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PATH3305 > 9 - Detection of Genetic Variation > Flashcards

9 - Detection of Genetic Variation Flashcards

1
Q

Mutation

A

A change in a DNA sequence that arises in an individual

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2
Q

Polymorphism

A

A germline DNA sequence variation that can be stably inherited

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3
Q

Genotyping/mutation detection

A

Determination / identification of a particular genetic variation

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4
Q

Critical techniques in genotyping

A
  • Amplification of selected sequence
  • Restriction enzyme digestion
  • Separation of amplified fragments
  • Detection
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5
Q

Amplification of selected sequence

A
  • PCR
  • Automated sequencing
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6
Q

Restriction enzyme digestion

A
  • Bacterial enzymes
  • Recognition of specific sequences (palindromes)
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7
Q

Separation of amplified fragments

A
  • Electrophoresis
  • Chromatography
  • Mass spectrometry
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8
Q

Detection

A
  • General detection
  • Specific detection
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9
Q

DNA sequencing

A
  • Direct, absolute identification
  • Essential to identify mutations
  • Expensive, time consuming
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10
Q

AFLP analysis

A
  • Simple
  • Cheap
  • Easy to interpret
  • Limited to indels >15bp
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11
Q

What does AFLP stand for

A

Amplified fragment length polymorphism

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12
Q

Microsatellite genotyping

A
  • Primers sited outside repeat region
  • Length allows determination of number of repeats
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13
Q

RFLP analysis

A
  • Use of internal control
  • Quick
  • Easy to interpret
  • Cheap
  • Limited to SNP/mutations in RE sites
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14
Q

Fluorescent probes and primers

A
  • 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
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15
Q

Quencher

A

Stops dye from fluorescing

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16
Q

Fluorescent detection with 1 colour (FRET assay)

A
  • 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)
17
Q

Genotyping by Real-Time PCR on the Lightcycler vs RFLP method for genotyping

A

Lightcycler method eliminates problems of contamination as tube contain reaction is opened less

18
Q

Fluorescent detection with 2 different colours (Taqman assay)

A
  • 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
19
Q

Advantages of fluorescent detection

A
  • 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.
20
Q

Disadvantages of fluorescent detection

A
  • Requires some expertise in molecular biology
  • Probe design requires careful thought
  • Probes are expensive
  • High initial capital outlay
21
Q

MALDI-TOF Mass Spectrometry

A
  • PCR
  • Anneal sequencing primer
  • Extend sequencing primer with ddNTPS
  • Discern which ddNTP added by measuring mass
22
Q

analysis by MALDi TOFF

A
  1. Sample mixed with matrix, dried on plate
  2. Plate placed in high vacuum of MS
  3. Sample irradiated with laser, energy absorbed volatises
    sample/matrix, ionised matrix protonates oligonucleotides
  4. Ions strike detector, time of flight determines mass
23
Q

SSCP analysis

A
  • SNP can change conformation and mobility of ssDNA
  • PCR products heat denatured to create ssDNA
  • Electrophoresis conditions strictly controlled
  • Requires silver staining
  • Time consuming
24
Q

Advances in SSCP analysis

A
  • Capillary electrophoresis, using a buffered polymer solution (easier control of run conditions)
  • High capital outlay precludes its use in small laboratories.
25
Q

Disadvantages of QF-PCR

A

cannot detect any changes that lie outside the target sequence of the markers and will not detect balanced rearrangements

PATH3305 (25 decks)

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