Main Difference – PCR vs RT-PCR
PCR and RT-PCR are two important techniques in molecular biology. PCR was developed by Kary Mullis in 1980s. It is capable of increasing the number of copies of a particular gene in an exponential manner, facilitating the detection of that particular DNA fragment. Taq Polymerase is the enzyme most frequently used in PCR systems. It is a thermostable enzyme. In RT-PCR, reverse transcription is followed by PCR. The enzyme, reverse transcriptase is involved in the synthesis of complementary DNA from RNA. The main difference between PCR and RT-PCR is that PCR uses double-stranded DNA as the template whereas RT-PCR uses RNA as the template.
Key Areas Covered
Key Terms: PCR, RT-PCR, Polymerase Chain Reaction, Reverse Transcription-Polymerase Chain Reaction, DNA Template, DNA Polymerase, Denaturation, Annealing, Primer Extension
What is PCR
PCR (Polymerase Chain Reaction) is a relatively simple but revolutionary method. PCR uses the ability of the enzyme, DNA polymerase to synthesize new strands of DNA in a complementary manner to the offered template strand. PCR is an indispensable technique used in both clinical and research laboratories for functional analysis of genes, diagnosis, and monitoring of hereditary diseases, DNA cloning, sequencing, and ancient DNA amplification. DNA template, nucleotides, primers, and DNA polymerase are the four major components of PCR. The DNA template is usually a double-stranded DNA with the target sequence, which is to be amplified. Taq DNA polymerase which is isolated from Thermus aquatics is commonly used in PCR. The Pfu DNA polymerase is another type of DNA polymerase with high fidelity. Both Taq and Pfu polymerases are heat resistant. The nucleotides added by the DNA polymerases are adenine (A), guanine (G), cytosine (C), and thymine (T). Since DNA polymerase can only add a new nucleotide into a 3′ end of a pre-existing DNA strand, an oligonucleotide primer is required for the initiation of DNA synthesis. The requirement of a primer in PCR allows delineating only a specific region in the template. The target sequence is flanked by forward and reverse primers. At the end of a PCR, new copies called amplicons of a specific DNA sequence are accumulated in billions. The components of the PCR should be optimized in such a way to improve PCR performance while minimizing failure.
PCR is an automated process done by thermal cyclers, which are capable of switching between different temperatures. PCR is a three-step process.
- Denaturation – The double-stranded DNA template is separated into two single strands by heating to 94-95 °C.
- Annealing – The forward and reverse primers bind to the complementary sequences in the template. The temperature of this step depends on the melting temperature of the primer combination.
- Primer extension – DNA polymerase enzyme extends each of the primers at their 3’end by adding complementary bases to the growing strand. The optimum temperature of Taq polymerase, 72 °C is used as the temperature in the extension step. The time of the extension depends on the number of base pairs in the template strand.
The three steps are repeated for 28-35 times.
The products of the PCR are size fractionated by agarose gel electrophoresis in comparison with a DNA ladder. The visualization of DNA on an agarose gel is done by staining with ethidium bromide and observing under UV. The amplified DNA bands under UV in an ethidium bromide-stained gel are shown in figure 2. DNA ladder is shown in the leftmost well.
What is RT-PCR
RT-PCR (Reverse transcription-polymerase chain reaction) is one of the most sensitive techniques used for the detection of mRNA. RNA is the appropriate template for gathering information either on the gene expression of a normal tissue or the gene expression of an infected tissue. The template for the RT-PCR can be either total RNA or viral or bacterial RNA respectively. RNA is reverse transcribed into complementary DNA (cDNA) by the enzyme, reverse transcriptase. The optimum temperature of reverse transcriptase is 46 °C. The cDNA is used in PCR in order to obtain the product. Steps in the reverse transcription PCR are shown in figure 3.
RT-PCR can be performed in a two-step or one-step reactions. In the two-step reaction, the reverse transcription and PCR are performed in two separate steps. In one-step RT-PCR, reverse transcription is followed by PCR in a single tube in a sequential manner. Both cDNA and final PCR product can be run on an agarose gel for the size fractionation and visualization purposes. One-step and two-step RT-PCR are shown in figure 4.
Difference Between PCR and RT-PCR
PCR: PCR is a technique used in molecular biology to amplify a segment of DNA generating millions of copies of a DNA sequence.
RT-PCR: RT-PCR is a variant of PCR used in the detection of gene expression in molecular biology.
PCR: Denaturation, annealing, and extension are the three steps in PCR.
RT-PCR: In RT-PCR, reverse transcription is followed by PCR.
PCR: A double-stranded DNA molecule serves as the template for PCR.
RT-PCR: A single-stranded RNA molecule serves as the template for the reverse transcription. A single-strand DNA molecule serves as the template for the PCR.
PCR: DNA polymerase is used as the enzyme in PCR.
RT-PCR: Reverse transcriptase and DNA polymerase are used as enzymes in RT-PCR.
PCR: Forward and reverse primers are used in the PCR.
RT-PCR: Only the reverse primer is used for the reverse transcription and both forward and reverse primers are used in the PCR.
PCR: PCR is a sensitive method.
RT-PCR: RT-PCR is more sensitive that PCR.
PCR: PCR is used in functional analysis of genes, diagnosis, and monitoring of hereditary diseases, DNA cloning, DNA sequencing, and ancient DNA amplification.
RT-PCR: RT-PCR is used in the detection of gene expression.
PCR and RT-PCR are two the important techniques used in molecular biology. Both PCR and RT-PCR are automated techniques capable of increasing exponentially the number of copies of a target DNA sequence, which is defined by the forward and reverse primers. In PCR, double-stranded DNA is used as the template. By PCR, DNA cloning, DNA sequencing, and functional analysis of the genes can be done. In RT-PCR, gene expression in a particular tissue can be observed by amplifying RNA. The main difference between PCR and RT-PCR is in their templates used in each reaction and their applications.
1.”Polymerase Chain Reaction (PCR).” National Center for Biotechnology Information. U.S. National Library of Medicine, n.d. Web. Available here. 01 June 2017.
2.”Polymerase Chain Reaction (or PCR).” Cloning and Molecular Analysis of Genes. N.p., n.d. Web. Available here. 01 June 2017.
3. Biolabs, New England. “CDNA Synthesis & RT-PCR.” CDNA Synthesis & RT-PCR | NEB. N.p., n.d. Web. Available here. 01 June 2017.
1.”Polymerase chain reaction” By Enzoklop – Own work (CC BY-SA 3.0) via Commons Wikimedia
2. “DNA fragmendid etiidiumbromiidiga värvitud agaroosgeelis.” By Rainis Venta – Own work (CC BY-SA 3.0) via Commons Wikimedia
3. “Reverse transcription polymerase chain reaction” By Jpark623 – Own work (CC BY-SA 3.0) via Commons Wikimedia
4. “One-step vs two-step RT-PCR” By Jpark623 – Own work (CC BY-SA 3.0) via Commons Wikimedia