Molecular diagnostics I Flashcards
What does the bacterial ‘immune system’ consist of?
Restriction/modification enzymes
How do restriction/modification enzymes protect the bacterial genome?
Nucleases start cutting viral DNA upon activation –> recognition sites for restriction enzymes are methylated
What do most restriction enzymes recognize?
4/6 bp palindrome
Why do plasmid vectors make it easier to manipulate genetics?
Can exist as many copies per cell
How do you select for the presence of a certain plasmid?
Antibiotic resistance gene incorporation
Why is there very little crossover between various eukaryotic cell types with respect to promoters for transcription?
These various cell types require unique promoters
What is a critical regulatory step during transcription?
The transition of RNA polymerase from a closed to an open confirmation
Which factors help RNA polymerase bind to the promoter in these regions? (in bacteria)
Sigma factors
How is the start site of translation marked in both prokaryotes and eukaryotes?
Triplets in RNA
Name similarities of translation between prokaryotes and eukaryotes (6)
- 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
What are the differences between prokaryotic and eukaryotic translation? (5)
- 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
Why would you perform a forward genetic screen?
Discover gene underlying a specific phenotype
Describe the process of a forward genetic screen (3)
- Pick phenotype
- Induce DNA alterations -> change the phenotype
- Investigate in which genes DNA alterations have occurred
Which technique is often used to cause DNA alterations? (forward genetics)
Transposons
What is the advantage of using transposons?
Can be used to identify which gene had been altered
What is reverse genetic screen?
Phenotype resulting from alteration of a known gene
Describe the process of a reverse genetic screen (3)
- Start with known gene of interest
- Inhibit this gene in the genome
- Observe the resulting phenotype alteration
Which technique is often used in reverse genetic screens?
Homologous recombination
How does homologous recombination result in modification of the target gene?
Gene targeting vector against a selection marker
Name two revolutions of bacterial genetic alteration of the past decade
- Gibson assembly
- CRISPR/Cas9
What is the purpose of Gibson assembly?
To couple several DNA pieces in one single cloning step
What is the process of Gibson assembly? (3)
- Adjacent DNA fragments with complementary ends are synthesized
- Overlapping fragments are added to Gibson complementary master mix and incubated
- Result: fully-sealed dsDNA
Gibson assembly: during incubation, the master mix’ enzymes perform several processes (3)
- 5’-3’ exonuclease activity –> ss-3’ overhangs
- Anneal complementary strands –> dsDNA of interest
- DNA polymerase extends 3’ ends + DNA ligase seals remaining nicks
Gibson assembly: fully-sealed dsDNA can serve as a template for…? (3)
- PCR
- RCA
- Direct transformations
What are the components of the CRISPR system?
- Cas9: DNA-cutting protein
- Single guide RNA: recognizes specific section of DNA
What is the single guide RNA made up of in CRISPR/Cas9?
- Crispr RNA: 17-20 nucleotide sequence complementary to target DNA
- TracrRNA: binding scaffold for Cas nuclease
Describe the process of CRISPR/Cas9 (4)
- 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
Name three ways to use CRISPR/Cas9
- Deleting genes (KO)
- Editing genes
- Inserting genes
How is deleting genes ensured using CRISPR/Cas9?
NHEJ after DNA cutting
How is editing genes ensured using CRISPR/Cas9?
Introducing an exogenous repair template containing a GENE CORRECTION
How is inserting genes ensured using CRISPR/Cas9?
Introducing an exogenous repair template that contains LONG STRETCH OF DNA
Via which methods can DNA be visualized? (4)
- Direct
- Staining (fluorescent labels)
- Hybridisation (binding of antibodies)
- In case of small amounts: PCR
What are the steps of PCR? (3)
- Target DNA denaturation
- Separated stands are accessible for forward/reverse primers (annealing)
- Loose nucleotides/DNA pol are added
PCR: How does the target DNA get denatured?
Hydrogen bonds broken by temperature
PCR: at which temperature are the loose nucleotides added?
72C
How can DNA be separated based on size?
Put on agarose gel with buffer –> apply electricity –> small fragments migrate faster
Which substance can be used to visualize DNA on agarose gels?
Ethidium bromide
What is the problem with PCR? What is the solution?
Cannot determine the amount of material at the start –> Real-time PCR
How is PCR positivity measured during real-time PCR?
