We have seen in a previous post, that the PCR is one of the most used techniques in molecular biology. It is used to amplify a specific DNA region.

PCR can be divided into three different steps; Denaturation, Annealing and Extension. These three steps are repeated in cycles to synthesize a large number of copies of the required DNA region.

(Just for info: Read our post on PCR.)

Fig 1: Amplification of the specific DNA region (Image Source: Rodriguez-Lazaro & Hernández, 2013)

In the traditional PCR, the products are detected, after the completion of all the cycles of PCR in thermocycler, in form of bands in the electrophoresis gel. Therefore, it is also known as end-point PCR. It’s use is limited as qualitative and semi-quantitative analysis.

In a modification of the PCR, named real time PCR, the increase in the product can be detected at the same time as the PCR process is going on, that is, the detection is happening real time.

(Just for info: According to Oxford dictionary, real-time means the actual time during which a process or event occurs.)

For the real-time detection, the copy of target DNA synthesized during PCR cycle, is labelled using a fluorescent reporter. The fluorescent reporters have fluorescent dye in their structure, that emit fluorescense.

The fluorescent reporters are designed such that they fluoresce on binding the DNA, hence the fluorescent signal is in direct proportion to the number of DNA copy. That is, as the number of DNA copies increases the fluorescent signal increases. Hence, the fluorescent signals allow the real time quantification of the amplified PCR products, and the technique is also known as quantitative PCR (qPCR).

For the simultaneous detection of the fluorescence, the thermal cycler has a detector attached. The fluorescent signal is then plotted against the cycle number to generate an amplification curve or plot.

Fig 3: LightCycler® 96 Instrument by Roche for real time PCR.

The amplification curve allows the visualisation of progression of RT-PCR, that can be divided into four stages: linear ground phase, exponential phase (log phase) and plateau phase.

 

 

Fig 4: Amplification Curve: Fluorescense (Relative Fluorescense Unit) on Y axis is plotted against the number of cycles on X axis (Image Source: Medina-Hernandez et al, 2013).

In the first phase (see red curve in fig 5), the linear ground phase, there is minimum background fluorescence emitted by the fluorescent reporters, so called baseline fluorescence. This baseline fluorescence is emitted even in the absence of the DNA template. The second phase, the exponential phase, is where fluorescent signal increases significantly above the background, due to the increase in the copies of the target DNA (amplicon) as the amplification takes place in each cycle. The plateau phase occurs as substrates and other additives get exhausted and fluorescent signal no long increases.

 

 

Fig 5: The typical amplification plot of the real time PCR. The fluorescence (RFU) or the amount of DNA (Y axis) is plotted against the cycle number (X axis) (Image Source: Modified from Nassiri et al, 2017).

Some important terms in the analysis of qPCR are:

– Baseline

As mentioned above, the baseline in real time PCR is the low level signal detected during the few initial cycles. It may be considered as background noise It occurs due to the presence of the fluorescent dyes in the solution and exists even in the absence of the template DNA (blue horizontal line in Fig 5).

– Threshold

The threshold is the level of fluorescent signal, which marks that the fluorescence is due to amplification product, and is not baseline signal. It helps distinguish the statistically significant fluorescent signal due to amplification, from the background noise (dotted horizontal line in fig 5).

– Threshold cycle (Ct)

The cycle number at which the fluorescent signal rises above the threshold value, is known as the threshold cycle (Ct). It is used to calculate the initial DNA copy number as Ct value is inversely proportional to the initial amount of the DNA copies in the solution/ sample. That is higher the amount of target DNA, faster the threshold is reached. (It is the point where the amplification curve meets the threshold, fig 5).

– Standard curve

Standard curve is obtained when the log of known concentration of DNA in a series of the dilution (x-axis) is plotted against the Ct value for that concentration (y-axis) (see fig 6). It helps in calculating the concentration of the DNA in unknown/test sample from its Ct value obtained from the qPCR.

