Session 2 - testing methods Flashcards
What factors influence method of mutation detection?
Is the mutation known? Type of mutation. Tissue type tested Cost Hazardous 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) SSCP CSGE dHLPC High Resolution Melt Curve Analysis (HRM) MALDI-TOF MutS
What are the advantages of MALDI-TOF?
High throughput
Rapid
Can 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 throughput Can use leftover PCR products Low risk of contamination
What are the disadvantages of HRM?
Doesn’t detect actual mutation.
Not 100% sensitive
Need all DNA samples to be prepped in the same way
All DNA samples must be run at the same concentration
Not suitable for highly polymorphic genes
Lots of optimisation needed
What are the advantages of the PTT?
Fast Cheap Good for genes with nonsense mutations Large coding regions covered in one fragment Can detect mutations at 5-10%
Give some disadvantages of PTT
Costly reagents Won't detect missense variants Time consuming Radiolabels required Large deletions may be missed
Give some methods of known mutation detection
ARMS Allele-specific PCR OLA Pyrosequencing Minisequencing Restriction Enzyme digest (needs mutation to create a new restriction site)
What methods are available to size DNA fragments?
Long range PCR Electrophoresis Southern blotting Fluroescent PCR TP-PCR Molecular Combing Nanochannel technology
Name the 6 types of electrophoresis that can be used for fragment length detection
Capiliary Agarose Gel Polyacrylamide gel Nanowire Pulse-phase Bioanalyzer
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 gDNA Gel electrophoresis to separate Transfer to membrane Apply label Hybridise Wash Detect
List some bisulphite-dependent methods of methylation detection
ms-MLPA
ms-PCR
Pyrosequencing (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.
List some bisulphite independent methods of methylation detection
Restriction enzyme digest at unmethylated sites
Southern blotting
Which two types of primer can be used for methylation detection, and what is the difference between the two?
Methylation-Indepedent PCR primer - these amplify methylated and unmethylated sequence
Methylation-Specific primers - these primers are specific to the methylated target DNA
What are the sizes of the digested fragments seen in a normal female’s FRAX blot?
5.2kb and 2.8kb.
Draw expected patterns of bands for normal/pre-mutation/full mutation females and males.
Andrew: see revision notes :) 14m2.02 Methylation Detection page 5
What are the two band sizes you would expect to see on a normal Prader-Willi Angelman msPCR gel?
What is amplified to produce these products?
164bp for the paternal allele
131bp for the maternal allele.
Exon 1 of SNRPN has been amplified.
List methods of copy number detection.
G-banding aCGH FISH MLPA MAPH BAC array Oligo array SNP array NGS QF-PCR Real Time QPCR
What are the three main components of SNP arrays?
Slide labelled with allele-specific probes
Fragmented nucleic acid labelled with fluorescent dye
Detection system
What does the signal density of a SNP array depend on?
The copy number of the target sequence.
The affinity between the DNA and the probe.
What are the two types of SNP array?
Illumina.
Affymetrix
What are the differences between the Affymetrix array and an Illumina array?
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.
What can SNP arrays be used to detect?
Loss of Heterozygosity (LOH) and copy number changes.
List some applications of SNP arrays
LOH detection in imprinted regions (e.g. Prader Willi)
DNA copy number changes
Methylation analysis - DNA is fragmented using methylation sensitive or methylation specific enzymes.
Gene expression analysis - use cDNA
Why are SNP arrays good for prenatal testing?
A sex-matched control isn’t required.
What are some limitations of SNP arrays?
Can’t detect balanced rearrangements.
Not highly sensitive.
List the three types of microarray
BAC
Oligo
SNP
What are the advantages of BAC arrays?
Low res, so fewer variants of uncertain significance
High signal to noise ratio due to long fragment length
Cheap
Dye-swap to check results
Follow-up FISH is easy and FISH probes are also BACs
What are the disadvantages of BAC arrays?
Low res - may miss smaller abnormalities Fiddly Expensive as only 1 patient/slide Array design limited by BAC availability Determining precise breakpoints is not possible Lots of batch-batch variation.
List advantages of oligo arrays
Higher res than BAC arrays
Cheaper to run - multiple samples per slide
Calls are dependent on multiple consecutive probes - more acurate
List disadvantages of oligo arrays
No UPD/LOH
Need sex-matched control
increased number of variants of uncertain significance
Poor signal to noise ratio.
Some abnormalities are too small to be verified by FISH.
List some applications of arrays.
CNV detection
LOH detection
Gene expression - compare tissues or tumour/normal
Epigenetic expression
What kit is available for expression profiling in tumours, and what is it used to profile?
Mammaprint, breast tumours.
What array methods can be used to investigate epigenetic expression in tumours?
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.
DamID
Bisulphite modification and appropriate oligos on the array
What factors influence classification of CNVs?
Gene content Any known microdeletions/duplications in the region Size Presence in a control population Inheritance Presence in a disease population Zygosity CNV location - imprinting regions etc.
What does the EACH study recommend is reported in prenatal aCGH?
Only report de novo, fully penetrant CNVs, or those which correspond to a significant imbalance.
Avoid reporting VUSs.
What factors need to be taken into consideration when analysing prenatal arrays?
Prenatal phenotypes of disease may be different or poorly characterised compared with postnatal phenotypes
Mosaicism could be maternal cell contamination or confined placental mocaisism.
Name the two forms of detection chemistry used in RQ-PCR, and give examples of each.
Non-specific fluorescent dyes (SYBR green)
Specific fluorescent dyes (TaqMan, FRET, Hairpin, Scorpion).
How does SYBR green work?
Small dye that binds to the dsDNA groove. Fluorescence emitted when bound to dsDNA.
What can SYBR green be used to detect?
Accumulation of dsDNA during PCR cycles.
How does TaqMan work?
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.
How does FRET work?
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.
How is material quantified using RQ-PCR?
As signal reaches threshold (Ct) the signal is above the baseline measurement and can be quantified.
Product in measured during the exponential phase.
