PCR or polymerase chain reaction, is a biotechnological tool to amplify a gene of interest. Simple PCR is used for the detection and amplification of the gene. There are various advanced methods of PCR, which are used for various biotechnological, diagnostics and research purposes. qPCR or quantitative PCR is also known as real-time PCR. qPCR is used to quantify the nucleic acids. The amplification of the DNA molecule can be monitored during the PCR, i.e. in real time. RT-PCR is referred to as a reverse transcription polymerase chain reaction. It utilises both processes, i.e. reverse transcription and polymerase chain reaction. It is used to detect and measure the amount of RNA.
Difference between PCR, RT-PCR and qPCR
The table below shows the main differences between PCR and qPCR or real-time PCR.
|PCR is used to analyse a short stretch of DNA by amplification||It is an advanced PCR method which is used to amplify as well as quantify the amount of DNA|
|In PCR, primer is used for polymerisation||In qPCR, primer, as well as fluorescent probes or dyes, are used|
|Results are investigated by gel electrophoresis||Fluorescence emitted by dye or probe is recorded during the PCR process|
|The data is recorded at the end of the process||The data is recorded during the amplification process at the exponential phase|
|Ethidium bromide is used to stain the DNA fragments||Fluorescent dyes or DNA probes labelled with a fluorescent reporter are used|
|It is a low-resolution technique||It is a high-resolution technique|
|Distinct bands of various DNA fragments can be seen on agarose gel||Different peaks related to different DNA fragments are seen during qPCR|
|Conventional PCR takes more time to generate a result||qPCR takes less time to deliver the result|
|It is used to detect the presence or absence of DNA in the sample, detect gene mutations and amplify the template for DNA sequencing||It is used to quantify the DNA present in the sample. It is used to analyse gene expression, identification and detection of pathogens, quantification and identification of mutation|
In RT-PCR, RNA is used as a template. It is used to analyse and amplify a short stretch of RNA. RNA is first converted to complementary DNA or cDNA by reverse transcription, followed by the standard PCR method. In RT-qPCR, RNA transcripts are quantified by first performing reverse transcription, and then the standard qPCR is carried out.
What is PCR?
Polymerase chain reaction or PCR is used to amplify or produce multiple copies of a gene or short stretch of DNA. It was invented by Kary Mullis. This technique is a very useful tool in molecular biology and biotechnology. It is used in labs to make billions of copies of DNA for research and diagnostics. There are three steps in PCR: Denaturation, Primer annealing and Extension of primers.
It requires two sets of primers complementary to both ends of the DNA template and a thermostable DNA polymerase. The polymerase cycle is repeated repeatedly to get multiple copies of the DNA segment.
The three steps of the polymerase chain reaction are:
- Denaturation: In this step, the double-stranded DNA is separated. Single strands of DNA are formed.
- Annealing: The reaction temperature is reduced to allow the complementary base pairing and annealing of the primer.
- Extension: Taq polymerase, which is a thermostable DNA polymerase, is commonly used for this purpose. The DNA polymerase adds nucleotides to the primer and extends the complementary strand in the 5’-3’ direction.
What is qPCR?
qPCR or quantitative PCR is also referred to as real-time PCR. It gives an additional quantitative analysis of the DNA or RNA in RT-qPCR.
In qPCR or real-time PCR, as the name suggests, the amplification can be monitored as the PCR progresses. In qPCR, fluorescent labelling allows real-time data collection as a polymerase chain reaction is performed.
PCR products can be detected in real-time in qPCR by two methods:
- Using non-specific fluorescent dyes helps in detecting double-stranded DNA. The dsDNA binding dye gives fluorescence signals as the DNA is amplified, and it increases after each cycle. The disadvantage of this method is that only one target can be examined at a time as it binds to all dsDNA fragments.
- Using fluorescent-labelled DNA probes, which are sequence-specific. They can be detected after hybridization with its complementary sequence. As the DNA probes are target specific, many targets can be analysed simultaneously.
What is RT-PCR?
RT-PCR or reverse transcription PCR comprises two steps, the first is the reverse transcription process, and the second is the amplification of the desired DNA sequence by polymerase chain reaction or PCR. RT-PCR is used to detect the RNA in a sample. The amount of RNA can be measured by using RT-qPCR. RT-PCR combined with qPCR (RT-qPCR) is very useful in doing quantitative analysis of viral RNA and gene expression.
The steps of RT-PCR are the same as PCR, with the additional first step of reverse transcription. In RT-PCR, complementary DNA (cDNA) is produced first using reverse transcriptase (RT). The cDNA, thus formed, is then used as a template for the standard amplification process by PCR.
To sum up, RT-PCR and qPCR are the advanced methods of PCR or polymerase chain reaction. qPCR gives faster, more detailed real-time results and is used to quantify nucleic acids. RT-PCR is used to detect and amplify cDNA.
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