2: PCR Variants (MIDTERMS) Flashcards
Identify the PCR variant:
a process that involves a reverse transcriptase (RTase), an enzyme that uses RNA as the template to make complementary DNA (cDNA)
Reverse Transcription
For how long does Reverse Transcription last?
3-4 hours
T or F
Reverse Transcription-PCR is the same with RT-PCR COVID-19 swab test
F (COVID uses Real-Time PCR)
What enzyme does Reverse Transcription-PCR use?
Reverse Transcriptase
T or F
PCR can amplify both complementary DNA and RNA
F (PCR can only amplify DNA because the polymerase being used is DNA polymerase which can only detect a DNA sample)
What is the needed temperature to activate Reverse Transcriptase
37°C
What is the main mechanism of Reverse Transcription?
use RNA Template → complementary DNA
What are the 2 primers used in Reverse Transcriptase-PCR?
- Oligo (dT) primers
- Random Primers
Primers used in RT-PCR:
→ choosy
→ used when the template is messenger RNA
(mRNA)
→ anneal when the template contains a polyadenylated tail (poly-A tail) which is present in most eukaryotic RNA at the 3’
→ 18-base-long single stranded poly dT sequences
Oligo (dT) primers
Primers used in RT-PCR:
→ Can be random hexamers or random decamers
→ Used if RNA is degraded RNA or has no poly- A tail
→ Any RNA template is used
→ For prokaryotic RNA or degraded RNA
→ 6 or 10-base long single-stranded
oligonucleotides of random sequences
Random primers
Which primer for RT-PCR is used when template is messenger RNA (mRNA)
Oligo (dT) primers
Oligo (dT) primers only anneal to the template when there is the presence of?
polyadenylated tail (poly-A tail)
This primer for RT-PCR is 18-base-long single-stranded poly dT sequences
Oligo (dT) primers
Which primer for RT-PCR is used when RNA is degraded RNA or has no poly- A tail?
Random primers
This primer for RT-PCR is 6 or 10-base long single-stranded oligonucleotides of random sequences
Random primers
What are the 2 ways to perform Reverse Transcriptase PCR?
- One-step
- Two-step
A way to perform RT-PCR where the primers, reverse transcriptase, and standard PCR reagents (buffer magnesium, dNTPs) are all combined in an epender tube and placed in PCR machine
One-Step
Identify what way to perform RT-PCR:
Advantage: quicker, less prone to contamination
Disadvantage: If error occurs, process is repeated
One-Step
A way to perform RT-PCR that involves 2 tubes and 2 steps:
1st tube is where RNA is converted to cDNA while 2nd tube if getting aliquot of DNA and adding standard PCR reagents
Two-Step
IDENTIFY WHAT TUBE IS USED in Two-step way of performing RT-PCR:
RNA is converted to cDNA; only the
primer and reverse transcriptase is added
1st tube
IDENTIFY WHAT TUBE IS USED in Two-step way of performing RT-PCR:
Getting an aliquot of the cDNA then adding standard PCR reagents
2nd tube
Two-step way of performing RT-PCR:
T or F
In the 1st tube of two-step, dNTP, buffer, primer, and reverse transcriptase is added
F (only the PRIMER and REVERSE TRANSCRIPTASE is added)
Identify what way to perform RT-PCR:
Advantage: more sensitive, easier to
troubleshoot, you already have a stock of cDNA
Disadvantage: prone to contamination
Two-Step
Identify the PCR variant:
→ Is a technique used to amplify multiple target sequences in a single PCR reaction using multiple primer sets.
→ This method is used to detect deletions, polymorphisms, mutations, etc.
→ This method is also used to detect different viral, bacterial, and other pathogens in a single tube.
→ consumes less time and effort in obtaining the results.
Multiplex PCR
T or F
In a traditional/conventional PCR, the ratio of sample and primer is 1:1
T bading ka
T or F
In a Multiplex PCR, there can be either a:
Multiple samples, 1 primer, 1 reaction tube w/ standard PCR reagents
OR
Multiple primers, 1 sample, 1 reaction tube w/ standard PCR reagents
T
Multiplex PCR:
Uses several sets of primers to amplify specific regions within a template
Single-template PCR reaction
Multiplex PCR:
Uses multiple templates and several primer sets in the same reaction tube.
multiple-template PCR reaction
→ This method is used to detect deletions, polymorphisms, mutations, etc.
