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I&I SC2 > Molecular diagnostics I > Flashcards

Molecular diagnostics I Flashcards

1
Q

What does the bacterial ‘immune system’ consist of?

A

Restriction/modification enzymes

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2
Q

How do restriction/modification enzymes protect the bacterial genome?

A

Nucleases start cutting viral DNA upon activation –> recognition sites for restriction enzymes are methylated

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3
Q

What do most restriction enzymes recognize?

A

4/6 bp palindrome

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4
Q

Why do plasmid vectors make it easier to manipulate genetics?

A

Can exist as many copies per cell

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5
Q

How do you select for the presence of a certain plasmid?

A

Antibiotic resistance gene incorporation

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6
Q

Why is there very little crossover between various eukaryotic cell types with respect to promoters for transcription?

A

These various cell types require unique promoters

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7
Q

What is a critical regulatory step during transcription?

A

The transition of RNA polymerase from a closed to an open confirmation

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8
Q

Which factors help RNA polymerase bind to the promoter in these regions? (in bacteria)

A

Sigma factors

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9
Q

How is the start site of translation marked in both prokaryotes and eukaryotes?

A

Triplets in RNA

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10
Q

Name similarities of translation between prokaryotes and eukaryotes (6)

A
  • mRNA template
  • mRNA is synthesized from DNA
  • Ribosome = protein synthesis machinery
  • All 20 amino acids are the same
  • All 61 codons are similar
  • Protein synthesis occurs in cytoplasm
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11
Q

What are the differences between prokaryotic and eukaryotic translation? (5)

A
  • Transcription/translation are continuous vs. separated processes
  • Ribosome 70S vs. 80S type
  • Freely moving vs. attached to ER ribosomes
  • Polycistronic vs monocistronic
  • No introns/no splicing vs. introns/splicing
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12
Q

Why would you perform a forward genetic screen?

A

Discover gene underlying a specific phenotype

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13
Q

Describe the process of a forward genetic screen (3)

A
  • Pick phenotype
  • Induce DNA alterations -> change the phenotype
  • Investigate in which genes DNA alterations have occurred
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14
Q

Which technique is often used to cause DNA alterations? (forward genetics)

A

Transposons

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15
Q

What is the advantage of using transposons?

A

Can be used to identify which gene had been altered

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16
Q

What is reverse genetic screen?

A

Phenotype resulting from alteration of a known gene

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17
Q

Describe the process of a reverse genetic screen (3)

A
  • Start with known gene of interest
  • Inhibit this gene in the genome
  • Observe the resulting phenotype alteration
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18
Q

Which technique is often used in reverse genetic screens?

A

Homologous recombination

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19
Q

How does homologous recombination result in modification of the target gene?

A

Gene targeting vector against a selection marker

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20
Q

Name two revolutions of bacterial genetic alteration of the past decade

A
  • Gibson assembly
  • CRISPR/Cas9
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21
Q

What is the purpose of Gibson assembly?

A

To couple several DNA pieces in one single cloning step

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22
Q

What is the process of Gibson assembly? (3)

A
  • Adjacent DNA fragments with complementary ends are synthesized
  • Overlapping fragments are added to Gibson complementary master mix and incubated
  • Result: fully-sealed dsDNA
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23
Q

Gibson assembly: during incubation, the master mix’ enzymes perform several processes (3)

A
  • 5’-3’ exonuclease activity –> ss-3’ overhangs
  • Anneal complementary strands –> dsDNA of interest
  • DNA polymerase extends 3’ ends + DNA ligase seals remaining nicks
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24
Q

Gibson assembly: fully-sealed dsDNA can serve as a template for…? (3)

A
  • PCR
  • RCA
  • Direct transformations
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25
Q

What are the components of the CRISPR system?

A
  • Cas9: DNA-cutting protein
  • Single guide RNA: recognizes specific section of DNA
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26
Q

What is the single guide RNA made up of in CRISPR/Cas9?

A
  • Crispr RNA: 17-20 nucleotide sequence complementary to target DNA
  • TracrRNA: binding scaffold for Cas nuclease
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27
Q

Describe the process of CRISPR/Cas9 (4)

A
  • Cas9 binds to PAM
  • sgRNA unwinds part of DNA double helix and anneals to complementary sequence in the DNA
  • Two nuclease domains of Cas9 make dsDNA break
  • NHEJ or template to repair DNA
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28
Q

Name three ways to use CRISPR/Cas9

A
  • Deleting genes (KO)
  • Editing genes
  • Inserting genes
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29
Q

How is deleting genes ensured using CRISPR/Cas9?

A

NHEJ after DNA cutting

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30
Q

How is editing genes ensured using CRISPR/Cas9?

A

Introducing an exogenous repair template containing a GENE CORRECTION

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31
Q

How is inserting genes ensured using CRISPR/Cas9?

A

Introducing an exogenous repair template that contains LONG STRETCH OF DNA

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32
Q

Via which methods can DNA be visualized? (4)

A
  • Direct
  • Staining (fluorescent labels)
  • Hybridisation (binding of antibodies)
  • In case of small amounts: PCR
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33
Q

What are the steps of PCR? (3)

A
  • Target DNA denaturation
  • Separated stands are accessible for forward/reverse primers (annealing)
  • Loose nucleotides/DNA pol are added
34
Q

PCR: How does the target DNA get denatured?

A

Hydrogen bonds broken by temperature

35
Q

PCR: at which temperature are the loose nucleotides added?

A

72C

36
Q

How can DNA be separated based on size?

A

Put on agarose gel with buffer –> apply electricity –> small fragments migrate faster

37
Q

Which substance can be used to visualize DNA on agarose gels?

A

Ethidium bromide

38
Q

What is the problem with PCR? What is the solution?

