Lecture 9: Detection of Genetic Variation Flashcards
What is Genetic Variation?
* Mutation :4
- ‘Known Unknowns and Unknown Unknowns’
2 * a change in a DNA sequence that arises (de novo) in an individual
leads to
….3. – Tissue restriction
….4. – Independent mutations in the same gene in different individuals (not necessarily in the same place)
What is Genetic Variation?
Polymorphism : 4
- (literally “many shapes”) ‘Known Knowns’
- a germline DNA sequence variation that can be stably
inherited
…3. – All tissues the same
….4. – Position of the variation same in different individuals
What is Genotyping/Mutation detection??
Determination and/or identification of a particular genetic (ie. DNA) variation
Genotyping/Mutation detection used in? 2
- Research:
identification of disease genes - Diagnostics
- confirmation
- prognostic value
Genotyping vs phenotyping = 2
- Some variations cause physical changes to proteins
- Most genetic variations dont change physical properties
Genotyping vs phenotyping
explain:
‘Some variations cause physical changes to proteins’ = 3
1 – Proteins can be easily obtained
2 – Relatively simple techniques
3 – Need to infer genotype from phenotype
Genotyping vs phenotyping…explain …
‘Most genetic variations dont change physical properties’ = 3
1 – Expression level
2 – Spatial/temporal expression
3 – Splicing changes
Genotyping vs phenotyping…explain …
‘Most genetic variations dont change physical properties’
SO WE: 3
SO : ‘We examine DNA to determine the exact nature of genetic variation.’
- Genotyping allows us to PREDICT an outcome
-Phenotyping forces us to INFER A CAUSE
Work flow in practice = 4
- Decision to determine
genotype/mutation (later lectures) - Collection of genetic material (DNA)
– Blood
– Other tissues - Appropriate genotyping method
– Controls
– Accuracy - Interpretation and reporting of results
– Homozygous/heterozygous
Critical Techniques in Genotyping = 4
- Amplification of selected sequence
- Restriction Enzyme Digestion
- Separation of amplified fragments
4 .Detection
Critical Techniques in Genotyping
Amplification of selected sequence = 4
- – PCR (Polymerase Chain Reaction)
- Specificity determined by oligonucleotide primers
- 2^ 35 fold amplification in 2-3 hrs
– Automated sequencing
Critical Techniques in Genotyping:
Restriction Enzyme Digestion - 2
– Bacterial enzymes
– Recognition of Specific sequences (palindromes)
Critical Techniques in Genotyping:
Separation of amplified fragments = 4
– Electrophoresis (agarose vs acrylamide)
- Non-denaturing PAGE or Agarose Gel, can discriminate 5-10% difference in weight - not suitable for small indels.
– Chromatography
– Mass Spectrometr
Critical Techniques in Genotyping:
DETECTION -2
– General Detection (eg. Ethidium Bromide)
– Specific Detection (eg. Radioactive/Fluorescent probes)
Example 1: DNA Sequencing = 5
1 * Direct, absolute identification
2 * Essential to identify mutations
3 * Expensive, time consuming
….4. – Expensive equipment
….5. – Skilled operators
Example 2: AFLP Analysis
- 5
- (Amplified fragment length Polymorphism)
process:
Sample 1 and sample 2 (with section)
goes into PCR
THEN PAGE ELECTROPHORESIS/EtBr staining
(s1 and s2)
– full length, shorter product (travels faster) - Simple to perform
3 * Cheap
4 * Easy to interpret
5 * Limited to indels >15bp
what is Microsatellite genotyping? 8…
- (A special case of AFLP)
2.* Primers sited outside repeat region
3 * Length allows determination of no. of repeats
4 * Requires acrylamide gels (neurotoxin)
….5. *Capillaries used now
- Problems
…7Shadow bands
…8Microsatellite heterogeneity
- Problems
Example 3: RFLP Analysis 8
- (Restriction fragment length Polymorphism)
process
- sample 1 and sample 2
- PCR
- All products are the same size
- ‘Rsa I’ digestion
- electrophoresis
2.Variations
* Use of mutagenic PCR primers to introduce RE sites
3.RsaI only cuts at GTAC
- *Use of internal control
- *Quick
- *Easy to interpret
- *Cheap
- *Limited to SNP/Mutation in RE sites
problems with the examples = 3
1 *Multiple handling stages
*DNA Extraction
*PCR
*Post-PCR handling
2 *Can be slow (relatively)
*Clinical applications need rapid answers sometimes
- How can data integrity be maintained?
*1000s of samples
*Contamination!!!!!
We try to make the detection as simple as possible, but this imposes other limitations on the method……..
One possible solution….for the problems…
1 * Change the detection method
– Still generic/easy to detect
– Reduce focus on specific
amplification and focus more on specific detection
…..2 * Fluorescent probes/primers
– Detection in tubes
(elimination of manual
handling)
– Equipment that can perform amplification and detection at the same time
understanding: Fluorescent probes and primers
= 4
1 * Single stranded DNA oligonucleotides (20-40 bp long),
….2 * Mutation detection can based on differences in affinity of probes for PCR products of the normal/mutant genes
….3. * Mutation detection can be based on binding of different probes, labelled with different coloured fluorophores
- Oligos often contain a “Quencher” as well as the fluorescent dye
* Quencher stops the dye from fluorescing
* Why is it needed??
- Oligos often contain a “Quencher” as well as the fluorescent dye
Fluorescent detection with 1 colour =
- (often referred to as “FRET” assay)
2.*Uses two probes which bind to adjacent sites around the SNP
2 *Both probes bind to both the wild-type and mutant gene products in the annealing phase of PCR
3 *One has a fluorescein tag at its 3’ end, the other a LC Red at is 5’ end
4 *The two probes form a fluorescent complex by FRET (so a signal only occurs when the two probes are near each other)
diagram on slide 19
Excitation and Emission Spectra of Donor/Acceptor
Fluorophores of FRET Probes ..diagram on slide 20
Donor
Fluorescein492,520
Acceptor
- Cy5 643, 667
- LC640 625,640
- LC705 685,705
fluorescence vs wavelength
The complexes have different stabilities = 2
- Probe + A Allele Complex
= Melting Temperature of 62C - Probe + G Allele Complex
= Melting Temperature of 55C