PCR

Question 1

What are the advantages of real-time pcr?

Answer 1

The advantages of real-time PCR include:

The ability to monitor the progress of the PCR reaction as it occurs in
real time

The ability to precisely measure the amount of amplicon at each cycle

An increased dynamic range of detection

The combination of amplifi cation and detection in a single tube, which eliminates post-PCR manipulations

Question 2

What are the main steps of PCR?

Answer 2

There are three major steps that make up a qPCR reaction. Reactions are generally run for 40 cycles.

Denaturation - The temperature should be appropriate to the poly-merase chosen (usually 95°C). The denaturation time can be increased if template GC content is high.

Annealing - Use appropriate temperatures based on the calculated
melting temperature (Tm) of the primers (5°C below the T
mof the primer).

Extension - At 70–72°C, the activity of the DNA polymerase is optimal,
and primer extension occurs at rates of up to 100 bases per second.
When an amplicon in qPCR is small, this step is often combined with the annealing step using 60°C as the temperature

Question 3

What is two-step PCR?

Answer 3

Reverse transcriptase first used to convert RNA to DNA

Then PCR happens as normal

This can happen in a separate process, creating a two-step process.

Alternatively this can be done as a one-step process, in which reverse transcriptase and DNA polymerase are in the same reaction tube. RT is eventually inactivated by high-temperature used during DNA polymerase stage

Question 4

What are the ingredients required for PCR?

Answer 4

DNA template

DNA polymerase

dNTPs nucleotides

primers

Magnesium chloride/ sulphate buffer

Reverse transcriptase - if RNA needs converted to DNA template

Question 5

What are key points when designing primers for PCR?

Answer 5

Need to be specific to the target

Should be short in length and be compatible (within 5degC) with melting temperature of DNA template

Question 6

What is baseline PCR signal?

Answer 6

The baseline of the real-time PCR reaction refers to the signal level during the initial cycles of PCR, usually cycles 3 to 15, in which there is little change in fluorescent signal. The low-level signal of the baseline can be equated to the background or the “noise” of the reaction

The baseline in real-time PCR is determined empirically for each reaction, by user analysis or automated analysis of the amplification plot. The baseline should be set carefully to allowaccurate determination of the threshold cycle (Ct), defined below. The base-
line determination should take into account enough cycles to eliminate the background found in the early cycles of amplification, but should not include the cycles in which the amplification signal begins to rise above background

Question 7

What is the PCR threshold?

Answer 7

The threshold of the real-time PCR reaction is the level of signal that reflects a statistically significant increase over the calculated baseline signal. It is set to distinguish relevant amplification signal from the background. Usually, real-time PCR instrument software automatically sets the threshold at 10 times the standard deviation of the fluorescence value of the baseline. However, the positioning of the threshold can be set at any point in the exponential
phase of PCR

Question 8

What is the PCR Ct - threshold cycle

Answer 8

The threshold cycle (Ct) is the cycle number at which the fluorescent signal of the reaction crosses the threshold. The Ct is used to calculate the initial DNA copy number, because the Ct value is inversely related to the amount of starting template. For example, in comparing two real-time PCR reactions, one with twice as much starting template as the other, the reaction with the 2X starting amount will have a Ct one cycle earlier (Figure 3). This assumes that
the PCR is operating at 100% efficiency (i.e., the amount of product doubles perfectly during each cycle) in both reactions

Question 9

What is a PCR standard curve?

Answer 9

A dilution series of known template concentrations can be used to establish a standard curve for determining the initial starting amount of the target template or for assessing the reaction efficiency

The log of each known concentration in the dilution series (x-axis) is plotted against the Ct value for that concentration (y-axis).

From this standard curve, information about the performance of the reaction as well as various reaction parameters (including
slope, y-intercept, and correlation coefficient) can be derived.

The concentrations chosen for the standard curve should encompass the expected concentration range of the target.

Standard curve means we can do absolute quantification - we know precisely how many copies are in a sample

Question 10

What is the correlation coefficient R2?

Answer 10

The correlation coefficient is a measure of how well the data fi t the standard curve. The R2 value reflects the linearity of the standard curve. Ideally, R2= 1, although 0.999 is generally the maximum value

Question 11

Why does PCR quantification need to occur during the exponential phase?

Answer 11

It is important to quantify your real-time PCR reaction in the early part of the exponential phase as opposed to in the later cycles or when the reaction reaches the plateau. At the beginning of the exponential phase, all reagents are still in excess, the DNA polymerase is still highly efficient, and the product,
which is present in a low amount, will not compete with the primers’ annealing capabilities. All of these things contribute to more accurate data

Question 12

What is PCR efficiency?

Answer 12

Maximal efficiency means that each PCR cycle results in duplication of amplicon

However most reactions are satisfactory at 90% amplification. Can be due to various issues such as PCR inhibitors, non-optimal reagent concentrations, enzyme quality

Question 13

What is a melting curve?

Answer 13

A melting curve charts the change in fluorescence observed when double-stranded DNA (dsDNA) with incorporated dye molecules dissociates, or “melts”, into single-stranded DNA (ssDNA) as the temperature of the reaction is raised.

For example, when double-stranded DNA bound with SYBR®
Green I dye is heated, a sudden decrease in fluorescence is detected when the melting point (Tm) is reached, due to dissociation of the DNA strands and subsequent release of the dye.

Melting temperature is affected by DNA template length, so can identify if you have a mutant or similar target in sample

Question 14

What is a common PCR fluorescent agent?

Answer 14

SYBR-green binds to dsDNA, and intensity increases with the amount of dsDNA present

as dsDNA accumulates, the dye generates a signal that is proportional to the DNA concentration and can be detected using real-time PCR instruments

lacks some specificity, as binds to all dsDNA present, not just the target DNA

Question 15

As opposed to using a fluorescent agent for detection, what are other options for detection?

