If you are a geneticist or a student of genetics, probably you know about the polymerase chain reaction. The PCR or The polymerase chain reaction is an in vitro process of DNA replication. In this article, I will Give you the complete guide of the polymerase chain reaction process.
The Polymerase chain reaction is one of the emerging scientific techniques in recent days and it has infinite opportunities in research as well as diagnostics. Different variations in the native PCR helps in the development of different techniques for different applications.
Allelic specific PCR, Real-time PCR, reverse transcriptase PCR, Hot start PCR and nested PCR are some of the types of PCR routinely used in the molecular labs.
Overview of the article,
History and overview
Principle of PCR
Application of PCR
Limitations of PCR
History and overview of the polymerase chain reaction
In 1983, Kary Mullis described the technique of in vitro gene amplification and called it as polymerase chain reaction. Later on, he was awarded the Nobel prize for this finding.
However, the story of PCR was begun when the Taq DNA polymerase was isolated from the thermostable bacteria. Thomas D Brook discovered a bacteria in 1966. He isolated the bacteria from the hot spring of water and named it as Thermus aquaticus. Later on, in the year 1976, Chien et al., isolated polymerase from Thermus aquaticus named it as Taq DNA polymerase.
The overall idea of the Polymerase Chain Reaction is to obtain millions of copy for the gene of our interest because single gene fragment cannot be visualized into the gel. So for analysis of particle gene, we required millions of copy of that particular gene.
Replication is a process in which the genetic material viz DNA becomes doubled however, we cannot replicate DNA in vitro.
After the isolation of thermostable Taq DNA polymerase, the idea of temperature dependent amplification came in the picture.
The PCR, polymerase chain reaction is a temperature dependent process of DNA amplification. It is the same as DNA replication, Yet DNA replication is an enzyme dependent process and it can not possible in vitro.
The machine used in the PCR technique is called as a thermocycler. Let’s understand each terminology properly.
The word PCR is made up of Polymerase + chain + reaction. Enzyme Taq DNA polymerase is used in this technique and it generates the chain of reaction for multiple copies of the DNA.
Also, it is a cyclic biochemical reaction, each step is followed by another step. Hence the word polymerase chain reaction is derived from the nature of this technique.
Thermocycler: as we already discussed, the technique is temperature dependent; the denaturation, annealing and extension steps are governed by a particular temperature in a continuous cycle in each step. So the machine is called as a thermocycler.
Principle of PCR
The denaturation of the template DNA occurs at 94ºC which creates two single-stranded DNA, the DNA primer binds to the single-stranded DNA in the annealing step at 55ºC to 65ºC temperature and Taq DNA polymerase amplifies the DNA strand with the help of the PCR enhancer in the extension step at 72ºC.
Each step is repeated for almost 30 to 35 times in a cyclic manner which results in the millions of copy of the DNA fragment. The entire process of PCR explained in the table below.
After the denaturation of the template DNA, the Taq polymerase comes in the picture. The Taq required 3’OH group for amplification as like the replication. The Taq can only add new dNTP if free 3’ OH group is present.
For that, the primer provides the free 3′ OH group for binding of the dNTPs. Now Taq adds dNTPs by forming the hydrogen bond with the complementary nucleotides.
Do you know?
The PCR machine was not always an automated machine.
The first PCR machine was a series of three different water baths with three different temperatures. The traditional machine did not have a digital display or the temperature controller. In those days, scientists have to transfer PCR tubes in each water bath manually for at least 35 times.
Each water bath had a thermometer for monitoring temperature. Karry Mullis had achieved PCR amplification through this process. However, in the Year 1985, PerkinElmer introduced the first automated PCR machine. Because of that PerkinElmer is one of the pioneers and tech giant company in making PCRs.
Template DNA, DNA primers, dNTPs, Taq DNA polymerase and PCR buffer are the major reagents used in the PCR reaction. The composition and quantity of each reagent are very important. A μL variation in any of the reagents leads to reaction failure.
The template must be DNA only. Plasmid DNA, bacterial DNA, cDNA or gDNA can be utilized as a template. The template DNA is a highly purified DNA which has the purity of around 1.80 and quantity of up to 200ng. The DNA works as a substrate for enzyme when it denatured.
The good quality of extracted DNA can boost the resulting efficiency of the polymerase chain reaction. The ideal concentration of gDNA for PCR reaction is 30ng with 260/280 absorbance ration of ~1.80.
Another important PCR ingredient is a DNA primer. Generally, the primer which is used in the replication process is RNA primers but in PCR, DNA primers are used instead of RNA primers because of the lack of proofreading activity of Taq DNA polymerase.
More detail on DNA replication please read the article: DNA Replication class 1: General process of DNA replication
The uracil bases of RNA prime is replaced during the proofreading activity in the replication which is not possible in case of Taq DNA Polymerase.
For more detail on properties of Taq DNA polymerase read the article: Function of taq DNA polymerase in PCR
The PCR primers are synthetic oligonucleotides of single-stranded DNA ranging from 18 to 22 bases long, short DNA sequences which anneals at the single-stranded template DNA at its exact complementary position.
For increasing the efficiency of primer, we should follow proper guidelines such as the length of the primer, GC content of the primer, annealing temperature of the primer etc.
For more detail on primer design guide, read the article: PCR primer design guidelines
Generally, 10pmol of each primer is sufficient for a PCR reaction.
