Lecture 9: Detection of Genetic Variation Flashcards

1
Q

What is Genetic Variation?
* Mutation :4

A
  1. ‘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)

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2
Q

What is Genetic Variation?
Polymorphism : 4

A
  1. (literally “many shapes”) ‘Known Knowns’
  2. a germline DNA sequence variation that can be stably
    inherited

…3. – All tissues the same
….4. – Position of the variation same in different individuals

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3
Q

What is Genotyping/Mutation detection??

A

Determination and/or identification of a particular genetic (ie. DNA) variation

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4
Q

Genotyping/Mutation detection used in? 2

A
  1. Research:
    identification of disease genes
  2. Diagnostics
    - confirmation
    - prognostic value
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5
Q

Genotyping vs phenotyping = 2

A
  1. Some variations cause physical changes to proteins
  2. Most genetic variations dont change physical properties
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6
Q

Genotyping vs phenotyping

explain:
‘Some variations cause physical changes to proteins’ = 3

A

1 – Proteins can be easily obtained

2 – Relatively simple techniques

3 – Need to infer genotype from phenotype

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7
Q

Genotyping vs phenotyping…explain …

‘Most genetic variations dont change physical properties’ = 3

A

1 – Expression level

2 – Spatial/temporal expression

3 – Splicing changes

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8
Q

Genotyping vs phenotyping…explain …

‘Most genetic variations dont change physical properties’

SO WE: 3

A

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

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9
Q

Work flow in practice = 4

A
  1. Decision to determine
    genotype/mutation (later lectures)
  2. Collection of genetic material (DNA)
    – Blood
    – Other tissues
  3. Appropriate genotyping method
    – Controls
    – Accuracy
  4. Interpretation and reporting of results
    – Homozygous/heterozygous
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10
Q

Critical Techniques in Genotyping = 4

A
  1. Amplification of selected sequence
  2. Restriction Enzyme Digestion
  3. Separation of amplified fragments

4 .Detection

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11
Q

Critical Techniques in Genotyping

Amplification of selected sequence = 4

A
  1. – PCR (Polymerase Chain Reaction)
  • Specificity determined by oligonucleotide primers
  • 2^ 35 fold amplification in 2-3 hrs

– Automated sequencing

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12
Q

Critical Techniques in Genotyping:

Restriction Enzyme Digestion - 2

A

– Bacterial enzymes

– Recognition of Specific sequences (palindromes)

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13
Q

Critical Techniques in Genotyping:

Separation of amplified fragments = 4

A

– 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

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14
Q

Critical Techniques in Genotyping:

DETECTION -2

A

– General Detection (eg. Ethidium Bromide)

– Specific Detection (eg. Radioactive/Fluorescent probes)

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15
Q

Example 1: DNA Sequencing = 5

A

1 * Direct, absolute identification

2 * Essential to identify mutations

3 * Expensive, time consuming
….4. – Expensive equipment
….5. – Skilled operators

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16
Q

Example 2: AFLP Analysis
- 5

A
  1. (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)
  2. Simple to perform

3 * Cheap

4 * Easy to interpret

5 * Limited to indels >15bp

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17
Q

what is Microsatellite genotyping? 8…

A
  1. (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
      …8
      Microsatellite heterogeneity
18
Q

Example 3: RFLP Analysis 8

A
  1. (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

  1. *Use of internal control
  2. *Quick
  3. *Easy to interpret
  4. *Cheap
  5. *Limited to SNP/Mutation in RE sites
19
Q

problems with the examples = 3

A

1 *Multiple handling stages
*DNA Extraction
*PCR
*Post-PCR handling

2 *Can be slow (relatively)
*Clinical applications need rapid answers sometimes

  1. 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……..

20
Q

One possible solution….for the problems…

A

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

21
Q

understanding: Fluorescent probes and primers
= 4

A

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??
22
Q

Fluorescent detection with 1 colour =

A
  1. (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

23
Q

Excitation and Emission Spectra of Donor/Acceptor
Fluorophores of FRET Probes ..diagram on slide 20

A

Donor
Fluorescein492,520

Acceptor
- Cy5 643, 667
- LC640 625,640
- LC705 685,705

fluorescence vs wavelength

24
Q

The complexes have different stabilities = 2

A
  1. Probe + A Allele Complex
    = Melting Temperature of 62C
  2. Probe + G Allele Complex
    = Melting Temperature of 55C
25
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
26
Melting Curve Analysis in Practice
diagram on slide 23
27
FIRST METHOD = 'RFLP method for Genotyping' = 1-5 SECOND METHOD 'Genotyping by Real-Time PCR on the Lightcycler' = 6-8
1. percipitate DNA 2. DNA 3. DNA + probe 4. PCR amplification ...thermocycler 5. Dyes + primers 6. LIGHTCYCLER STEP ..I THINK? 7. PCR AMPLIFICATION AND MELTING CURVE ANALYSIS ...VIA THERMOCYCLER AND FLUORIMETER 8. Melting curves ...D(Flu)/temp vs temp
28
explain Fluorescent detection with 2 different colours = 7
1. 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 6. *During PCR, polymerase 5’ nuclease activity cleaves the dye ...7. *Fluorescence!!!!
29
Fluorescent detection with 2 different colours (often referred to as “Taqman” assay)
diagram on slide 26
30
TaqMan Probe Assay Output
C vs T CC homozygote Blanks Outliers TT homozygote CT heterozygote DIAGRAM ON SLIDE 27
31
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.
32
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.
33
MALDI-TOF MS EXPLAIN =
1. Matrix Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry 2.PCR 3. Anneal sequencing primer 4. Extend sequencing primer with 'ddNTPs' 5. Discern which 'ddNTP' added by MALDI-TOF MS 6. A = 297.3Da C = 273.2Da G = 313.2Da T = 288.2Da 7. Sometimes referred to as “Single Nucleotide Extension”
34
MALDI-TOF MS (Matrix Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry)
EXAMPLE AND DIAGRAM ON SLIDE 30
35
Overview of Analysis by MALDI-TOFF = 6
1. Sample mixed with matrix, dried on plate 2. Plate placed in high vacuum of MS 3. 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
36
Analysis by MALDI-TOFF output = diagram on slide 32
intensity vs mass (m/2)
37
Searching For Mutations (Unknown unknowns) SIMPLEST APPROACH
1. PCR everything THEN 2. Sequence everything
38
Searching For Mutations (Unknown unknowns) = More efficient approach = 3
1. PCR everything 2. 'Simple Method to show which samples are different' 3. Sequence different sample ONLY
39
Example : SSCP analysis = 7
1. 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
40
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 ....4*Rapid 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.
41
Example : Thermal Curve Analysis ...CAN WE AVOID ELECTROPHORESIS?
1. – Yes we can………..Fluorescent analysis 2. * Tm of a product determined by sequence – Different sequences result in different Tm 3. * Detection with Double-stranded DNA-binding molecule (Eg. SYBRGreen, EvoGreen) DIAGRAM ON SLIDE 37