specifically target a gene implicated in disease pathogenesis

Answer 214

delivery is a major obstacle| non-specific off target affects as lack the ability for spatial and temporal control

Answer 215

Clustered regularily interspersed short palindromic repeatsRNA guided nuclease to enable RNA targeted cleavage and editing- highly specific, once a cut has been made any nearby DNA with matching sticky ends will be incorporated by endogenous DNA repair mechanisms

Answer 216

amnioglycoside antiobiotics can be used to allow ribosome to read through a PTC to produce the full length protein- amnioglycosides can lead to read through by binding a tRNA to the nonsense codon and insertion of an a.a. - 10% of human disease due to a PTClevel of read through generated is variable depending on nature of the PTC- best suited to diseases where low levels of functional protein are sufficient to restore function