Lab3 Flashcards
What is PCR used to?
To amplify a single or a few copies of DNA, generating thousands of copies of that particular DNA sequence.
What is the advantages of PCR?
- fast method
- specific
- sensitive
- robust
- valuable data
What is the disadvantages of PCR?
- targeted investigation
- needs specific equipment
- false negative result may be obtained
- false positive result may be obtained
Types of false negative result of PCR
damaged NA in the sample, prescence of inhibitory complex in the sample, mutations in the virus influencing primer annealing
Types of false positive result of PCR
Annealing of not virus-specific NA;
In chromatography method the virus suspension (sample or virus isolate) is mixed with?
a lysis buffer containing proteinase K for the lysis of the cells and virus particles.
What is the proteinase K for?
lysis of cells and virus particles.
What is added to the precipitation of the NA?
chromatography
Ethanol
Why is ethanol added after the incubation time? (chromatography)
for the precipitation of the NA,
What happens during centrifugation? (chromatography)
The fluid goes through the filter, to which the NA is bound, but the digested protein molecules (peptides, amino acids) and the fluid goes through
What is the function of the elution buffer? (chromatopgraphy)
It releases the NA from the filter and is also used as restoration buffer.
The PCR method relies on?
thermal cycling
What does the thermal cycling consist of? (PCR)
consisting of cycles of repeated heating and cooling of the reaction mixture
Why is the thermal cycling steps necessary? (PCR)
first to physically separate the two strands in a DNA double helix at a high temperature (DNA melting)
How does each strand act at a lower temperature? (PCR)
each strand act as a template in DNA synthesis by the DNA polymerase to selectively amplify the target DNA
What happens as the PCR progresses?
the DNA template is exponentially amplified.
What are the optimal conditions for PCR?
if there are no limiting factors such as substrates or reagents at each extension step
What happens under optimal conditions of PCR?
the amount of DNa target is doubled in each cycle.
The selectivity of PCR results from the us of?
primers that are short, usually 18-25 nucleotides long, single -stranded oligodeoxynucleotide (DNA) molecules, complementary to the (viral) DNA region targeted for amplification
The primers are designed using?
computer programs that select and evaluate short sequences of the viral DNA that are suitable for being usd as primers
Primer design is based on?
the genome sequence of the target virus (available in gene bank databases)
What is the primer necessary for?
the polymerase enzyme as an initiator of the polymerisation
A basic PCR set up requires several components and reagents:
- DNA template that contains the DNA region (target) to be amplified.
- Two primers that are complementary to a short segment of the sense and anti-sense strands
of the DNA target. - Heat resistant polymerase enzyme (i. e. Taq polymerase) with a temperature optimum at
around 72 °C. In case of RNA PCR, the enzyme mix also contains reverse transcriptase
enzyme, that transcribes DNA from the template RNA. - Deoxynucleoside triphosphates (dNTPs), the monomers from which the DNA polymerase
synthesizes a new DNA strand. - Buffer solution, providing a suitable chemical environment for optimum activity and
stability of the DNA polymerase. - NAase-free water
- In case of RNA template RNase inhibitor is added to the mixture to prevent the degradation
of the sample RNA by RNase enzymes.
What is Taq polymerase?
Heat resistant polymerase enzyme
In case of RNA PCR, the enzyme mix also contains?
reverse transcriptase enzyme, that transcribes CNA from the template RNA
What are the monomers from which the DNA polymerase synthesizes a new DNA strand?
Deoxynucleoside triphosphates (dNTPs)
What is the function of the buffer solution?
providing a suitable chemical environment for optimum activity and stability of the DNA plymerase
What happens in case of RNA template?
RNase inhibitor is added to the mixture to prevent the degradation of the sample RNA by RNase enzymes
What is added to the mixture to prevent the degradation of the sample RNA by RNase enzymes in case of RNa template?
RNase inhibitor
The PCR is commonly carried out in a reaction volume of?
10-200 microliter in small reaction tubes (0.2-0.5 ml volumes)in a thermal cycler
What is the function of the thermal cycler?
it heats and cools the reaction tubes to achieve the temperatures required at each step of the reaction
What are the reaction mixtures (master mix) used for DNA viruses?
- master-mix (25microl/reaction)
- H2O
- 5 x buffer
- dNTPs
- Primer F+R
- Taq polymerase
- Template DNA
What are the primers used for DNA viruses?
- Equine herpes virus 1: EHV1_if-EHV1_2r
- canine parvovirus: CPV_1f-CPV_23
- Aujeszky´s disease virus: PHV1_1f-PHV1_2r
what does PCR stand for? and in what virus is it used?
