Definitions and Explanations
What is the ‘thermal protocol’?
Thermal protocol is a PCR program/protocol required to carry out the reaction for denaturation and amplification of the DNA. It consists of repeating heating and cooling cycles
What is the ‘Ct’?
The Ct value is defined as the number of cycles required for fluorescent signal to cross the threshold (exceeds background level) for being detectable.
What is the ‘double-labeled probe’?
It is a segment of DNA, with a sequence complementary to the target DNA region. It is known as a TaqMan probe that is labeled with a fluorescent reporter molecule at the 5’end and with a quencher molecule at the 3’end. It is hydrolyzed by the 5' endonuclease activity of Taq Polymerase during PCR and results in increased fluorescence.
What is the ‘Tm (melting temperature)’?
The melting temperature(Tm) is the temperature at which one half of the DNA duplex will dissociate to become single stranded. It mainly depends on the base sequence and different DNA molecules with different base sequences have differ in their Tm (melting temperature) values.
What is the ‘melting curve analysis’?
‘Melting Curve Analysis’ is applied in the analysis where SYBR-Green and similar fluorescent dyes used, in order to confirm the true amplification of target DNA region. In a melting curve analysis; a graphic representing the change of fluorescent signal due to the dissociation-characteristics of double-stranded DNA is generated during heating. Denaturation of the DNA stands is monitored with the help of peaks caused by the sudden decrease of fluorescent signal during melting curve analysis.
What is the ‘negative control’?
It is named as a control which includes each reaction ingredients excluding the target DNA template. Every real-time PCR assay needs to include a negative control reaction to check the possible contamination and, confirm the reliability of the positive results.
What is the ‘positive control’?
It is a control reaction which the positive result is definitely expected. It helps to find out the problems due to application and used material. İf positive control reaction does not come out with positive results, that experiment would be no reliable and repeated.
What is the ‘Nucleic acid Standard’?
It is the known amount of nucleic acid sample, to be used in quantitative analysis. Standards only represent the amount of target gene/DNA/RNA of pathogen that is aimed to be quantified. As sometimes it is being confusing, standards have no relation with internal control. The standard curve cannot be achieved through the filter where internal control detected.
What is the ‘internal control’?
It is to amplify a different region then the target site. Two different PCR reactions are carried out in a single tube. By applying an internal control, the reliability of the test is checked and the false negative results are prevented. Internal control controls the reliability of nucleic acid isolation and/or PCR reaction depending in which process it is used. Not to obtain the detectable fluorescent signal from test probe assay while having detectable signal from internal control assay proves the samples as a negative. In a PCR assay, if there is no amplification of target pathogen/gene or no signal increase in internal control, the experiment should be repeated. If target pathogen/gene is amplified while internal control is not, there is no need to repeat the experiment. Because the internal control is designed for smooth increases to not affect the specificity of test. As both target and internal control PCR compete in the same tube, it is predicted that high positive test samples dominate the internal control. It is acceptable and satisfying to have increased signal of internal control in low positive and negative samples for the fourth standard.
Are there differences about fluorescent measurement and product detection between the double labeled probe and SYBR-Green kits?
Yes. For kits that are based on double labeled hydrolysis probes (a.k.a. TaqMan probes), the fluorescent measurement is applied during the ‘annealing+elongation’ step when the Taq Polymerase enzyme cuts the probe during the annealing + elongation’ step of the PCR. As SYBR-green kits don’t have particular sequence specificity, they may monitor undesirable double stranded DNA structures like primer dimers rather than target product. Because of that, in SYBR-green kits, a measurement must be carried out in temperature when primer dimers are in single stranded form and target PCR product is in double stranded form. This temperature comes up to the melting temperature ™ or 1-2 degrees below to it. So on, fluorescent signal increases caused by primer dimers are prevented as much as possible and the product increase is monitored specifically.
In kits based on double-labeled probes; base sequence of the PCR product is monitored by sequence specific fluorescent labeled probes resulting in specific detection of the amplified products while there is no specific detection of PCR products in kits using SYBR-green, as it anneals to all double stranded DNA molecules without sequence specificity. Those, in order to detect specifically the signal increase caused by PCR product, primers without seconder structures should be selected or analysis must be carried out in a temperature range higher than the melting temperature of primer-seconder structures. Specific detection of the PCR product can be definitely obtained by melting curve analysis.
At least how many standards required for quantitative kits?
To generate the standard curve, at least 3 standards required.
What is the reason to have 15 minutes denaturation step in some thermal protocols?
Some of the Taq polymerase enzymes used in various kits are known as ‘HotStarTaq Polymerase’ and need 95°C to be activated. This polymerase provides an advantage in one step RT-PCR kits:
HotStarTaq Polymerase” is modified to stay inactive during the reverse transcription (RT) step. So that the amplification of wrong paired bases or primer dimers are prevented. The 95°C denaturation step which is named as ‘Hot Start’ also causes to the total inactivation of reverse transcription enzyme resulting in exact differentiation between PCR and reverse transcription steps.
How the PCR efficiency is calculated?
PCR efficiency is directly proportional with the slope of standard curve (Efficiency= [10(-1/slope)] – 1). According to this equation, a slope value of ‘-3,332’ means 100% of efficiency. If the mean value of slope is greater than 3,332, efficiency will be lower than 100% and if the mean value of slope is lower than 3,332, efficiency will be greater than 100%. In both cases, the reaction must be optimized. The amount of template will be doubled with an efficiency of 100%. But in reality, none of the PCR reactions complete this value. The length of the target site (amplicon), seconder structures and the quality of primers can have effect on PCR efficiency.
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