II. Post-transcription | 29. Investigation of gene expression by real-time PCR and DNA-microarray methodology Flashcards
What is the aim of study gene expression?
- By studying gene expression, the aim is to determine how genes and the proteins they encode function in the intact organism.
- One of the best ways to find out about that, is to take out a gene and see what happens when the organism misses it.
- For this process to occur, we need to know which gene and how much we have of it – and if we have the right one.
II. Real-time PCR
1. What is Real-time PCR?
- Real-time PCR is a polymerase chain reaction (PCR), where DNA polymerase makes copies of the investigated DNA that we were able to isolate
- Instead of the end-product electrophoresis, we quantify this after each thermocycle.
=> we get our result during the PCR and not after it
II. Real-time PCR
3. What is the mechanism of real-time PCR?
- The real-time PCR uses a fluorescent dye which labels sequence-specific primer (probes)
- Specialized thermal cyclers with fluorescence detection are used to monitor the fluorescence during each cycle
- The measured fluorescence is proportional to the total amount of amplified DNA product (amplicon); the change of the intensity of fluorescence over time can be used to calculate the amount of amplicon produced in each cycle
II. Real-time PCR
4. Compared to the regular PCR, what can we do?
- Be more efficient. Because we can detect the investigated DNA after each thermocycle
- Determine the DNA product quantitatively: by the regular PCR we can only determine if the gene is there or not by using markers, but for the real-time PCR we can determine how much (how many grams) are there
II. Real-time PCR
5. Describe Real-time PCR graph
- Threshold level is defined. Only fluorescence in higher intensity is to be analyzed
- During the exponential phase, the amount of PCR product approximately doubles in each cycle
- As the reaction proceeds, reaction components (enzymes/substrates) are consumed, and they eventually limit the reaction. At this point, the reaction slows down and enters the non-exponential phase, followed by a plateau phase
II. Real-time PCR
6. What are the 2 techniques of fluorescent detection?
The fluorescent detection can either be specific or non-specific
II. Real-time PCR
7A. What is the mechanism of Non-specific fluorescent detection?
DNA-binding dyes (such as SYBR green) are dyes that bind to all the double-stranded DNA in the PCR mixture -> causing fluorescence of the dye when bound
- An increase in DNA products during PCR therefore leads to increase in fluorescence intensity, which can be measured with a detector at each cycle
- However, this method is somewhat inaccurate,
since it binds all dsDNA – also primer dimers (ssDNA primers that have dimerized -> dsDNA primers) = prevent us from accurately monitoring the target sequence
II. Real-time PCR
7B. Why is Non-specific fluorescent detection somewhat inaccurate?
This method is somewhat inaccurate, since it binds all dsDNA – also primer dimers (ssDNA primers that have dimerized -> dsDNA primers) = prevent us from accurately monitoring the target sequence
II. Real-time PCR
8A. What is the mechanism of specific fluorescent detection?
Specific detection: reporter probe method
- Specific detection of PCR products is done with a fluorescent reporter probe
- These probes are fragments of DNA labeled with a fluorescent reporter at one end, and a quencher at the other end
- The proximity of the quencher prevents detection of the fluorescence, but during annealing (when the probe is connected to the complementary ssDNA strand by DNA polymerase), the quencher is removed by the 5’ exonuclease activity of the (Taq) DNA polymerase
- The removal of the quencher allows the fluorescence, emitted from the reporter, to be detected once it is illuminated = no quencher in close proximity to prevent the fluorescence
- This is a more accurate way of measuring PCR products, because the probe will not be part of the primer dimers
- The fluorescence will be detected and measured in a real-time PCR machine
II. Real-time PCR
8B. What are probes in specific fluorescent detection?
These probes are fragments of DNA labeled with a fluorescent reporter at one end, and a quencher at the other end
II. Real-time PCR
9. What can we determine with real-time PCR?
With real-time PCR we are able to determine the template DNA both relative and absolute:
- Relative: where we compare the DNA with one another – the aim is to identify the differences between them. F.ex: to identify tumor cells in the tissue (qualitative)
- Absolute: the quantitative determination of the DNA template – identify the exact number of DNA in molecules of grams (quantitative)
II. Real-time PCR - Principle of quantitative analysis
10A. What are the 2 important factors in quantitative analysis of PCR?
- Threshold intensity
- Threshold cycle (CT)
II. Real-time PCR - Principle of quantitative analysis
10B. What is threshold intensity in quantitative analysis of PCR?
Intensity (amount of PCR-product) can be reliably detected in the efficient phase
II. Real-time PCR - Principle of quantitative analysis
10C. What is Threshold cycle (CT) in quantitative analysis of PCR?