Multiplex PCR
→ This method is also used to detect different viral, bacterial, and other pathogens in a single tube.
Multiplex PCR
Identify what PCR variant:
Advantages: Rapid, Save costly polymerase and template in short supply
Disadvantages: Difficult to optimize, Nag-aagawan reagents, Unable to mix organism with same amplicaon size, More complicated to develop, often is less sensitive than single-primer-set PCR
Multiplex PCR
T or F
In Multiplex PCR, if it is positive, 1 solid band forms with the 3 primers added
F (multiple bands form with 3 primers added)
T or F
In Conventional/Traditional PCR, if it is positive, 2 solid bands form
F (1 solid band)
In Multiplex PCR, if the sample if COVID-19, what band/s are present?
Red band only
T or F
Primers used in multiplex reactions must be selected carefully to have similar annealing temperatures and must be complementary
F (must NOT BE complementary to each other)
What temperature should be checked first in Multiplex PCR?
Melting temperature
T or F
Melting temperature should be close to annealing temperature, if not, the primer will not bind to the target sequence
T
Identify the PCR variant:
→ Is a modification of PCR that was designed to improve sensitivity and specificity
→ use of two primer sets (external primers and nested primers) and two successive PCR reactions.
Nested PCR
Nested PCR involves the use of primer sets which are?
- External primer
- Nested primer
What does Nested PCR require?
2 primer sets (external primers/nested primers) and 2 successive PCR reactions
T or F
Nested PCR is repeated 2x to increase sensitivity and specificity
T
Mechanism of Nested PCR?
1ST ROUND:
Outer primer bind to target template → Annealing → Extension → Amplified product
2ND ROUND:
Template for 2nd round is the amplified product of 1st round → Inner/Nester binds to target sequence → Annealing → Extension → Specified nested product
Identify the correct answer: Nested PCR
- Primer in 1st round _______
- Primer in 2nd round _______
a. Inner/Nested primer
b. Outer primer
c. Primase
d. Normal primer
- Primer in 1st round: b. Outer primer
- Primer in 2nd round: a. Inner/Nested primer
Nested PCR:
Increased sensitivity is attributed to what?
increased/high cycle (20 cycles on 1st round plus 20 cycles → It monitors the amplification of a targeted DNA on 2nd round)
Nested PCR:
Increased specificity is attributed to what?
increased/added primer (2 primers are used: outer primer and nested primer)
What are the 2 types of nested PCR?
- Traditional Nested PCR
- Semi-nested Asymmetrical PCR
Identify what type of nested PCR:
The target is amplified. Open the tube and use the first product for the 2nd round. Add the inner primer then amplify.
Traditional Nested PCR
Identify what type of nested PCR:
Almost the same as traditional but less time and less cycle because the inner and outer primers are combined in 1 reaction or tube
Semi-nested Asymmetrical PCR
Use this card to familiarize the application of Nested PCR
- Detection of Rickettsia, Bartonella, and similar organisms in blood (bacteremia) and tissues
- Detection of herpesvirus and enterovirus in the CSF
- Detection of M. tuberculosis in sputum sample
What is the recommended PCR variant when the quality of template is not that great? (Its concentration is low and less pure)
Nested PCR (will improve its sensitivity and specificity)
Identify the PCR variant:
→ It monitors the amplification of a targeted DNA molecule during the PCR (i.e., in real time).
→ Quantifying the amplification of a DNA as it occurs
qPCR
qPCR:
implies that data collection and
analysis occur as a reaction proceeds
Real time
T or F
Mechanism amplification of DNA in qPCR detects the accumulation of amplicon after each thermal cycle in real time
T
T or F
Process and primer used for qPCR is the same as traditional PCR
F (Process is same, but qpCR uses FLUOROMETRIC PROBES to bind target DNA)
What is used in qPCR to bind target DNA during the annealing phase of PCR?
Fluorometric probes
qPCR:
are then displaced and cleaved, which allows the emission of fluorescent dye upon target extension.
fluorometric probes
qPCR:
What happens fluorometric probes after binding to target DNA?
Displaced and cleaved–allowing emission of fluorescent dye upon target extension
T or F
The dye in qPCR cannot ultimately be detected, it is a slow process.
F (CAN BE DETECTED in order to determine the success of the amplification reaction)
qPCR:
Initial quantity of nucleic acid present in the sample is depicted in?
Graphical form
Identify the pCR variant:
Advantages:
→ Starting DNA concentration can be determined with accuracy and high sensitivity.
