Main Difference – PCR vs QPCR
PCR (polymerase chain reaction) and qPCR (quantitative PCR) are two techniques used in biotechnology to amplify DNA for various purposes. PCR is a relatively a simple technique. qPCR is also known as real-time PCR or digital PCR. The main difference between PCR and qPCR is that PCR is a qualitative technique whereas qPCR is a quantitative technique. PCR allows reading the result as “presence or absence’. But in qPCR, the amount of DNA amplified in each cycle are quantified. If RNA is used in PCR, the technique is known as RT-PCR (reverse transcription PCR), and if RNA is used in qPCR, the technique is known as qRT-PC.
Key Areas Covered
1. What is PCR
– Definition, Processes, Uses
2. What is QPCR
– Definition, Processes, Uses
3. What are the Similarities Between PCR and QPCR
– Outline of Common Features
4. What is the Difference Between PCR and QPCR
– Comparison of Key Differences
Key Terms: Agarose Gel Electrophoresis, Amplicons, DNA polymerase, Fluorescent Dye, PCR, Probes, qPCR, RT-qPCR
What is PCR
PCR refers to a technique in biotechnology that allows the analysis of a short sequence of DNA by amplifying a selected segment of DNA. It is comparatively a sensitive method as very small volumes are required by a single reaction. The technique is based on the ability of DNA polymerase to synthesize new strands of DNA to the offered template strand in a complementary manner. The reaction mixture of PCR is composed of DNA polymerase, DNA nucleotides, primers, the DNA template to be amplified, and magnesium. The amplification is carried out inside a thermocycler. DNA polymerase should be heat resistant as high temperatures are used in this reaction. The two types of DNA polymerases used in PCR are Taq DNA polymerase and Pfu DNA polymerase. Taq DNA polymerase is widely used in PCR.
DNA polymerase requires a pre-existing DNA strand at the 3′ end to synthesize a new strand. Hence, an oligonucleotide primer is added to the reaction mixture for the initiation of DNA synthesis. The requirement of a primer in PCR allows the amplification of 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 of a specific DNA sequence, which are called amplicons, are accumulated in billions. The components of the PCR should be optimized in such a way to improve PCR performance while minimizing failure. The standard PCR reaction is shown in figure 1.
Steps of PCR
The three steps of a PCR is described below.
- Denaturation – The double-stranded DNA template is separated into two single strands by heating to 94-95 °C.
- Annealing – Forward and reverse primers bind to the complementary sequences in the template. The temperature depends on the melting temperature of the primer combination.
- Primer extension – DNA polymerase enzyme extends each primer at their 3’end by adding complementary bases to the growing strand. The optimum temperature of Taq polymerase, i.e., 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. Agarose gel electrophoresis is used in the size fractionation of PCR products. The product is stained by ethidium bromide and is observed under UV. The PCR product or the amplified DNA can be used in cloning, sequencing or genotyping.
What is QPCR
QPCR refers to a technique in biotechnology that allows the detection, characterization, and quantification of nucleic acids for various applications. Hence, it is a type of quantitative PCR. Both DNA and RNA can be used as qPCR. If RNA is used as the template, it should be first reverse transcribed into cDNA. Thus, this type of qPCR is known as RT-qPCR. The traditional PCR is carried out for cDNA or usual DNA sample. However, in qPCR, fluorescent dyes are used to label the PCR product in each step of the PCR cycle. This enables the collection of data as PCR progresses, allowing the quantification of the amplicons during the exponential phase of the PCR. The main type of dye used in qPCR is SYBR Green. The dye binds to the double-stranded DNA. As the fluorescence is increased proportionally with the amount of amplified DNA, the quantification can be done in “real time”. The main disadvantage of the use of the dye is that it allows the quantification of one specific product in the sample. In addition to dyes, probes can also be used in the quantification process. TaqMan probes are one of the main types of oligonucleotide probes used in qPCR, and the process of fluorescence emission is shown in figure 2.
Probes can be designed in such a way to detect several PCR products within the same sample. TaqMan probe is one of the main types of hydrolysis probes; the incorporation of this probe into the PCR product exposes the fluorophore, emitting the fluorescence. Fluorescent dyes are more specific to the PCR product. Hence, they are used in most diagnostic assays to detect the PCR product.
Similarities Between PCR and QPCR
- PCR and qPCR are two types of techniques used in biotechnology to amplify DNA for various purposes.
- The traditional polymerase chain reaction is performed as the core technique in both PCR and qPCR.
- RNA can be used in both PCR and qPCR by using reverse transcription as the first reaction.
Difference Between PCR and QPCR
PCR: PCR is a technique in biotechnology that allows the analysis of a short sequence of DNA by amplifying a selected segment of DNA.
QPCR: QPCR is a technique in biotechnology that allows the detection, characterization, and quantification of nucleic acids for various applications.
PCR: PCR is qualitative technique.
QPCR: QPCR is a quantitative technique.
Detection of the Product
PCR: The product is detected by the agarose gel electrophoresis in PCR.
QPCR: The product can be detected in each amplification cycle in qPCR.
Collection of Data
PCR: The data is collected at the end of the reaction in PCR.
QPCR: The data is collected during the exponential phase of the reaction in qPCR.
PCR: PCR has a very poor resolution.
QPCR: QPCR has a very high resolution.
PCR: PCR uses ethidium bromide to stain the product during PCR.
QPCR: QPCR uses fluorescent dyes to detect the product.
PCR: PCR is a more time-consuming method.
QPCR: QPCR is less time-consuming.
PCR: RT-PCR is the type of PCR that uses RNA as the template.
QPCR: RT-qPCR is the type of qPCR that uses RNA as the template.
PCR: PCR is used to detect the presence or absence of certain genomic fragments.
QPCR: QPCR is used to quantify a particular fragment in a sample.
PCR and qPCR are two types of techniques used in biotechnology to amplify DNA for various purposes. PCR is the traditional amplification method used to identify the presence or absence of a DNA fragment. QPCR is used to quantify a particular fragment in a sample. Thus, PCR is a qualitative technique whereas qPCR is a quantitative technique. This the main difference between PCR and qPCR.
1. “QPCR vs. Digital PCR vs. Traditional PCR.” Thermo Fisher Scientific, Available here.