Lab tech Flashcards
Question
Answer
describe the ARMS process?
paired PCR reaction involving common primer and allele specific primers• The allele specific primers differ in their 3’ end nucleotides. amplification does not occur if there is a mismatch allowing different alleles to be distinguished (by size or different flourescence)useful for screening large number of samples for known panel of mutations
describe advantages of ARMS
quick, cheap, simple, sensitive, detects SNVs, ins/dels, can multiplex, doesn’t require specialized equipment
describe disadvantages of ARMS
unable to detect rare/unknown variants, cross-reactivity e.g. CF p.(Phe508del)/p.(Phe508Cys), NAFNAP, often needs commercial kit, confirmation via another method often necessary, MCC in prenatals,
describe restriction enzyme digest
• Restriction enzymes make DS breaks in DNA at specific recognition sites• Can be used when a base substitution creates or abolishes a recognition site of a restriction endonuclease- fragment amplified, product is digested with the relevant restriction enzyme and the products separated by gel or capillary electrophoresis• Variation includes methylation specific restriction digests eg. Methylation-Sensitive Restriction Enzymes are not able to cut methylated-cytosine residues
what are the advantages of restriction enzyme digests?
cheap, simple, quick, no specialist equipment
what are the disadvantages of restriction enzyme digests?
NAFNAP, only confident of exact variant if it creates a site, if it destroys a site then we dont know the exact change, need r.enzyme to be available for site of interest, enzyme may be expensive or poor quality if rare site, Partial or over digestion can affect interpretation, non-specific activity, heteroduplexes (fragment re-anneals to non-complimentary fragment eg. G:T) will not cut
what is FRET (Fluorescence resonance energy transfer) hybridisation?
fluorogenic Minor groove binder probes for rtPCR are specific for SNVs. Polymerisation of a new DNA strand is initiated from the primers, and once the polymerase reaches the probe, its 5’-3’-exonuclease degrades the probe, physically separating the fluorescent reporter from the quencher, resulting in an increase in fluorescence which is detected and measured in the real-time PCR machine eg. Jak2 V617F mutation in Myeloid disease
what are advantages of FRET?
highly sensitive, highly stable, can detect very low levels of mutant DNA in a background of wild-type genomic DNA, useful for large sample numbers
what are disadvantages of FRET?
costly, no multiplexing, PH-sensitive
describe Droplet digital (ddPCR)?
sample fractionated into thousands of droplets, each containing single DNA template, run PCR using WT and mutant TaqMan probes, software then reads + and - droplets distinguishing WT from mutant.eg. BRAF V600E and EGFR mutation testing
what are advantages of Droplet digital (ddPCR)?
detects low level mutations, quantitative
what are disadvantages of Droplet digital (ddPCR) for FFPE testing?
DNA quality obtained from tumour blocks is often of poor quality and can result in poor amplification- Accuracy of the results depends on the quality of sample (e.g. containing a high percentage of tumour cells).- Fixation of the sample causes DNA damage and can result in PCR artefacts.
what is the function of the centromere?
chromosome segregation - Microtubules of the spindle attach to the centromere via the kinetochore. acentric chromosomes fail to attach to spindle and are lost from the cell
describe the structure of the centromere?
constitutive heterochromatin consisting of repetitive satellite DNA. pericentric heterochromatin facilitates sister chromatin adhesion
describe the 2 groups of centromeric proteins (CENPs)?
1) constitutively associated with the centromere such as CENPA, CENPB and CENPC, which are thought to have structural roles in kinetochore formation2) passenger proteins associate transiently throughout the cell cycle
which diseases are associated with centromere malfunction?
- Premature centromere division (PCD) – age-dependent phenomenon occurring in women. may result in increase of x chr aneuploidy- Premature chromatid separation (PCS) - separate chromatids with no discernable centromere. no known phenotypic effect- Roberts syndrome (chr breakage) - LOF mutations in ESCO2 cause delayed cell division and increased cell death. growth retardation, Limb malformations (reduction), craniofacial (microcephaly, clefting), intellectual disability and renal and cardiac abnormalities
what is the kinetochore?
multiprotein complex that assembles on centromere acting as point of contact for spindle fibers. inner kinetochore tightly associated with centromere DNA. outer kinetochore interacts with microtubules. a pair of kinetochores appears on each chromatid in late prophase.