→ Can either be qualitative or quantitative
→ Less contamination
→ Detection of PCR inhibitors
→ Specificity
Disadvantages:
→ Expensive, sample volume is expended
qPCR
T or F
The concentration of the unknown nucleic acid sample is determined by comparing a peak curve of known concentrations to the amplification plot of the sample.
F (The concentration of the unknown nucleic acid sample is determined by comparing a STANDARD CURVE of known concentrations to the amplification plot of the sample.)
T or F
Conventional PCR is quantitatively accurate
F (SEMIQUANTITATIVE ONLY; evaluate the end product of a PCR reaction, which can be qualitatively but NOT quantitatively accurate)
What are the 2 common methods for detection in qPCR/real-time pcr?
- Non-specific fluorescent dyes
- Sequence-specific DNA probes
2 common methods for detection in qPCR/real-time pc:
intercalate with any double-stranded DNA.
Non-specific fluorescent dyes
2 common methods for detection in qPCR/real-time pc:
consisting of oligonucleotides that are labelled with a fluorescent reporter, which permits detection only AFTER hybridization of the probe with its complementary sequence
Sequence-specific DNA probes
2 common methods for detection in qPCR/real-time pc:
T or F
Sequence-specific DNA probes consisting of oligonucleotides that are labelled with a fluorescent reporter, which permits detection only during hybridization of the probe with its complementary sequence.
F (permits detection only AFTER HYBRIDIZATION)
Mechanism of qPCR/Real-time PCR?
Fluorometric probes bind target DNA → Fluorometric probes are then displaced and cleaved → Emission of fluorescent dye upon target extension.
What are the terms to remember in qPCR/Real-Time PCR
- Baseline
- Threshold
- Cycle Threshold
Definition of terms in qPCR/real-time PCR:
Signal level during the initial cycles of PCR, usually cycles 3 to 15, in which there is little change in fluorescent signal
Baseline
Definition of terms in qPCR/real-time PCR:
Can be equated to the background or the “noise” of the reaction.
low-level signal of the baseline
T or F
In baseline of qPCR/real-time PCR, there is fluorescent signal and it is counted
F (not counted because it’s only bg noise)
Definition of terms in qPCR/real-time PCR:
→ level of signal that reflects a statistically significant increase over the calculated baseline signal
→ above the baseline/background noise and below the plateau phase.
Threshold
What are the different parts of graph in qPCR?
- Exponential phase
- Linear phase
- Plateau phase
Different parts of graph in qPCR:
start of amplification
Exponential phase
Different parts of graph in qPCR:
amplification is equal to the amount of reagents
Linear phase
Different parts of graph in qPCR:
all reagents have been consumed; hence the flat line
Plateau phase
Definition of terms in qPCR/real-time PCR:
→”what cycle number did the threshold cross”
→ cycle at which the amplification plot crosses the threshold
→ significant detectable increase in fluorescence
→ can be a fractional number and allows calculation of the starting template amount.
Cycle Threshold
T or F. For each gene that passes thru the threshold, it produces a numerical value and an indicator that it’s positive.
T
Every time you run a qPCR, what do you need to add in order for it to tell if the sample is valid.
internal control
The amplicons are quantitated by the qPCR using a?
fluorescent signal
T or F. The lower the concentration of the target, the higher the fluorescent signal.
F (directly proportional)
T or F. If the concentration target is higher, the cycle needed to reach the threshold value is LOWER.
T (always inverse)
For example, Patient A has a CT value of 65. Patient B has a CT value of 59. Both of them has SARS-Cov. Who among them has more virus in their body?
Patient B
What are the Real Time PCR Detection Methods?
- dye-based
- probe-based
Where does the dye-based bind to?
ds DNA
T or F. The probe-based attach theirselves between the bases to emit fluorescence.
