9 - Detection of Genetic Variation Flashcards
Mutation
A change in a DNA sequence that arises in an individual
Polymorphism
A germline DNA sequence variation that can be stably inherited
Genotyping/mutation detection
Determination / identification of a particular genetic variation
Critical techniques in genotyping
- Amplification of selected sequence
- Restriction enzyme digestion
- Separation of amplified fragments
- Detection
Amplification of selected sequence
- PCR
- Automated sequencing
Restriction enzyme digestion
- Bacterial enzymes
- Recognition of specific sequences (palindromes)
Separation of amplified fragments
- Electrophoresis
- Chromatography
- Mass spectrometry
Detection
- General detection
- Specific detection
DNA sequencing
- Direct, absolute identification
- Essential to identify mutations
- Expensive, time consuming
AFLP analysis
- Simple
- Cheap
- Easy to interpret
- Limited to indels >15bp
What does AFLP stand for
Amplified fragment length polymorphism
Microsatellite genotyping
- Primers sited outside repeat region
- Length allows determination of number of repeats
RFLP analysis
- Use of internal control
- Quick
- Easy to interpret
- Cheap
- Limited to SNP/mutations in RE sites
Fluorescent probes and primers
- Single stranded DNA oligonucleotides
- Mutation detection can be based on differences in affinity of probes for PCR products of the normal/mutant genes
- Or can be based on binding of different probes, labelled with different coloured fluorophores
- Contain quencher
Quencher
Stops dye from fluorescing
Fluorescent detection with 1 colour (FRET assay)
- Uses two probes which bind to adjacent sites around the SNP
- Both probes bind to both the wild-type and mutant gene
products in the annealing phase of PCR - One has a fluorescein tag at its 3’ end, the other a LC-Red at its 5’ end
- The two probes form a fluorescent complex by FRET (so a signal only occurs when the two probes are near each other)
Genotyping by Real-Time PCR on the Lightcycler vs RFLP method for genotyping
Lightcycler method eliminates problems of contamination as tube contain reaction is opened less
Fluorescent detection with 2 different colours (Taqman assay)
- Use two different proves (one for each allele)
- Each probe has a different coloured fluorophore at 5’ end
- Bind in the annealing phase
- A Quencher at the 3’ end prevents fluoresence
- During PCR, polymerase 5’ nuclease activity cleaves the dye leading to fluorescence
Advantages of fluorescent detection
- Homogenous closed tube assay (No post PCR processing, tube transfers or RE digestions)
- Quick, simple, amenable to streamlined workflow
- Decreased potential for sample mix-ups in post PCR processing.
- Reduced potential for contamination with PCR products.
Disadvantages of fluorescent detection
- Requires some expertise in molecular biology
- Probe design requires careful thought
- Probes are expensive
- High initial capital outlay
MALDI-TOF Mass Spectrometry
- PCR
- Anneal sequencing primer
- Extend sequencing primer with ddNTPS
- Discern which ddNTP added by measuring mass
analysis by MALDi TOFF
- Sample mixed with matrix, dried on plate
- Plate placed in high vacuum of MS
- Sample irradiated with laser, energy absorbed volatises
sample/matrix, ionised matrix protonates oligonucleotides - Ions strike detector, time of flight determines mass
SSCP analysis
- SNP can change conformation and mobility of ssDNA
- PCR products heat denatured to create ssDNA
- Electrophoresis conditions strictly controlled
- Requires silver staining
- Time consuming
Advances in SSCP analysis
- Capillary electrophoresis, using a buffered polymer solution (easier control of run conditions)
- High capital outlay precludes its use in small laboratories.
Disadvantages of QF-PCR
cannot detect any changes that lie outside the target sequence of the markers and will not detect balanced rearrangements