Polymerase chain reaction, DNA viruses
What does RT-PCR stand for? and in what viruses is it used?
Reverse transcription polymerase chain reaction, RNA viruses
What are the reaction mixtures (master mix) used for RNA viruses?
- master-mix
- H2O
- Buffer
- dNTPs
- Primer F+R
- RN-ase inhibitor
- Enzyme-mix
- Template RNA
What are the primers used for RNA viruses?
- Parainfluenza virus 3
- Newcastle disease virus
- Avian influenza virus
Why is positive control test used in the PCR?
- that the reaction is performed well
- the identification of the DNA produced in the sample tube
What does the positive control contain?
NA from a previously identified, specific viral NA
What happens if the positive control provides negative result?
the reaction should be repeated, and the result of the current test cannot be accepted.
How does the positive control identify the DNa produced in the sample tube?
during agarose gel electrophoresis, the size of the DNA product amplified in the sample tube is compared to the size of the DNA product amplified in the positive control tube.
What does it mean if the size of the DNA product in the sample tube is similar to the positive control?
means that the sample most likely contains the same viral Na as the positive control.
What does it mean if the size of the DNA product in the sample tube differs from the positive control?
that not virus-specific DNA was amplified in the sample.
What is the initialization step of the PCR procedure?
It is a single temperture step at a high temperature, that consists of heating the reaction to a temperature of 94-96*C for 1-15min, as required for heat activation of DNA polymerases
What are the 3 steps of the three-step cycle of the PCR?
- denaturation step
- annealing step
- extension/elongation step
Explain the Denaturation step:
at 94–98 °C for 20–60 seconds results in the unwinding of the DNA template helix, by disrupting the hydrogen bonds between complementary bases, yielding single-stranded DNA molecules (separation of the complementary DNA strands).
Explain the Annealing step:
The reaction temperature is lowered to 50–65 °C for 20–60 seconds allowing the annealing of the primers to the single-stranded DNA template. The temperature depends on the primers’ characteristics (melting temperature [Tm] determined by the nucleotide content). Stable DNA-DNA hydrogen bonds are formed only when the primer sequence very closely matches the template sequence.
Explain the Extension/elongation step:
the temperature at this step depends on the DNA polymerase used; Taq polymerase has its optimum activity temperature at 70–80 °C, commonly a temperature of 72 °C is used for this enzyme. At this step the DNA polymerase binds to the primer-template hybrid and synthesises a new DNA strand complementary to the DNA template strand by adding dNTPs that are complementary to the template in 5’ to 3’ direction. The extension time depends both on the DNA polymerase used and on the length of the DNA fragment to be amplified: at its optimum temperature, the DNA polymerase can polymerize approximately a thousand bases per minute.
How does the inwinding of the DNA template helix happen?
at 94-98*C for 20-60seconds (in the denaturation step). By disrupting the hydrogen bonds btw complemetary bases, yielding single-stranded DNA molecules
When are stable DNA-DNA hydrogen bonds formed?
only when the primer equence very closely matches the template sequence (in the annealing step)
The temperature of the Annealing step depends on?
the primers characteristics
The temperature of the Extension/elongation step depends on?
the DNA polymerase used; Taq
What does the extension time depend on?
both on the DNA polymerase used and on the length of the DNA fragment to be amplified.
How is the final elongation performed?
It is the next step after the last cycle is finished.
it is performed at 70-74*C for 5-15min to ensure that any remaining single-stranded DNA is fully extended.
How is the final hold performed?
it is the final step.
at 4*C for short-term storage of the reaction mixture
What happens with the RNA in case of the RT-PCR?
The RNA of interest is transcribes into DNA complement (cDNA) by the use of reverse transcriptase enzyme in the initial step performed at 50*C for 30 minutes before the polymerase activation step.
Steps of the PCR program used for DNA viruses:
- activation of polymerase
- seperation of DNA strains
- Primer annealing
- extension
- final extension
- storage
Steps of the PCR program used for RNA viruses:
(RT-PCR)
- RT
- denaturation of RTase, activation of polymerase
- separation of DNa strains
- primer annealing
- extension
- final extension
- storage
How can you check whether the PCR generated the anticipated DNA fragment?
i.e the sample is positive, agarose gel electrophoresis is used for seperation and visualization of the amplification products.
why is agarose gel electrophoresis ised in PCR?
used for seperation and visualization of the amplification products.
