Cases
H2: PCR
1) How can you detect SARS-CoV2 presence (an RNA virus; and the genome is
known!) in a nasopharyngeal swab? How can you determine the virus titer
(hoeveelheid virus)?
- RNA is first transcribed into cDNA via Reverse transcriptase. The cDNA is then
amplified using q-PCR (more specific than regular PCR). When a fluorescent
signal is measured, the SARS-CoV2 gene is present.
- It can be determined by q-PCR using a standard curve. This allows
quantification of viral RNA copies. (Can also be done with ddPCR)
2) In a freight of chocolate from Brazil, you suspect the presence of GMO soy
containing the Bt gene (a bacterial gene). However, this freight is not labeled as
GMO. How to detect if there is GMO material in this freight? And what about
detection in sugar from GMO sugar beet?
- After extracting DNA from the chocolate, you first check if there is soy-DNA in
the chocolate with qPCR. Then you design a specific primer pair for the Bt
gene and perform qPCR. If both the soy-specific qPCR and the Bt qPCR are
positive, this indicates the presence of GMO soy containing the Bt gene.
- There is no DNA in sugar, so we can’t detect GMO
3) You want to detect if the tumor suppressor gene p53 is expressed in a patient
with colon cancer. How would you do that? How can you check if this gene is
lower/higher expressed in patients with cancer in comparison with healthy
persons?
- Extract RNA and convert into cDNA using reverse transcriptase. cDNA
quantified by qPCR using p53-specific primers.
- The comparison of expression between healthy and sick persons is performed
by relative quantification using qPCR. The Ct values need to be normalized
using reference genes.
4) You have done a PCR to amplify the SMN1 gene. To your surprise you see 5 bands
on the agarose gel. How is this possible? What can you do to improve specificity
of this PCR?
- This can be due to aspecific amplification. The primers are not specific for the
SMN1 gene and can bind on multiple DNA regions resulting in multiple bands.
- Increase annealing temperature, buffer: optimize Mg2+ concentration, hot
start PCR, touch down PCR, nested PCR
, H3: DNA hybridization
1) In a library of dog DNA, you want to search for homologues of the human BRCA1
gene. How can you do that? Which conditions will you use? How do you design
and label your probe?
- You can screen library of genes with colony or plaque hybridization.
- Non stringent conditions to allow mismatch. The dog and human BRCA1 will
be similar but not identical
- Design probe based on the known BRCA1 gene (human) with PCR and label
with fluorescent nucleotides
2) The muscle disease Spinal Muscular Atrophy is in 95% of the cases caused by a
homozygous deletion of exon 7 of the survival motor neuron 1 (SMN1) gene. a.
How can you identify in DNA samples of a couple and their unborn child whether
they are having the mutant allele in heterozygous or homozygous form? b. How
can you do this in high throughput, for screening? c. How to do the same, but now
to screen for 10.000 SNPs (all linked to different diseases)?
- a) Dot blot hybridization: DNA samples are spotted onto a membrane. 2
labeled ASO probes are used (1 tested per hybridization): one for the wild type
and one for the mutated allele.
- b) Macro/micro array: DNA from many individuals can be spotted on the
membrane.
- c) Reverse dot blot: 10000 allele-specific probes are spotted onto a
membrane. Labeled DNA from one individual is then added in solution.
3) You want to silence an essential gene in stinkbug, an important pest in
greenhouses. How can you do that? How can you check if the target gene is
indeed silenced?
- The gene is silences using RNAi for pest control. Via feeding, injection or
spraying, you can deliver dsRNA specific to the target gene
- Gene silencing can be verified with western blot (check presence of protein →
should be gone) or with northern blot (check if RNA is still there → should be
less)
H2: PCR
1) How can you detect SARS-CoV2 presence (an RNA virus; and the genome is
known!) in a nasopharyngeal swab? How can you determine the virus titer
(hoeveelheid virus)?
- RNA is first transcribed into cDNA via Reverse transcriptase. The cDNA is then
amplified using q-PCR (more specific than regular PCR). When a fluorescent
signal is measured, the SARS-CoV2 gene is present.
- It can be determined by q-PCR using a standard curve. This allows
quantification of viral RNA copies. (Can also be done with ddPCR)
2) In a freight of chocolate from Brazil, you suspect the presence of GMO soy
containing the Bt gene (a bacterial gene). However, this freight is not labeled as
GMO. How to detect if there is GMO material in this freight? And what about
detection in sugar from GMO sugar beet?
- After extracting DNA from the chocolate, you first check if there is soy-DNA in
the chocolate with qPCR. Then you design a specific primer pair for the Bt
gene and perform qPCR. If both the soy-specific qPCR and the Bt qPCR are
positive, this indicates the presence of GMO soy containing the Bt gene.
- There is no DNA in sugar, so we can’t detect GMO
3) You want to detect if the tumor suppressor gene p53 is expressed in a patient
with colon cancer. How would you do that? How can you check if this gene is
lower/higher expressed in patients with cancer in comparison with healthy
persons?
- Extract RNA and convert into cDNA using reverse transcriptase. cDNA
quantified by qPCR using p53-specific primers.
- The comparison of expression between healthy and sick persons is performed
by relative quantification using qPCR. The Ct values need to be normalized
using reference genes.
4) You have done a PCR to amplify the SMN1 gene. To your surprise you see 5 bands
on the agarose gel. How is this possible? What can you do to improve specificity
of this PCR?
- This can be due to aspecific amplification. The primers are not specific for the
SMN1 gene and can bind on multiple DNA regions resulting in multiple bands.
- Increase annealing temperature, buffer: optimize Mg2+ concentration, hot
start PCR, touch down PCR, nested PCR
, H3: DNA hybridization
1) In a library of dog DNA, you want to search for homologues of the human BRCA1
gene. How can you do that? Which conditions will you use? How do you design
and label your probe?
- You can screen library of genes with colony or plaque hybridization.
- Non stringent conditions to allow mismatch. The dog and human BRCA1 will
be similar but not identical
- Design probe based on the known BRCA1 gene (human) with PCR and label
with fluorescent nucleotides
2) The muscle disease Spinal Muscular Atrophy is in 95% of the cases caused by a
homozygous deletion of exon 7 of the survival motor neuron 1 (SMN1) gene. a.
How can you identify in DNA samples of a couple and their unborn child whether
they are having the mutant allele in heterozygous or homozygous form? b. How
can you do this in high throughput, for screening? c. How to do the same, but now
to screen for 10.000 SNPs (all linked to different diseases)?
- a) Dot blot hybridization: DNA samples are spotted onto a membrane. 2
labeled ASO probes are used (1 tested per hybridization): one for the wild type
and one for the mutated allele.
- b) Macro/micro array: DNA from many individuals can be spotted on the
membrane.
- c) Reverse dot blot: 10000 allele-specific probes are spotted onto a
membrane. Labeled DNA from one individual is then added in solution.
3) You want to silence an essential gene in stinkbug, an important pest in
greenhouses. How can you do that? How can you check if the target gene is
indeed silenced?
- The gene is silences using RNAi for pest control. Via feeding, injection or
spraying, you can deliver dsRNA specific to the target gene
- Gene silencing can be verified with western blot (check presence of protein →
should be gone) or with northern blot (check if RNA is still there → should be
less)