Cycle threshold (Ct-)level
Real-time PCR: What can be said about the amount of fluorescence and DNA in the first cycle?
No fluorescence, very little DNA
Real-time PCR: At what is point is the laser able to detect fluorescence?
Cycle threshold
Real-time PCR: What causes the plateau?
Reagents running out
Real-time PCR: Less DNA put in, more/less cycles are needed
More
Real-time PCR: More DNA put in, more/less cycles are needed
Less
Real-time PCR: What does the 2nd derivative max calculate?
Difference between the fluorescence in the different cycles
Why would one want to determine the exact amount of virus particles using real-time PCR?
To determine treatment success
Which three detection formats for PCR exist?
- Probe based
- Non-probe based
- Transcription-mediated amplification (TMA)
Name an example of a probe-based detection format
Taqman technology -> specific double-dyed fluorescent hydrolysis probes
How does Taqman technology work? (5)
- 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
Taqman: Why is there more signal per cycle?
In every cycle, more DNA is available for probes to bind to
Taqman: When is the signal measured?
In the middle of the annealing/elongation step
Name examples of non-probe-based PCR detection methods (2)
- Intercalating dyes (Sybr green) -> non-specific dye, specific primers
- MultiCode technology
How do intercalating dyes bind to dsDNA?
Non-specifically
Which problem arises when using a non-probe-based detection method?
Non-specific –> doesn’t allow quantification of specific fragments
How can you control the non-specific nature of non-probe-based detection methods?
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
On what is transcription-mediated amplification (TMA) based?
Isothermal reaction
What is the process of TMA? (2)
- Every target made is turned into RNA using reverse transcriptase machinery
- RNA as template for next target
PCR: How can you account for differences in sequences that make it difficult to design primers for those sequences? (3)
- Degenerate oligos –> slightly different primer mixture
- Dual target-assays
- Dual-probe assays
Describe the principle of a dual target assay
Two PCRs on one gene –> small mutations might prevent one probe from binding, but the other can still bind
DNA sequencing: What is the smallest building block?
Nucleoside -> pentose molecule with base connected
With what is the pentose base connected in DNA/RNA?
DNA: hydroxyl group
RNA: hydrogen atom
Name two historical DNA sequencing techniques
- Maxam-Gilbert
- Sanger
What is the principle of Maxam-Gilbert sequencing?
By degrading the DNA molecule into small pieces and identifying the end –> all DNA lengths –> identification of sequence
Why is Sanger sequencing easier to read-out than Maxam-Gilbert sequencing?
Leads to one result per lane
What is the principle of Sanger sequencing?
Primer is extended by DNA pol -> dideoxy nucleosides -> act as chain terminators
What is the function of the primer and template in Sanger sequencing?
Primer = start of DNA synthesis
Template = DNA to be sequenced
Sanger: What is the result of a proper dNTP/ddNTP ratio?
Chain will terminate throughout the length of the template
Sanger: how are the single stranded fragments separated?
By denaturing gel electrophoresis to determine length + final nucleotide
Sanger: fragments of four tubes are loaded in 4 different lanes of polyacrylamide gel. This leads to?
Sequencing ladder -> each fragment shows up on a different point on the gel
What are two main developments in DNA sequencing?
- Fluorescent dyes
- Cycle sequencing
Why is cycle sequencing linear PCR amplification?
There is only a primer on one side –> one single copy per reaction
What does linear PCR amplification allow for?
To start from small amount of DNA -> lowers amount of DNA required for sequencing
What are the main advantages of pyrosequencing? (2)
- Very targeted approach
- Fast and real-time -> useful if looking for a specific mutation/gene in humans or pathogens
Describe the process of pyrosequencing
Primer/template lead to polymerase extention of fragment. During building in -> enzymatic steps -> production of oxyluciferin –> light signal
Pyrosequencing: How are the nucleotides build in?
Sequential (A -> C -> T -> G) –> leads to different light signals
Pyrosequencing: What happens when two of the same nucleotides are built in in a row?
Doubling of the signal
For what is pyrosequencing particularly useful?
The determination of SNPs
Pyrosequencing: how long can the DNA fragment be? What happens to this fragment?
<200 bp -> amplified by PCR -> sequencing primer added to PCR fragment -> dNTPs added sequentially
What does parallel sequencing refer to?
Production of all different templates of your sample
What does parallel sequencing allow for?
Identification of single differences between cells, which would have been lost in bulk sequencing
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