Threshold cycle (CT): calculated cycle number, at which the amount of PCR- product equals the threshold
II. Real-time PCR
11. What are the features of Absolute quantification?
Absolute quantification: determine grams or exact amounts of DNA fragments (ex: the amount of viruses in a sample)
- This requires a calibration curve (by series of dilutions) with a set threshold intensity and the number of cycles needed to reach this intensity
- Analysis of the unknown (ex: amount of virus), can then be obtained from the calibration curve
II. Real-time PCR
12. What are the features of Relative quantification?
Relative quantification: compare samples by investigating the effect of a compound on transcriptional activity
- for example: a tumor sample may have 10 times more PCR products than a healthy sample -> measure the amount of mRNA
- mRNA is present as complementary DNA (cDNA) by reverse transcriptase
- the quantification is expressed as the change in levels of cDNA compared to the amount of the control reference gene
II. Real-time PCR
13B. How does SNP genotyping work?
SNP genotyping: detected after PCR is done
- Detected by probes that are specific for 2 alleles, and these probes are labelled with different dyes (colors)
- If there for instance is a C/T polymorphism, these 2 probes – one containing C and other containing T, will have different colors -> allows us to determine the SNP
II. Real-time PCR
13C. How does Identification of novel polymorphisms work?
Identification of novel polymorphisms: ‘’melting curve analysis’’
- Also used in the identification of polymorphism
- The PCR product is slowly heated, and the melting point is the point where the dsDNA -> ssDNA, which means at this point the fluorescent signal will decrease (since it is only emitting fluorescence when bound to dsDNA)
- The melting point of DNA molecules with different sequences of bases differs, due to the amount of H-bond, and therefore the amount of energy required to break them
- So by investigating the melting point, the point where the signal decreases, we can investigate the DNA sequence (polymorphism)
III. DNA microarray
1. What are the features of DNA microarray?
- DNA microarray (also called DNA chip) are used in measuring the expression levels of a large number of genes at the same time or to genotype regions of a genome.
- DNA microarrays are microscope slides that are printed with thousands of tiny spots in defined positions (rows and columns), with each spot containing a known DNA sequence or gene.
III. DNA microarray
8. What are the applications of DNA microarray?
DNA microarray can be used in
- SNP detection
- genotyping
- forensic analysis
- drug discovery
- measuring pre-disposition to diseases
…and many other things
III. DNA microarray
3. What is the 6-step mechanism of DNA microarray?
1) Both mRNA samples are converted into complementary DNA (cDNA) by reverse transcriptase – cDNA = basically a copy of mRNA, but is chemically DNA
2) The experimental cDNA can be labeled with a red fluorescent dye, while the reference cDNA is labeled with a green dye -> the two samples are then mixed together and allowed to bind to the microarray slide
3) The specific DNA sequences (probes) in the spots of the microarray are ssDNA, meaning that they will find the cDNA strands and bind to each other = hybridization (forming of H-bonds between complementary nucleotide base pairs)
4) The more complementary the two strands are, the tighter they will be bound to each other -> allowing us to wash off non-specific binding sequences
5) If the expression of a particular gene is higher in the experimental sample than in the reference sample -> corresponding spot on microarray appears red
- In contrast, if the expression in the experimental sample is lower than in the reference sample -> spot appears green
- If there is equal expression in the two samples -> spot appears
yellow
6) The data gathered through microarrays can be used to create gene expression profiles, which show simultaneous changes in the expression of many genes in response to a particular condition or treatment
III. DNA microarray
4. The specific DNA sequences (probes) in the spots of the microarray are ssDNA
=> What does it mean?
The specific DNA sequences (probes) in the spots of the microarray are ssDNA, meaning that they will find the cDNA strands and bind to each other = hybridization (forming of H-bonds between complementary nucleotide base pairs)
III. DNA microarray
5. What happen If the expression of a particular gene is higher in the experimental sample than in the reference sample?
If the expression of a particular gene is higher in the experimental sample than in the reference sample
=> corresponding spot on microarray appears red
III. DNA microarray
6. What happen if the expression in the experimental sample is lower than in the reference sample ?
Spot appears green
III. DNA microarray
7. What happen If there is equal expression in the two samples?
spot appears yellow
III. DNA microarray
2. How do we perform DNA microarray?
- To perform a microarray analysis, mRNA molecules are typically collected from both an experimental and a reference sample.
- For example, the reference sample can be collected from a healthy individual, while the experimental sample is collected from someone with a disease.