what is a neocentromere?
new centromere that forms in non-standard centromere location as a result of disruption to natural centromere. they lack repetitive α satellite DNA sequences .
what are telomeres?
highly conserved gene-poor DNA-protein complexed that cap the ends of chromosomes. if lost the chr is unstable and can fuse with other chrs. prevents shortening > cell death. aids chr pairing.
describe the structure of telomeres
consists of tandem repeats associated with telomere-binding proteins. next to these are telomere associated repeats (unknown function). also has ss-DNA 3’ overhang protects chr end particulary in replication of lagging strand (back-stitching synthesis creates okazaki fragments)
what is telomerase?
RNA-protein enzyme that extends synthesis of leading strand to use as template for lagging strand. has 2 subunits: protein subunit and RNA subunit consisting of complimentary hexanucleotide sequence to telomere. cells that lack telomerase shorten progressively > cell death and ageing
what is cri du chat?
5p deletion. including cat-like cry, microcephaly, distinct facies and palmar creases. Deleted region includes the hTERT gene – telomerase reverse transcriptase, which helps maintain telomere ends
what is the Nucleolar organizing region (NOR)
on short arm of acrocentrics. contains rRNA genes 5.8S, 18S and 28S. responsible for organising the nucleolus structure and contain the approx. 200 rRNA genes necessary for protein synthesis, if transcriptionally active it stains dark with Ag-NOR staining
what info is required about the gene for a uv investigation?
NAME?
how do you carry out a SNV investigation?
- 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
how can RNA studies help UV investigation and describe limitations?
- 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
what is LOH in tumour tissue & how does it influence pathogenicity investigation?what limitations are there to this?
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
what is ChiP-seq? (Chromatin immunoprecipitation followed by sequencing)-
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.
what is the transcriptome?
the complete set of transcripts in a cell and their quantity at a specific developmental stage or physiological condition
what is transcriptomics?
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
how does RNA sequencing work?
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
what are the advantages of RNA sequencing?
NAME?
what are challenges of RNA-sequencing?
- 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
how is DNA fragmented before sizing if it is not being amplified by PCR?
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. )
what are issues with PCR amplification before sizing?
NAME?
describe how gel electrophoresis is used to size DNA fragments?
NAME?
how is capillary electrophoresis is used to size DNA fragments?
- 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- • 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
describe fluorescent PCR for sizing? what are its limitation
- • PCR with one primer with a fluorescent tag- • Products are analysed by capillary electrophoresis• able to resolve products 1 bp apart• limited by size of fragment able to be amplified by PCR ~ 5kb• Preferential amplification of smaller fragments means that large alleles may not be detected when present with smaller ones
describe long range PCR for sizing?
- 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’ 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.
describe TP-PCR for sizing? what are disadvantages?
-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
describe southern blotting for sizing?
- 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
describe how MLPA works?
- 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
what is reverse transcriptase MLPA?
NAME?
what two things does a SNP array look for?
NAME?
what are the general principles of a SNP array?
- 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).• 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
what is the theory behind the B-allele SNP array chart?
- 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
is SNP array able to detect triploidy?
yes
what ratios will SNP array give for triploidy?
ABB = 0.66 AAB = 0.33 AAA = 0 and BBB = 1
can snp array detect MCC and non amplification?
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.
can nullisomy be detected with SNP array?
yes - seen as a large drop in the LogR ratio and so spots/features appear randomly between 0.0 and 1.0
how is UPD detected on SNP array?
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
what might Copy number neutral LOH indicate on a SNP array?
- 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.
how does SNP array coverage compare to aCGH? what can be done to compensate?what are other limitation of SNP compared to aCGH?
NAME?
what is the first-line test for dev delay/dysmorphism/ID and congenital abnormalities in most uk labs?what is the diagnostic yield?
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%
what are the considerations for prenatal SNP array testing?
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,
how are SNP array and CNV analysis useful for tumour studies?
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
what are the limitations of SNP arrays?
- 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
how does array CGH work?
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.
what are different arrays used for?
NAME?
what are the first and second line tests for prenatal ab scan and pregnancy loss?
NAME?
what are the advantages of an oligo array?
- multiple patients on one slide- • 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- • 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.
what are the disadvantages of an oligo array?