Answer 15

Primer-based detection - fluoresces when attaches to specific DNA primer. More expensive, but can be more specific

Probe-base detection system - TaqMan. More expensive, but can be more specific as only binds to probe

Question 16

Why is Uracil DNA glycosylase (UDG) sometimes used in PCR reactions?

Answer 16

Uracil DNA glycosylase (UDG) is used to reduce or prevent DNA carryover contamination between PCR reactions by preventing the amplification of non-template DNA in real-time PCR. The use of UDG prior to a PCR reaction reduces false positives, in turn increasing the efficiency of the real-time PCR reaction and the reliability of your data

Carryover contamination of amplification products and primers from
previous PCRs; this is considered to be the major source of false positive PCR results

The contaminants may be carried over from previous amplification reactions due to aerosolizaton, contaminating pipettes, surfaces, gloves and reagents

Question 17

How does Uracil DNA glycosylase (UDG) work at reducing contamination in PCR?

Answer 17

The use of UDG for carryover prevention begins with the substitution of dUTP for dTTP in real-time PCR assays. Subsequent real-time PCR reaction mixes are then treated with UDG, which degrades any contaminating uracil-containing PCR products, leaving the natural (thymine-containing) target DNA template unaffected.

With standard UDG, a short incubation at 50°C is performed prior to
the PCR cycling to allow the enzyme to cleave the uracil residues from any contaminating DNA.

The removal of the uracil bases causes fragmentation of the DNA, preventing its use as a template in PCR. The UDG is then inactivated in the ramp up to 95°C in PCR. A heat-labile form of the enzyme is also available, which is inactivated at 50°C, allowing its use in one-step qRT-PCR reaction mixes

Question 18

Before PCR begins, extraction occurs

What is a common method of purifying nucleic acid?

Answer 18

Phenol-based organic extraction

During sample lysis, phenol and guanidine disrupt cells and dissolve cell components, while maintaining integrity of nucleic acids by protecting them from RNases

Question 19

What are the benefits of one-step PCR?

Answer 19

The benefits of one-step qRT-PCR include the following:

Contamination prevention
—The closed-tube system prevents the introduction of contaminants between the RT and PCR stages.

Convenience
—The number of pipetting steps is reduced and hands-on time is minimized.

High-throughput sample screening
—For the reasons mentioned above.

Sensitivity
—The one-step reaction may be more sensitive than the
two-step because all the first-strand cDNA created is available for real-time PCR amplification

Question 20

What are the draw backs of one-step PCR?

Answer 20

The drawbacks of one-step qRT-PCR are:

Increased risk of primer-dimers
—Forward and reverse gene-specific primers, present from the start in one-step reactions, have a greater
tendency to dimerize under the 42°C to 50°C reverse transcription conditions. This can be especially problematic in reactions involving DNA-binding dyes.

Unavailability of cDNA for other real-time PCR reactions
—One-step reactions use all the cDNA from the RT step, so if the reaction fails, thesample is lost

Question 21

What are the benefits of two-step PCR?

Answer 21

The benefits of two-step qRT-PCR include:

cDNA may be archived and used for additional real-time PCR reac-
tions—Two-step qRT-PCR produces enough cDNA for multiple real-
time PCR reactions, making it optimal for rare or limited samples.

Sensitivity
—The two-step reaction may be more sensitive than the
one-step because the RT and qPCR reactions are performed in their
individually optimized buffers.

Multiple targets
—Depending on the RT primers used, two-step qRT-PCR can allow you to interrogate multiple genes from a single RNA sample

Question 22

What are the draw backs of two-step PCR?

Answer 22

The drawbacks of two-step qRT-PCR are:

RT enzymes and buffers can inhibit real-time PCR
—Typically, only 10% of cDNA synthesis reaction is used in real-time PCR, because the reverse transcriptase and associated buffer components may inhibit the Taq DNA polymerase if not diluted properly. The specific level of inhibition will depend on the reverse transcriptase, the relative abundance of the target, and the robustness of the amplification reaction.

Less convenient
—Two-step reactions require more handling and are less amendable to high-throughput applications.

Contamination risk
—The use of separate tubes for each step increases the risk of contamination.

Question 23

What is PCR sensitivity?

Answer 23

PCR sensitivity is sometimes incorrectly thought of as how early a target Ct appears on amplification plot

However, true gauge of sensitivity of an assay is whether a given low amount of template fits to the standard curve while maintaining a desirable efficiency. The most dilute sample that fits determines reaction sensitivity

Question 24

What are causes of poor PCR efficiency?

Answer 24

Inhibition

poor RNA/ DNA quality

high template concentration - not enough primers/ enzymes for this

carryover from nucleic acid purification

Question 25

Why should you not compare PCR results between different systems?

Answer 25

Different systems might have different PCR efficiencies, different fluorescent analysis, and different standard curves

So results between labs may not be 100% comparable

Question 26

What are the phases of PCR?

Answer 26

Linear phase

Exponential phase

Plateau phase

Question 27

PCR results for covid has high CT value

What are possible explanations for this?

Answer 27

Inadequately collected sample or degraded sample

Pre-symptomatic - about to develop symptoms

Asymptomatic -unknown infectivity risk

Symptomatic -
recovery phase
chronic infection due to immunocompromised

Question 28

PCR run with late CT result detected

What are possible explanations for this?

Answer 28

if you use sequence-specific probes than any ct-value (no matter how large) tells you that the specific sequences has been amplified (and tus was present in the sample).

If you use dsDNA-specific probes (like SYBR-Green) a ct value may stem from some unspecific amplification product.

One would perform a melting curve analysis to check if the correct sequence was amplified.