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I&I SC2 > Molecular diagnostics IV > Flashcards

Molecular diagnostics IV Flashcards

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1
Q

Maxam-Gilbert sequencing is based on…

A

Chemical degradation

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2
Q

How is degradation performed in Maxam-Gilbert sequencing?

A

Addition of several chemicals

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3
Q

What chemicals cause certain degradations in Maxam-Gilbert sequencing? (4)

A
  • Formic acid = A+G
  • Dimethylsulfide = G
  • Hydrazine = C+T
  • Hydrazine + NaCl = C
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4
Q

What are chain terminators in Sanger sequencing?

A

ddNTPs -> next nucleotide cannot be coupled

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5
Q

What includes the Sanger sequencing reaction mix? (2)

A
  • Primer
  • Template
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6
Q

How are strands separated during Sanger sequencing?

A

Denaturation

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7
Q

Sanger sequencing is still used for targeted/high-throughout screening

A

Targeted

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8
Q

NGS is still used for targeted/high-throughout screening

A

High-throughput screening

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9
Q

What is the advantage of using fluorescent dyes in replacing radioactive read-outs?

A

Everything can be run in a single lane

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10
Q

How can you identify nucleotides when using fluorescent dyes in sequencing?

A

Each nucleotide has a different colour

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11
Q

True or false: Capillary systems have a greater efficiency than gels to run fragments

A

True

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12
Q

What is a problem in serological HLA typing?

A

Antibodies are not specific for different subtypes –> cell lysis

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13
Q

What dictates total HLA expression?

A

Combination of both haplotypes (mom and dad)

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14
Q

Having HLA-B27 predisposes an individual to.. (3)

A
  • M.Bechterew
  • M. Reiter
  • Acute uveitis anterior
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15
Q

What kind of antibodies are used to type HLA-B27 using flow cytometry?

A

Monoclonal

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16
Q

What problems arise when typing HLA-B27 using flow cytometry? (2)

A
  • Most monoclonals cannot identify all B27 subtypes
  • Monoclonals against B27 are not fully specific
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17
Q

How can you combat that most monoclonals can not identify all subtypes of a certain HLA molecule?

A

Using multiple monoclonals to increase the coverage

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18
Q

How can you combat that monoclonals against B27 are not fully B27-specific?

A

Combination of multiple monoclonals -> increase specificity

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19
Q

What are the advantages of using flow cytometry for typing B27? (2)

A
  • Fast
  • Relatively cheap
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20
Q

What are the DISadvantages of using flow cytometry for typing B27? (2)

A
  • Sensitivity low for some B27 alleles
  • Specificity low due to cross-reaction with other HLA-B genes
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21
Q

Which flow cytometry characteristics make it useful for B27 screening? (3)

A
  • Negatives can be excluded with high certainty
  • Strong positive signals can be detected with high certainty
  • Weak positives can be typed further (PCR)
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22
Q

What are the steps of molecular typing of B27 using PCR? (2)

A
  • DNA isolation
  • PCR-SSP
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23
Q

What does SSP stand for?

A

Sequence specific primer

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24
Q

What are two frequently used methods for PCR-SSP? Which exon do they target?

