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I&I SC2 > Molecular diagnostics IV > Flashcards

Molecular diagnostics IV Flashcards

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
Q

Exon 2/Exon 3 is the exon with high allelic variation

A

Exon 2

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26
Q

How do you achieve the highest resolution of HLA-B27?

A

Sequence-based typing (SBT)

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27
Q

While achieving the highest resolution, sequence-based typing is not always used. Why not?

A

Not always necessary and far more expensive

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28
Q

With what disease is HLA-A29 associated?

A

Birdshot chorioretinopathy

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29
Q

Why is HLA typing used for HLA-A29?

A

To make diagnosis more clear

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30
Q

How is HLA-A29 determined?

A

Using HLA-A low resolution PCR-SSP

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31
Q

How many PCR reactions are required for HLA-A low resolution PCR-SSP typing for HLA-A29?

A

29 different PCR reactions

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32
Q

With which disease is HLA-B51 associated?

A

Behçet’s disease (BD)

33
Q

How is HLA-B51 determined?

A

Using HLA-A low resolution PCR-SSP –> many PCR reactions required -> expensive/laborious

34
Q

Which HLA molecule is associated with Coeliac disease?

A

HLA-DQ2- or DQ8

35
Q

Why is typing for HLA-DQ2/8 clinically relevant?

A

High negative predictive value

36
Q

What does MLPA stand for?

A

Multiplex Ligation-dependent Probe Amplification

37
Q

What do you investigate using MLPA? How?

A

DNA using a mixture of ~40 probes simultaneously -> targeting different sites on the DNA

38
Q

MLPA: Of how many parts does a probe consist?

A

2 -> one of them contains the stuffer sequence

39
Q

MLPA: What are the two types of probes?

A
  • Probes with hybridization sequences to which primer Y can bind
  • Probes with hybridization sequence + stuffer sequence + sequence to which primer Y can bind
40
Q

MLPA: What is a stuffer sequence?

A

Random nucleotide sequence of a certain length

41
Q

MLPA: All probes have/do not have the same PCR primer sequence?

A

Have

42
Q

MLPA: Probes hybridize to a specific target, true or false?

A

True

43
Q

MLPA: Each probe has a hybridization sequence. What is a characteristic of this sequence?

A

Complementary to target site

44
Q

What allows for multiplexing in MLPA?

A

Stuffer sequence length differs per probe

45
Q

MLPA: What leads to amplification of the probe?

A

Addition of primers directed against Y and X

46
Q

Describe the process of MLPA (4)

A
  • 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
Q

MLPA: What reflects the relative copy no. of target sequences?

A

Relative amounts of probe amplification products

48
Q

What is the strength of MLPA?

A

Amplification of different targets from the same DNA using the same primers

49
Q

How does abacavir interfere with HLA-B?

A

Abacavir is able to bind this HLA subtype -> prevents normal presentation of self-antigens by HLA-B

50
Q

Abacavir prevents normal presentation of self antigens by HLA-B. How are these antigen presented instead?

A

In a novel confirmation -> no longer recognized as self -> induced immune activation

51
Q

Abacavir is used to treat acute HIV-infection. What does a doctor do when this patient is positive for HLA-B57:01? Why?

A

Use different drug -> otherwise adverse drug reaction

52
Q

What are theories presented as to why abacavir interacts with HLA-B in this way? (3)

A
  • Normal presentation of constitutive self
  • Novel confirmation of constitutive self
  • Presentation of novel self
53
Q

Why are proteins harder to study than DNA/(m)RNA?

A

Harder to isolate, fractionate, etc.

54
Q

What is meant with proteotyping?

A

Defining cells/phenotypes by defining their proteome

55
Q

What is studies with immunopeptidomics?

A

Study of HLA peptides

56
Q

What are use cases for clinical proteomics (for analysis of tissues)? (2)

A
  • Biopsies of transplant organs or tumor tissues
  • Serum and plasma profiling
57
Q

What are advantages of mass spectrometry? (3)

A
  • Efficient
  • Sensitive
  • Accurate
58
Q

Why is plasma proteomics challenging?

A

Plasma contains a few high abundance proteins, and a lot of low abundance proteins

59
Q

Sensitivity of mass spectrometry is strongly dependent on..? (3)

A
  • Depletion
  • Fractionation
  • Use of nanoparticles
60
Q

Which types of mass spectrometers exist? (4)

A
  • TOF: time of flight
  • Magnetic sector deflection
  • Quadrupole
  • Ion traps
61
Q

What is the principle of time-of-flight mass spectrometry?

A

Particles of different masses are accelerated in a vacuum tube using an electrical field

62
Q

TOF: Smaller particles will be accelerated faster/slower than bigger particles

A

Faster

63
Q

What is the principle of magnetic sector deflection?

A

Particles are accelerated into an electric sector, in which they are deflected

64
Q

What is a frequently used mass spectrometry type (Ion trap)?

A

Orbitrap

65
Q

What is the principle of the orbitrap?

A

Ions are introduced into a vacuum chamber and orbit the metal rod and move along its length in a harmonic oscillation

66
Q

Orbitrap: what dictates the harmonic oscillation?

A

The mass of the peptide

67
Q

What has to happen to biomolecules in order to perform mass spectrometry on these molecules?

A

Convertion to gas phase

68
Q

What are the two ways to convert biomolecules into the gas phase?

A
  • ESI: electrospray ionization
  • MALDI: matrix-assisted laser desorption ionization
69
Q

What is the principle of ESI?

A

Solution with protein/peptide of interest is sprayed through a capillary with a tiny nozzle -> high voltage applied

70
Q

ESI: What is the result of applying high voltage on the process that occurs in the tiny nozzle?

A

Cone of charged liquid droplets

71
Q

ESI: What happens during spraying?

A

Solvent evaporates -> only charged particles stay behind

72
Q

What is the principle of MALDI?

A

Analyte is mixed in a co-crystal matrix and shot with laser beams

73
Q

What does the laser beam cause during MALDI?

A

Instant evaporation –> positively charged particles

74
Q

Describe the general proteomics workflow (3)

A
  • Digestion of proteins
  • Measures masses of peptides
  • Identification of peptides
75
Q

How do you identify peptides in proteomics?

A

Combination of fragments + intact mass of peptide –> use reference databases

76
Q

Which technique allows for the study of interactions between molecules/proteins?

A

Co-immunoprecipitation

77
Q

What are advantages of using proteomics over Western Blotting and/or ELISA? (3)

A
  • Lower detection limit
  • Quantification
  • Detection of specific post-translational modifications
78
Q

Why do ‘normal’ peptides that are analyzed with mass spectrometry always have an arginine or lysine at their end?

A

Use of trypsin during digestion of proteins

79
Q

Immunopeptidomics: Why is the analysis of antigens presented in the context of HLA more difficult?

A

These antigens don’t have an arginine or lysine at their end

I&I SC2 (13 decks)

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