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
Probe + A Allele Complex Probe + G Allele Complex Temperature diagram
slide 22
lower temp of 40C is ideal
at 60C Probe + G allele complex… denature
Melting Curve Analysis in Practice
diagram on slide 23
FIRST METHOD = ‘RFLP method for Genotyping’ = 1-5
SECOND METHOD
‘Genotyping by Real-Time PCR on the Lightcycler’
= 6-8
- percipitate DNA
- DNA
- DNA + probe
- PCR amplification …thermocycler
- Dyes + primers
- LIGHTCYCLER STEP ..I THINK?
- PCR AMPLIFICATION AND MELTING CURVE ANALYSIS …VIA THERMOCYCLER AND FLUORIMETER
- Melting curves …D(Flu)/temp vs temp
explain
Fluorescent detection with 2 different colours = 7
- often referred to as “Taqman” assay
2.Use two different probes (one for each allele)
…3 *Each probe has a different colored fluorophore at 5’ end
… 4 *Bind in the annealing phase
…5. *A Quencher at the 3’ end prevents fluoresence
- *During PCR, polymerase 5’ nuclease activity cleaves the dye
…7. *Fluorescence!!!!
Fluorescent detection with 2 different colours
(often referred to as “Taqman” assay)
diagram on slide 26
TaqMan Probe Assay Output
C vs T
CC homozygote
Blanks
Outliers
TT homozygote
CT heterozygote
DIAGRAM ON SLIDE 27
Fluorescent detection : Pros = 4
1 * Homogenous closed tube assay
– No post PCR processing , no tube transfers, RE digestions
2 * Quick, simple, amenable to streamlined workflow
– Reduced labour/reagent costs
– Automation?
3 * Decreased potential for sample
mix-ups in post PCR processing.
4 * Reduced potential for
contamination with PCR products.
Fluorescent detection : CONS = 4
1 * Requires some expertise in molecular biology
2 * Probe design requires careful thought
3 * Probes are expensive –if they don’t work
4 * High initial capital outlay* though cost-effectiveness is HIGHLY dependent on test-volume, test repertoire and other aspects of laboratory management.
MALDI-TOF MS EXPLAIN =
- Matrix Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry
2.PCR
- Anneal sequencing primer
- Extend sequencing primer with ‘ddNTPs’
- Discern which ‘ddNTP’ added by MALDI-TOF MS
6.
A = 297.3Da
C = 273.2Da
G = 313.2Da
T = 288.2Da
- Sometimes referred to as “Single Nucleotide Extension”
MALDI-TOF MS
(Matrix Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry)
EXAMPLE AND DIAGRAM ON SLIDE 30
Overview of Analysis by MALDI-TOFF = 6
- Sample mixed with matrix, dried on plate
- Plate placed in high vacuum of MS
- Sample spot irradiated with laser (UV), energy absorbed volatizes sample/matrix, ionised matrix protonates oligonucleotides
4.ons accelerated, with smaller ionised species being faster
….5.Ions strike detector which generates a signal
6.—- . Computer system controls instrument parameters and collects data
Analysis by MALDI-TOFF output = diagram on slide 32
intensity vs mass (m/2)
Searching For Mutations
(Unknown unknowns)
SIMPLEST APPROACH
- PCR everything
THEN
- Sequence everything
Searching For Mutations
(Unknown unknowns) =
More efficient approach = 3
- PCR everything
- ‘Simple Method to show which samples are different’
- Sequence different sample ONLY
Example : SSCP analysis
= 7
- Single Strand conformational polymorphism
2.* SNP can change conformation and mobility of single stranded DNA
3 * PCR products heat denatured to create
single stranded DNA
– Formamide
4 * Electrophoresis conditions strictly controlled
– Temp, % cross-linker, buffer composition
5 * Need different conditions for satisfactory sensitivity
6 * Requires silver staining/radioactive probes
7 * Difficult to automate, time consuming
Advances in SSCP analysis = 7
ADVANCE, DRAW BACK, FUTURE
1 *Capillary electrophoresis, using a buffered polymer solution.
….2 *Easier control of run conditions.
3 Fluorescent primers allow for easier detection
….4Rapid and easily automated.
5 *Drawback: high capital outlay precludes its use in small laboratories.
Future developments
…..6 Single-channel glass micro-chips.
….7.Ultra-fast (eg 4X). 100X faster than conventional SSCP.
Example : Thermal Curve Analysis …CAN WE AVOID ELECTROPHORESIS?
- – Yes we can………..Fluorescent analysis
- Tm of a product determined by sequence
– Different sequences result
in different Tm
- Tm of a product determined by sequence
- Detection with Double-stranded DNA-binding
molecule (Eg. SYBRGreen, EvoGreen)
- Detection with Double-stranded DNA-binding
DIAGRAM ON SLIDE 37