A
  • Olerup method (exon 2)
  • Dominquez (exon 3)
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25
Exon 2/Exon 3 is the exon with high allelic variation
Exon 2
26
How do you achieve the highest resolution of HLA-B27?
Sequence-based typing (SBT)
27
While achieving the highest resolution, sequence-based typing is not always used. Why not?
Not always necessary and far more expensive
28
With what disease is HLA-A29 associated?
Birdshot chorioretinopathy
29
Why is HLA typing used for HLA-A29?
To make diagnosis more clear
30
How is HLA-A29 determined?
Using HLA-A low resolution PCR-SSP
31
How many PCR reactions are required for HLA-A low resolution PCR-SSP typing for HLA-A29?
29 different PCR reactions
32
With which disease is HLA-B51 associated?
Behçet's disease (BD)
33
How is HLA-B51 determined?
Using HLA-A low resolution PCR-SSP --> many PCR reactions required -> expensive/laborious
34
Which HLA molecule is associated with Coeliac disease?
HLA-DQ2- or DQ8
35
Why is typing for HLA-DQ2/8 clinically relevant?
High negative predictive value
36
What does MLPA stand for?
Multiplex Ligation-dependent Probe Amplification
37
What do you investigate using MLPA? How?
DNA using a mixture of ~40 probes simultaneously -> targeting different sites on the DNA
38
MLPA: Of how many parts does a probe consist?
2 -> one of them contains the stuffer sequence
39
MLPA: What are the two types of probes?
- Probes with hybridization sequences to which primer Y can bind - Probes with hybridization sequence + stuffer sequence + sequence to which primer Y can bind
40
MLPA: What is a stuffer sequence?
Random nucleotide sequence of a certain length
41
MLPA: All probes have/do not have the same PCR primer sequence?
Have
42
MLPA: Probes hybridize to a specific target, true or false?
True
43
MLPA: Each probe has a hybridization sequence. What is a characteristic of this sequence?
Complementary to target site
44
What allows for multiplexing in MLPA?
Stuffer sequence length differs per probe
45
MLPA: What leads to amplification of the probe?
Addition of primers directed against Y and X
46
Describe the process of MLPA (4)
- Two parts of each probe hybridize to adjacent target sequences - Both parts are ligated through DNA ligase - Product of each probe is amplified - Amplification products are separated by electrophoresis
47
MLPA: What reflects the relative copy no. of target sequences?
Relative amounts of probe amplification products
48
What is the strength of MLPA?
Amplification of different targets from the same DNA using the same primers
49
How does abacavir interfere with HLA-B?
Abacavir is able to bind this HLA subtype -> prevents normal presentation of self-antigens by HLA-B
50
Abacavir prevents normal presentation of self antigens by HLA-B. How are these antigen presented instead?
In a novel confirmation -> no longer recognized as self -> induced immune activation
51
Abacavir is used to treat acute HIV-infection. What does a doctor do when this patient is positive for HLA-B57:01? Why?
Use different drug -> otherwise adverse drug reaction
52
What are theories presented as to why abacavir interacts with HLA-B in this way? (3)
- Normal presentation of constitutive self - Novel confirmation of constitutive self - Presentation of novel self
53
Why are proteins harder to study than DNA/(m)RNA?
Harder to isolate, fractionate, etc.
54
What is meant with proteotyping?
Defining cells/phenotypes by defining their proteome
55
What is studies with immunopeptidomics?
Study of HLA peptides
56
What are use cases for clinical proteomics (for analysis of tissues)? (2)
- Biopsies of transplant organs or tumor tissues - Serum and plasma profiling
57
What are advantages of mass spectrometry? (3)
- Efficient - Sensitive - Accurate
58
Why is plasma proteomics challenging?
Plasma contains a few high abundance proteins, and a lot of low abundance proteins
59
Sensitivity of mass spectrometry is strongly dependent on..? (3)
- Depletion - Fractionation - Use of nanoparticles
60
Which types of mass spectrometers exist? (4)
- TOF: time of flight - Magnetic sector deflection - Quadrupole - Ion traps
61
What is the principle of time-of-flight mass spectrometry?
Particles of different masses are accelerated in a vacuum tube using an electrical field
62
TOF: Smaller particles will be accelerated faster/slower than bigger particles
Faster
63
What is the principle of magnetic sector deflection?
Particles are accelerated into an electric sector, in which they are deflected
64
What is a frequently used mass spectrometry type (Ion trap)?
Orbitrap
65
What is the principle of the orbitrap?
Ions are introduced into a vacuum chamber and orbit the metal rod and move along its length in a harmonic oscillation
66
Orbitrap: what dictates the harmonic oscillation?
The mass of the peptide
67
What has to happen to biomolecules in order to perform mass spectrometry on these molecules?
Convertion to gas phase
68
What are the two ways to convert biomolecules into the gas phase?
- ESI: electrospray ionization - MALDI: matrix-assisted laser desorption ionization
69
What is the principle of ESI?
Solution with protein/peptide of interest is sprayed through a capillary with a tiny nozzle -> high voltage applied
70
ESI: What is the result of applying high voltage on the process that occurs in the tiny nozzle?
Cone of charged liquid droplets
71
ESI: What happens during spraying?
Solvent evaporates -> only charged particles stay behind
72
What is the principle of MALDI?
Analyte is mixed in a co-crystal matrix and shot with laser beams
73
What does the laser beam cause during MALDI?
Instant evaporation --> positively charged particles
74
Describe the general proteomics workflow (3)
- Digestion of proteins - Measures masses of peptides - Identification of peptides
75
How do you identify peptides in proteomics?
Combination of fragments + intact mass of peptide --> use reference databases
76
Which technique allows for the study of interactions between molecules/proteins?
Co-immunoprecipitation
77
What are advantages of using proteomics over Western Blotting and/or ELISA? (3)
- Lower detection limit - Quantification - Detection of specific post-translational modifications
78
Why do 'normal' peptides that are analyzed with mass spectrometry always have an arginine or lysine at their end?
Use of trypsin during digestion of proteins
79
Immunopeptidomics: Why is the analysis of antigens presented in the context of HLA more difficult?
These antigens don't have an arginine or lysine at their end

I&I SC2 (13 decks)

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