A

Cannot determine the amount of material at the start –> Real-time PCR

39
Q

How is PCR positivity measured during real-time PCR?

A

Cycle threshold (Ct-)level

40
Q

Real-time PCR: What can be said about the amount of fluorescence and DNA in the first cycle?

A

No fluorescence, very little DNA

41
Q

Real-time PCR: At what is point is the laser able to detect fluorescence?

A

Cycle threshold

42
Q

Real-time PCR: What causes the plateau?

A

Reagents running out

43
Q

Real-time PCR: Less DNA put in, more/less cycles are needed

A

More

44
Q

Real-time PCR: More DNA put in, more/less cycles are needed

A

Less

45
Q

Real-time PCR: What does the 2nd derivative max calculate?

A

Difference between the fluorescence in the different cycles

46
Q

Why would one want to determine the exact amount of virus particles using real-time PCR?

A

To determine treatment success

47
Q

Which three detection formats for PCR exist?

A
  • Probe based
  • Non-probe based
  • Transcription-mediated amplification (TMA)
48
Q

Name an example of a probe-based detection format

A

Taqman technology -> specific double-dyed fluorescent hydrolysis probes

49
Q

How does Taqman technology work? (5)

A
  • Fluorophores emit fluorescent signal when excited
  • Quencher added on same probe and takes up emitted light
  • DNA strand elongation on the end of the probe with quencher
  • DNA pol hydrolyses quencher -> fluorophore emits signal
  • Fluorophore hydrolysed
50
Q

Taqman: Why is there more signal per cycle?

A

In every cycle, more DNA is available for probes to bind to

51
Q

Taqman: When is the signal measured?

A

In the middle of the annealing/elongation step

52
Q

Name examples of non-probe-based PCR detection methods (2)

A
  • Intercalating dyes (Sybr green) -> non-specific dye, specific primers
  • MultiCode technology
53
Q

How do intercalating dyes bind to dsDNA?

A

Non-specifically

54
Q

Which problem arises when using a non-probe-based detection method?

A

Non-specific –> doesn’t allow quantification of specific fragments

55
Q

How can you control the non-specific nature of non-probe-based detection methods?

A

At the end of the reaction (sequence known) -> determine energy needed to break bonds –> measure how much fluorescence drops after breaking the dsDNA of interest

56
Q

On what is transcription-mediated amplification (TMA) based?

A

Isothermal reaction

57
Q

What is the process of TMA? (2)

A
  • Every target made is turned into RNA using reverse transcriptase machinery
  • RNA as template for next target
58
Q

PCR: How can you account for differences in sequences that make it difficult to design primers for those sequences? (3)

A
  • Degenerate oligos –> slightly different primer mixture
  • Dual target-assays
  • Dual-probe assays
59
Q

Describe the principle of a dual target assay

A

Two PCRs on one gene –> small mutations might prevent one probe from binding, but the other can still bind

60
Q

DNA sequencing: What is the smallest building block?

A

Nucleoside -> pentose molecule with base connected

61
Q

With what is the pentose base connected in DNA/RNA?

A

DNA: hydroxyl group
RNA: hydrogen atom

62
Q

Name two historical DNA sequencing techniques

A
  • Maxam-Gilbert
  • Sanger
63
Q

What is the principle of Maxam-Gilbert sequencing?

A

By degrading the DNA molecule into small pieces and identifying the end –> all DNA lengths –> identification of sequence

64
Q

Why is Sanger sequencing easier to read-out than Maxam-Gilbert sequencing?

A

Leads to one result per lane

65
Q

What is the principle of Sanger sequencing?

A

Primer is extended by DNA pol -> dideoxy nucleosides -> act as chain terminators

66
Q

What is the function of the primer and template in Sanger sequencing?

A

Primer = start of DNA synthesis
Template = DNA to be sequenced

67
Q

Sanger: What is the result of a proper dNTP/ddNTP ratio?

A

Chain will terminate throughout the length of the template

68
Q

Sanger: how are the single stranded fragments separated?

A

By denaturing gel electrophoresis to determine length + final nucleotide

69
Q

Sanger: fragments of four tubes are loaded in 4 different lanes of polyacrylamide gel. This leads to?

A

Sequencing ladder -> each fragment shows up on a different point on the gel

70
Q

What are two main developments in DNA sequencing?

A
  • Fluorescent dyes
  • Cycle sequencing
71
Q

Why is cycle sequencing linear PCR amplification?

A

There is only a primer on one side –> one single copy per reaction

72
Q

What does linear PCR amplification allow for?

A

To start from small amount of DNA -> lowers amount of DNA required for sequencing

73
Q

What are the main advantages of pyrosequencing? (2)

A
  • Very targeted approach
  • Fast and real-time -> useful if looking for a specific mutation/gene in humans or pathogens
74
Q

Describe the process of pyrosequencing

A

Primer/template lead to polymerase extention of fragment. During building in -> enzymatic steps -> production of oxyluciferin –> light signal

75
Q

Pyrosequencing: How are the nucleotides build in?

A

Sequential (A -> C -> T -> G) –> leads to different light signals

76
Q

Pyrosequencing: What happens when two of the same nucleotides are built in in a row?

A

Doubling of the signal

77
Q

For what is pyrosequencing particularly useful?

A

The determination of SNPs

78
Q

Pyrosequencing: how long can the DNA fragment be? What happens to this fragment?

A

<200 bp -> amplified by PCR -> sequencing primer added to PCR fragment -> dNTPs added sequentially

79
Q

What does parallel sequencing refer to?

A

Production of all different templates of your sample

80
Q

What does parallel sequencing allow for?

A

Identification of single differences between cells, which would have been lost in bulk sequencing

81
Q

HIER EVT ILLUMINA

A

I&I SC2 (11 decks)

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