Polymerase Chain Reaction (PCR), undoubtedly, is the best technique to replicate DNA.
In the process of DNA replication, a double-stranded DNA molecule is copied to generate two identical molecules of DNA. This is an essential process because the formation of two new daughter cells, for cell division, must contain the identical genetic information, or DNA, as the parent cell.
DNA replication process is regulated with much care that involves multiple components characterizing more than 20 genes parting significant roles in duplication of the genome. The process is constrained on the DNA structure by both DNA sequence and topology. It also requires specific protein–DNA interactions.
Polymerase chain reaction (PCR)
It is a powerful method of amplifying particular segment of DNA, distinct from cloning propagation within the host cell. This procedure is carried out biochemically, in vitro. PCR was invented by an American Biochemist Kary Mullis in 1983. The principle of PCR is DNA replication which consists of 20-25 cycles. The process of PCR takes place in a machine called ‘thermocycler’. It provides thermal condition for amplification. DNA duplicates at an exponential rate (2)n. and the final product of PCR is called amplicon.
Steps of PCR
The following steps are involved in PCR process:
Template is heated to 94°C which results in the breakage of hydrogen bonds and the production of single stranded DNA.
Mixture is cooled to 50°C to 70°C. This allows primers to anneal (blind) to the complementary sequence in DNA template.
Then reaction is heated to 72°C, the optimal temperature for DNA polymerase (enzymes that produce DNA molecules by assembling nucleotides) to perform its action. After each cycle, DNA becomes double in numbers. For the next cycle, these two pieces are then available for amplification. As these cycles are completed, more copies of the DNA are produce.
Ingredients and procedure
The ingredients of PCR are as follows and will be added in the exact same manner.
Take a PCR tube and add about 15.2 microliter water in it using micro-pippette. To make a final volume of 25 microliter including all ingredients nuclease free water will be added.
These are the bases or building blocks of DNA strand added about 2.5 microliter dNTPs solution in the tube.
A buffer maintaining pH of the solution is added in the tube with total quantity of 2 microliter.
This buffer provides Mg+2 ions which act as cofactors for the enzyme. Only 2.5 microliter MgCl2 buffer is added in the tube because excess amount can cause hyper activity of Taq polymerase.
In this step, two types of primers are used:
- Forward primer
- Reverse primer
These primers are short oligonucleotide sequences about 18 to 20 base pairs long. Primers are involved in the function of initiation and particulate the region of amplification. The amount of primers that are used after primer reconstitution is 0.75 microliter for each forward and reverse primer.
The quantity of template matters the most for a good amplification. Contamination can cause inhibition and can induce the non-amplification processes. DNA template is added in amount of 1 microliter.
Thermos Aquaticus (Taq) polymerase enzyme is used which has the ability of proofreading, polymerization, and exonuclease activity. 0.3 microliter enzyme is added in the tube deep at bottom.
After the addition of all ingredients, the tube is mixed on the PCR vortex machine and after that, the tube is placed into the thermocycler machine, which is set at 25 cycles. These cycles are completed in 2 hours and 40 minutes.
After the completion of cycles successfully, the amplicons are subjected into the gel electrophoresis chamber and then are observed under UV light machine.
Advantages of PCR
PCR has a large number of advantages including:
- It is quite simple to understand and easy to use.
- PCR produces results rapidly.
- The process is highly sensitive with the power to generate millions to billions of copies of a certain product for sequencing, cloning, and analysis.
- qRT-PCR has the same advantages as the PCR, with an additional function of quantification of the synthesized product. That is why, PCR has its exceptional uses analyzing the alterations of gene expression levels among tumors, microbes, or other disease states.
- PCR is an extremely powerful with the best practical research tool.
- The sequencing of unknown etiologies of many diseases can figure out with the help of PCR.
- This process can also help identifying the sequence of previously unknown viruses associated to those already known. Thus, it can give a better understanding of the disease itself.
Applications of PCR
Other than amplification of DNA, PCR has good number of applications, such as:
- Selective DNA isolation
- Forensic applications
- Quantification of DNA
- Medical and diagnostic applications
- Research applications
Which type of contaminants to deal with while working with PCR?
The sources of contaminants between samples are varied and can all contribute to the contamination of the amplicon, such as sample carryover, reagents, disposable supplies, improper handling procedures, etc. Some other type of contaminants in PCR may include:
1. Genomic DNA containing RNA samples
Complete genome consists of nucleotide sequences of DNA. Genome may include both the genes and non-coding DNA.
2. Cross-contamination among various nucleic acids may process simultaneously
In a laboratory, at a single time, all the equipments are handling more than one type of molecule which can cause virulent or non-virulent cross-contamination.
3. Laboratory contamination of the target genomic DNA sequences
Air borne contaminants in laboratory can cause vigorous hazards in the results of amplification.
4. DNA may intermix from the outside sources
This can happen in a variety of ways from someone sneezing or coughing over the tubes in laboratory, can result which can cause the adverse results of PCR.
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