Fig 6: Standard curve was plotted between Ct values obtained from dilution of standard plasmid (pTiLV) against calculated log copy number.(Image Source: Tattiyapong et al, 2017)

It is also used to derive other information like the slope, y-intercept and the correlation coefficient.

– Melting curve (dissociation curve):

A melting curve reports the change in fluorescence when dsDNA, “melts” into single-stranded DNA (ssDNA) as the temperature of the reaction is raised. The fluorescence decreases as the temperature increases due to dissociation of the fluorescent reporters.

The melting point (Tm) is the point at which half the probes (or dye) have melted off the DNA. This value is obtained by plotting the fluorescence against the temperature.

Each ds DNA molecule has characteristic Tm, as it depends on length, GC content, and the presence of base mismatches. Melting curve analysis also gives information about the primer-dimer and reaction specificity.

Fig 7: The melting curve as reported by Prada-Arismendy & Castellanos, 2011.

Shown in the fig 7, is the melting curve analysis performed by Prada-Arismendy & Castellanos, 2011, during a real time PCR assay. In their analysis, the dissociation temperature range was from 63°C to 91.9°C. The dotted line represents the fluorescent signal decrease during the heating. Some peaks can be seen; left peak at 72°C is the dissociation curve of primer dimers and ones in the right at 81.5°C are dissociation curve of two specific amplification products.

(Just for info: Read the paper by Prada-Arismendy & Castellanos, 2011 titled ‘Real time PCR. Application in dengue studies’.)

In the next post, we describe the various fluorescent reporters used in the real time PCR.

This is all for this post. Hope u like this post, if yes please comment, like and share!!

Also follow us on Facebook, Twitter, Instagram or send an email to thebiotechnotes@gmail.com.

Have a nice day!

Thank you!!

Read other posts by The Biotech Notes:

Mutation: Different Types.

Flow cytometer

Immunoprecipitation- P1

— —

References:

Bass et al (2010) The Vector Population Monitoring Tool (VPMT): High-Throughput DNA-Based Diagnostics for the Monitoring of Mosquito Vector Populations. Malaria research and treatment. 2010. 190434. 10.4061/2010/190434.

Fraga et al (2014) Current Protocols Essential Laboratory Techniques. 10pp.10.3.1-10.3.40 .1002/9780470089941.et1003s08.

Hart et al (2001). Novel Method for Detection, Typing, and Quantification of Human Papillomaviruses in Clinical Samples. Journal of clinical microbiology. 39. 3204-12. 10.1128/JCM.39.9.3204-3212.2001.

Medina-Hernandez et al (2013). Effects and Effectiveness of Two RNAi Constructs for Resistance to Pepper golden mosaic virus in Nicotiana benthamiana Plants. Viruses. 5. 2931-45. 10.3390/v5122931.

Nassiri et al (2017) Evaluation of different statistical methods using SAS software: an in silico approach for analysis of real-time PCR data. Journal of Applied Statistics, DOI: 10.1080/02664763.2016.1276890

Overbergh et al (2017) Chapter 4 – Quantitative Polymerase Chain Reaction. Molecular Diagnostics (Third Edition): 41-58.

Prada-Arismendy & Castellanos (2011) Real time PCR. Application in dengue studies. Colombia Médica 42(2).

Pryor & Wittwer. Real-time polymerase chain reaction and melting curve analysis. Methods Mol Biol. 2006;336:19‐32. doi:10.1385/1-59745-074-X:19

Rodriguez-Lazaro & Hernández (2013). Real-time PCR in Food Science: Introduction. Current issues in molecular biology. 15. 25-38.

Sugden (2007). Quantitative PCR.Medical Biomethods Handbook. DOI: 10.1385/1-59259-870-6:327.

Tattiyapong et al (2017). Development and validation of a reverse transcription quantitative polymerase chain reaction for tilapia lake virus detection in clinical samples and experimentally challenged fish. Journal of Fish Diseases. 41. 10.1111/jfd.12708.

Thelwell et al. (2000) Mode of action and application of Scorpion primers to mutation detection. Nucleic Acids Res. 28(19): 3752–3761.