Polymerase Chain Reaction (PCR)
Polymerase chain reaction (PCR) is a molecular biology technique that can exponentially amplify a specific DNA fragment in vitro. This is a widely used molecular biology technique that enables the amplification of specific DNA sequences.
It is employed significantly in genetic research, medical diagnostics, forensic science, and biotechnology. PCR works by replicating targeted DNA sequences through cycles of denaturation, annealing, and extension, allowing for the detection and analysis of genetic material even from minute samples. Essential components of PCRs The following components are required to set up PCRs:-
1) Template DNA: It consists of target DNA sequence to be amplified, which is a part of large and complex DNA of a genome. It can be either a DNA or RNA.
2) Primers: PCR primers are short, single-stranded pieces of DNA (typically 18 to 25 nucleotides long) that bind to the start and end of a target DNA sequence to direct its amplification Thus, making or designing of primers is a crucial step.
3) Deoxy nucleotide triphosphates (d NTP’s): These are dATPs, dTTPs, dCTPs and dGTPs present in equal amounts in a Standard PCR, which are added by the polymerase enzyme to the primer in extension step of the reaction.
4) DNA polymerase: To catalyse template-dependent synthesis of DNA, a thermostable DNA polymerase is required and for routine PCRs, Taq polymerase obtained from Thermus aquaticus is widely preferred.
5) Divalent and monovalent cations: Mg+2 is the divalent cation required by all thermostable DNA polymerases in order to react with dNTPs to form complexes to stabilize the primer-template complexes. Monovalent cations are provided by KCl in standard PCR.
6) Buffer: Tris-Cl is the commonly used buffer to adjust pH between 8.3 and 8.8 at room temperature.
7) Water
8) Thermal cyclers (thermocyclers): The instruments used for DNA amplification that raises and lowers the temperature of the samples in pre-programmed steps.
Three phases of the PCR
Three phases of the PCR
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1) Denaturation: It is the first step in which the two complimentary strands of template DNA are separated by heating at a temperature of 94oC for 2 minutes in the first PCR cycle, followed by 1 minute in subsequent cycles.
2) Annealing: In this step the mixture is cooled to a temperature of in between 40-60oC that allows the primers to anneal to the complementary 3’ ends of the target DNA segment to be amplified. The duration of this step 1 minute.
3) Extension: It is the final step where the primers bound to 3’ ends are extended by addition of nucleotides (dNTPs) catalysed by a thermostable DNA polymerase for synthesis of a new strand. This step of extension is carried out at a temperature of 70oC for 2 minutes.
One cycle will yield two daughter DNA strands, which act as templates for next cycle. This process is repeated for about 35-40 times in a thermal cycler, a programmable device that controls temperature and time of each step in the cycle. Thus, with doubling of target sequence in each cycle, PCR generates millions of copies of it suitable for further use.
Types of PCR Technique:
Types of PCR Technique:
There are several types of PCR methods used in genetic studies. Each type modifies the basic PCR principle to improve specificity, sensitivity, quantification, or to amplify particular types of DNA sequences.
Some important types of PCR include:- Conventional PCR
- Real-Time PCR (qPCR)
- Multiplex PCR
- Reverse Transcription PCR (RT-PCR)
- Nested PCR
- Hot Start PCR
- Touchdown PCR
- Thermal asymmetric interlaced PCR (TAIL-PCR)
- High-Resolution Melt (HRM) PCR
Real-time PCR or quantitative PCR (qPCR):
Real-time PCR or quantitative PCR (qPCR):
Real-time PCR also referred to as quantitative PCR (qPCR) is a type of polymerase chain reaction (PCR) that was introduced with the concept of monitoring DNA amplification in real time.
This method uses fluorescent dyes, such as Sybr Green, or fluorescence-containing DNA probes, such as TaqMan, to measure the amplification of DNA at each cycle of PCR in realtime by the use of Special thermal cyclers that monitor the amount of amplified product during amplification by measuring the fluorescence after each cycle. Initially the fluorescent material is attached with the template DNA strand, and as the polymerase synthesize new strand over template, this fluorescent material gets detached from the template strand. This indicates that polymerase enzyme attaching the nucleotides on template DNA strand and the synthesis of new DNA strand occurs. Increase in emitted fluorescence reflects the increase in DNA in each cycle. The release of fluorescent material is measured and corelated with the amount of new DNA synthesizes in real time.
Nested PCR:
Nested PCR:
Nested PCR is a modified form of polymerase chain reaction in which two sets of primers, external and internal, are used sequentially to amplify the same target DNA.
The first amplification is carried out using external or outer primers, followed by a second amplification using internal primers to minimizing the amplification of non-specific genes and to spurious PCR products. The specificity is increased by using two sets of primers in two successive runs (two rounds) of the PCR reaction for a single target DNA. In first round, one set of primers (outer primer) are used to amplify the target DNA products. This amplification takes place for 15-30 cycles. These outer primers anneal to the outside or flanking the target sequence. They are present upstream of the inner set of primers. These primers are 18-22 nucleotides long. The second round of amplification is performed by the second set of primers, i.e., internal primers or nested primers (These primers are up to 20 nucleotides long.). The product from the first round is diluted 100-1000 times and then utilized as a template for the second round of PCR. These inner primers are designed to bind inside the outer primer regions. Therefore, only the desired target DNA containing the internal primer sequences is amplified, resulting in highly specific amplification of the target DNA.
Asymmetric PCR:
Asymmetric PCR:
Asymmetric PCR is designed to preferentially amplify only one strand of the target DNA molecule. In this variant of PCR, primers are added in unequal molar ratio to favour the synthesis of the desired DNA strand as replication occurs arithmetically by using the excess primer.
It is called asymmetric PCR because the two primers are present in unequal (asymmetric) amounts, causing one DNA strand to be produced much more than the other. Initially, PCR works normally because both primers are available, and double-stranded DNA is produced. After a few cycles, the limiting primer (present in small amount) gets used up completely, and now only the excess primer remains. Since there is no opposite primer, only one DNA strand can continue to be synthesized. This kind of PCR has applications in molecular techniques such as DNA sequencing and hybridization probing where amplification of one of the complementary strands is required.