F (dye-based because they intercalate between the dsDNA during the extension phase so that it can go in the middle of the strand to emit)
This Real Time PCR detection method is for specific PCR product detection ONLY
Fluorophore-linked probes (probe-based)
This Real Time PCR detection method is for or both specific and nonspecific detection of amplified products
ds DNA binding dye chemistry (dye-based)
Which among does not belong
ds DNA binding dye chemistry (dye-based)
A. Cost-effective
B. Specific and nonspecific detection
C. Harmless
D. Toxic
C
SYBR Green or Eva Green
less inhibitory to PCR
E
SYBR Green or Eva Green
used to know if the specific product or there’s a presence of a nonspecific product
S
SYBR Green or Eva Green
unsuitable for high-resolution melt curve analysis (HRM)
S
SYBR Green or Eva Green
suitable for qPCR using a fast cycling protocol
E
SYBR Green or Eva Green
not suitable for alkaline medium
S
SYBR Green or Eva Green
causes mispriming if too much is add
S
this analysis consists of applying heat to the sample (from 50°C to 95°C) and monitoring the fluorescence emission during the process
Melting Curve Analysis
T or F. A single peak in the melting curve analysis means there’s a specific product while multiple peaks reveals nonspecific amplification
T
All of this are true about dye-based method except:
A. can intercalate with a primer dimer
B. melting curve analysis is not neccessary
C. more affordable than probe-based
D. not that specific since it binds to dsDNA
E. NOTA
B
small fluorescent molecules that are attached to oligonucleotides in order to function as probes
Fluorophores
Types of Fluorophores Used in qPCR:
Reporter (donor) and quencher (acceptor)
T or F. The reporter and donor needs to be in close proximity to produce fluorescence.
T
T or F. Fluorescence is caused by energy transfer.
T
These are oligonucleotides that combine a primer and probe in a single molecule.
primer probes
Fluorescence emitted from primer-probes is detected and measured during the what phase of the qPCR?
denaturation or extension
Oligonucleotides with an attached-donor and/or acceptor fluorophore.
Probes
mechanism of action relies on the 5–3′ exonuclease activity of Taq polymerase, which degrades the bound probe during amplification
Hydrolysis probes
the fluorescence emitted by binding ________________ can be measured either during the annealing or the extension phase.
Hybridization probes
Arrange in sequence
(A) Probe cleaved by 5’-3; nuclease activity of polymerase
(B) During PCR, probe hybridizes to the target
(C) Fluorescence signal form your reporter molecule will be detected
(D) Probe is displaced by your primer during extension
B D A C
T or F. qPCR enables the absolute and reproducible quantification of target
nucleic acids
F (dPCR)
ensures the amount of DNA after amplification is complete and then determines the fraction of replicates
dPCR
TOF. dPCR allows measurement of absolute quantification of amplicons
T
This is representative of an endpoint measurement as it requires the observation of the data after the experiment is completed.
dPCR
TOF. This “real-time” aspect (qPCR requires stops in the experimental process) of qPCR may theoretically affect results due to the stopping of the experiment.
T
This is mostly used if the experiment is about cancer or mutation.
dPCR
called as the “gold standard”
qPCR
enables absolute quantification (standard curves and reference ranges to count the concentration of the target are not needed, unlike in qPCR), high tolerance to PCR inhibitors (not easily affected if there are inhibitors or contaminants)
dPCR
this is accomplished by capturing or isolating each individual nucleic acid molecule present in a sample within many chambers, zones, or regions that are able to localize and concentrate the amplification product to detectable levels.
dPCR
All of this are Isothermal Amplification Techniques except:
- Helicase-Independent Amplification (HIA)
- Nucleic acid sequence-Based Amplification (NASBA)
- Strand Displacement Amplification (SDA)
- Rolling Circle Amplification (RCA)
- Loop-Mediated Isothermal Amplification (LAMP)
- Recombinase Polymerase Amplification (RPA)
- Helicase-Independent Amplification (HIA)
[it should be - Helicase-dependent Amplification (HDA)]
T or F. Other Isothermal Amplification Techniques different in terms of number of enzymes to be used, number of primers/probes, reaction time, portability, and availability
T
specific, simple, rapid and cost-effective nucleic acid amplification method.
LAMP PCR
The LAMP-PCR relies on the auto-cycling strand displacement deoxyribonucleic acid (DNA) synthesis which is carried out at a constant temperature water bath/heat block in the presence of what type of polymerase?
Bacillus stearothermophylus(Bst) DNA polymerase
What does the presence of turbid mean in a LAMP PCR?
positive because of the addition of Magnesium pyrophosphate
Without fluorescence, the LAMP-PCR cn be detected as?
negative
A change in color in the LAMP PCR indicates?
positive
LAMP usually uses 4-6 primers, these are?
FIP (Forward Inner Primer)
FP (Forward Primer)
BIP (Backward Inner Primer)
B3 (Outer Primer)
+ loop primers (FIP or etc)
qPCR: Primer Probes
Hairpin Primer-Probes involve what blocker?
hexaethylene glycol
