20.02.01 Mutation scanning Flashcards
What is mutation scanning
Searching for novel sequence variants within a define DNA fragment
Factors that influence the choice of scanning method used
- Detection sensitivity
- Suitability according to sample type (peripheral blood or tumour tissue)
- Suitability for predicted mutation type (PTT for polypeptide chain terminating mutations)
- Features of the DNA sequence analysed (presence of polymorphisms)
- Healthy and safety (toxic chemicals required)
- Sample throughput
- Equipment and running costs
- Post PCR manipulation requirements (lots of post PCR steps)
What is DHPLC
- Denaturing high performance liquid chromatography
- Relies on the creation of hetero- or homo-duplexes of paired DNA in order to identify SNPs.
- Products are then separated using a denaturing liquid chromatography column, which denatures the duplexes
What is MALDI-TOF
- Matrix-assisted laser desorption/ionisation time of flight mass spectrometry
- Determines the mass of a molecule by measuring the mass-to-charge ratio (m/Z) of it’s ions.
Steps of MALDI-TOF
- Sample is placed on a UV absorbing matrix pad and exposed to a laser pulse
- Ionised sample is accelerated and moved in an electric field towards detector
- Time of flight required to reach the detector depends on the m/z of individual molecules.
Uses of MALDI-TOF
- Used to measure DNA molecules up to 20kD
- Can size oligonucleotides 100bp faster than electrophoretic separation
- Can determine base composition
- Can analyse proteins transcribed from DNA to detect sequence variation
Benefits of MALDI-TOF
-Efficient ionisation of DNA molecules
-Accurate determination of mass
-Faster
-High throughput
Allele frequencies of a point mutation can be estimated in analysis of a pooled sample
Example of MALDI-TOF
- Sequenom iPLEX kit
- Contains mass-modified dideoxynucleotide terminators
Disadvantages of MALDI-TOF
- Expensive
- Large bits of specialist equipment required
What is HRM
- High resolution melt curve analysis
- Detects differences in sequence based on an altered melting temperature (Tm)
Steps in HRM
- PCR-based amplification of region of interest in presence of dsDNA binding dye which is fluorescent only when bound to dsDNA.
- Fluorescence is measured whilst amplicon is subjected to gradual increase in temperature. When Tm is reached DNA duplex separates and dye is released, fluorescence therefore decreases.
- Plot fluorescence vs temperature.
What intercalating dyes should be used in HRM
- Ones that don’t preferentially bind to specific bases, that do not inhibit PCR and do not influence Tm.
- SYBR green- unsuitable as inhibits polymerase and stabilises duplex
- SYT09. Doesn’t alter reaction dynamics even at high concentrations.
- Evagreen. Fluorescent signal is quenched when in solution. Quencher is released on binding.
What factors would affect HRM
- Extraction method. All DNAs being analysed should be extracted using same methodology
- DNA concentration. At low template concentration there is a greater chance of incorporating a mutation early in the PCR which will affect the melting behaviour. At high concentration there will be high background fluorescence due to increased intercalation of dye into dsDNA
- Higher concentration of MgCl2 increases number of mispaired heterozygote samples
Applications of HRM
Genotyping, mutation scanning and methylation analysis (bisulphite modification prior to PCR)
Advantages of HRM
- Fast
- Closed tube system, limits contamination
- Cheap (only high cost is the machine)
- High throughput
- PCR products can be used for other applications (e.g. sequencing if aberrant melt profile)
Disadvantages of HRM
- Not 100% sensitive
- Identifies presence of variant but does not characterise it (further work required, sequencing)
- Not suitable for highly polymorphic genes
- Need small fragments (50-150bp), with no more than 1-2 melt domains.
- Needs extensive optimisation
- Better for analysing large numbers of samples to enable accurate data analysis.
What happens in HRM
- Following PCR there will be a pool of 4 different amplicons (2 het duplexes and 2 hom duplexes). V/W, W/V, V/V, W/W
- 4 different melt profiles in one fluorescence signal output.
- hets are easily identified as a double peak. Due to more unstable heteroduplex pairing, they will melt at a relatively lower temperature.
What is PTT
- Protein truncation test
- Detects mutations that create premature termination codons (nonsense, splice, frameshift)
- Special primers that contain RNA pol promoter sequence and eukaryotic initiation sequences to allow in vitro transcription and translation
Steps in PTT
- Amplification of region using PCR/ RT-PCR
- In vitro transcription/ translation of region
- SDS-PAGE for protein analysis. Shorter protein products from mutated alleles are easily distinguished from wild type.
Advantages of PTT
- Suited to genes where majority of mutations are truncating. BRCA1/2, APC
- Large coding regions can be covered in one fragment
- Can determine approximate location of mutation
- Sensitivity of (5-10%)
Disadvantages of PTT
- Time consuming and outdated
- Use of radiolabels.
- Missense variants not detected.
- Deletions spanning segment being analysed or 5’ end will be missed
- Low throughput.
What is enzymic cleavage of heteroduplexes
-Use of endonucleases to cleave dsDNA at heteroduplexes
Steps in enzymic cleavage of heteroduplexes
- Amplification of region of interest using PCR or RT-PCR
- Digest with enzyme
- Gel electrophoresis
Advantages of enzymic cleavage of heteroduplexes
- Rapid, cheap
- Can analyse long range PCR products that are difficult to sequence
- Can detect multiple variants in a single PCR product
- Could be automated
Disadvantages of enzymic cleavage of heteroduplexes
- Detection rate is 92-95%
- Still need to sequence to characterise mutations