- 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- • In prenatal diagnosis, more expensive and slower than QF-PCR
how does an expression array work?
NAME?
how are expression arrays used in tumour profiling?
NAME?
what methods can be used to explore epigenetic changes in tumours?
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.
what is quantitative real-time PCR?
- 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
what are the applications for quantitative real-time PCR?
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
how do sequence-specific DNA probes labelled with fluorescent reporter work eg. Taqman? for sequence specific DNA probes used in quantitative pcr.
- 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
what are applications for low level mutation detection?
NAME?
what is Enrichment of low-level mutations?
the process that increases mutant allele concentration relative to wt alleles. may be for known or unknown mutants. known is easier to design for.
by what 3 methods do Allele-Specific Amplification (ASA) methodologies preferentially amplify the known variant? give examples
- 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.
what methods can be used to enrich for unknown mutations?
- 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.
what is the purpose of a western blot?
NAME?
what is the procedure for western blot?
NAME?
what is immunoprecipitation?
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).
what is IHC?
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
what is Mass spectrometry (MS)?
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.
what are advantages of IHC?
fast and provides positional info
what are disadvantages of IHC?
- 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
give examples for the use of IHC?
- 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
how does RNA differ from DNA?
NAME?
what 3 forms of RNA are there?
mRNA, tRNA and rRNA
what is northern blotting used for?what are pros and cons?
NAME?
what is the process of northern blot?
- isolate RNA2. gel electrophoresis 3. transfer to membrane4. detection with hybridised probe (often cDNA)
describe the process of RNA sequencing?PROS and CONS?
NAME?
what are the pros and cons of hybridisation-based microarray for RNA?
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
what are pros and cons of quantitative RT-PCR for RNA analysis?
PROS: can use mRNA or RNA (RNA best for relative quantification of targets). mRNA more sensitiveCONS: different yields for different mRNAs
what are pros and cons of sanger for RNA analysis?
NAME?
what are pros and cons of RNA-in situ hybridisation for RNA analysis?
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
what is the principles of sanger sequencing?
- 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
what are cons of sanger sequencing?
- lower sensitivity than NGS or qPCR- low level variants may be missed-
what are applications of sanger sequencing? what are pros and cons?
- 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
what are the advantages of using array over karyotype?
- 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
What factors influence method of mutation detection?
Is the mutation known?Type of mutation.Tissue type testedCostHazardous materials needed?Specialist equipment needed?Turnaround time?Throughput?Polymorphic region?
Name some methods that can be used to scan DNA when a known mutation is not present.
Protein Truncation Test (PTT)Restriction Fragment Length Polymorphism (RFLP)SSCPCSGEdHLPCHigh Resolution Melt Curve Analysis (HRM)MALDI-TOFMutS
What are the advantages of MALDI-TOF?
High throughputRapidCan determine base composition of DNA
What are the disadvantages of MALDI-TOF?
Expensive| Need a huge machine
What are the advantages of HRM?
Cheap,Easy,High throughputCan use leftover PCR productsLow risk of contamination
What are the disadvantages of HRM?
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
What are the advantages of the PTT?
FastCheapGood for genes with nonsense mutationsLarge coding regions covered in one fragmentCan detect mutations at 5-10%
Give some disadvantages of PTT
Costly reagentsWon't detect missense variantsTime consumingRadiolabels requiredLarge deletions may be missed
Give some methods of known mutation detection
ARMSAllele-specific PCROLAPyrosequencingMinisequencingRestriction Enzyme digest (needs mutation to create a new restriction site)
What methods are available to size DNA fragments?
Long range PCRElectrophoresisSouthern blottingFluroescent PCRTP-PCRMolecular CombingNanochannel technology
Name the 6 types of electrophoresis that can be used for fragment length detection
CapiliaryAgarose GelPolyacrylamide gelNanowirePulse-phaseBioanalyzer
What is chimeric PCR?
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.
What method can be used to detect changes in fragment size due to inversions?
Inverse Shifting PCR (IS-PCR).This is used to detect in Haemophilia A inversion of intron 22.
Describe the basic method of Southern blotting.
Restriction enzyme digest of gDNAGel electrophoresis to separateTransfer to membraneApply labelHybridiseWashDetect
List some bisulphite-dependent methods of methylation detection
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.