Deoxynucleotide triphosphates are artificially synthesized nucleotides which bind to the growing DNA strand. With the help of the Taq DNA polymerase, the dATP, dGTP, dCTP and dTTP binds at its complementary nucleotides on the growing DNA strand.
1mM to 2mM of each dNTPs are sufficient for 25μL of PCR reaction, For more detail on how to prepare working dNTP solution, read the article: The Function of dNTPs in PCR reaction
Taq DNA polymerase
The PCR technique is entirely based on the Taq DNA polymerase. If Taq DNA polymerase was not discovered, the PCR might not be discovered.
Amplification of DNA is possible due to the unique property of Taq DNA polymerase that is thermostability. The Taq DNA polymerase remains stable even at the higher temperature. That is why it can work properly at a higher temperature in the PCR.
The Taq DNA polymerase binds at the ssDNA- primer junction and utilizes it as a substrate for the enzymatic reaction. In the extension step, this will helps in the binding of dNTPs at growing DNA strand.
1 unit of Taq is sufficient for 25μL PCR reaction. For more detail on Taq DNA polymerase read the article: Function of taq DNA polymerase in PCR
PCR buffer is yet another important ingredient in the polymerase chain reaction. It contains all the enhancer which helps in proper amplification. Also, the PCR buffer maintains the constant pH of the reaction nearly 7.9 to 8.5 by maintaining the constant chemical environment for the PCR reaction.
The pH of the buffer is controlled by the addition of Tris.
Mgcl2, DMSO, KCl, albumin, betaine, BSA, glycerol, (NH4)2SO4 and formamide are some of the chemicals commonly used in the PCR buffer. The composition of each ingredient may vary from manufacturer to manufacturer.
However, in each PCR buffer, the MgCl2 must be included because it is worked as a cofactor for the Taq DNA polymerase. For more detail on PCR buffer ingredients read the articles:
The PCR machine
The PCR machine is called as a thermocycler. This machine is simply a heating block (just like our iron) which provides the constant temperature and even rapidly changes between two temperature states.
The machine has a lower block of metal having deep wells for putting PCR tubes. Also, the temperature of the inner environment is maintained by the heating block present on the upper side of the lead.
Further, the machine contains the display, power on and off switch and cooling assembly. The machine has the ability to heat and cool the PCR tube in a short period of time.
The PCR procedure is divided into three steps: denaturation, annealing and extension followed by initial denaturation and one final extension.
The denaturation is the process in which the double-stranded DNA becomes single stranded. At 94ºC temperature, the double-stranded DNA denatured. The process of denaturation is followed by the initial denaturation for 5 to 7 minutes.
After the denaturation, The primer binds to the single-stranded DNA at its exact annealing temperature. Each primer had its own annealing temperature, at that particular temperature the primer binds to its complementary sequence at the flanking region of the DNA.
Generally, the annealing temperature is ranging between 55ºC to 65ºC. Annealing temperature lower than that of the primer leads to non-specific bindings while higher temperature leads to failure in amplification.
The temperature for the annealing step is between 55ºC to 65ºC for 45 seconds. If the annealing step is performed for more than 45 seconds, it leads to non-specific bindings.
After the binding of the primer, its time to expand the DNA strand. Here in extension step the Taq DNA polymerase comes in action and adds dNTPs to the growing DNA strand. The temperature for extension is 72ºC for 45 seconds.
After the completion of all the cycles of denaturation- annealing- extension, one more time the final extension is performed by the PCR for 7 minutes.
The graphical representation of each PCR step is explained in the figure below:
Application of PCR
The PCR has numerous applications in biological research as well as diagnostics.
Diagnosis of inherited disease: the PCR is most routinely used in the diagnosis of some inherited disease such as sickle cell anaemia, thalassemia, MTHFR gene mutation etc. This technique is appropriate for single gene disorders. The result is 99% accurate as compared with other methods.
Microbial identification: the microbial culture technique is traditional and time-consuming also the chance of infection is also high in case of culturing. In modern days, PCR is used in the identification of microbes. The bacteria unique DNA sequence is targeted for the identification of particular bacteria and it will give a result within 3 to 4 hours.
Additionally, PCR is also applicable to the diagnosis of infectious diseases such as HIV or HPV. Again the method is the same as the identification of microbes. The unique DNA sequence of a particular virus is targeted for the identification. This will give a result within an hour.
PCR is used in the identification of genetic carriers as well. The heterozygous condition of the disease can be easily identified using PCR amplification.
DNA fingerprinting and genetic imprinting: the PCR is the first choice for DNA fingerprinting. For more detail on DNA fingerprinting read the article: DNA fingerprinting
The suspect, individual or parental verification is possible because of the PCR.
The PCR is one of the best technique for marker assistant selection. RFLP, AFP, RAPD, STS, VNTR and STR are some of the marker techniques based on the PCR.
PCR is applicable in the prenatal diagnosis of inherited disease as well.
Cancer, retroviral viral infection and other infection can be detected using the polymerase chain reaction.
Further PCR is applicable to sex determination and sex identification.
Apart form mutation detection PCR is useful in gene expression studies too. The expression of a particular gene can be measured using RT PCR. It is even applicable in gene cloning.
mRNA studies are also possible due to the reverse transcriptase PCR and we can calculate gene expression through it.
PCR amplification is one of the important steps in DNA sequencing and microarray.
The PCR is also useful in the validation of personalized medicines.
Limitations of PCR
Identification of new mutation is not possible with the help of PCR. For that, we have to go for DNA sequencing.
Also, Multigenic disorders cannot be detected using PCR.
We can not identify structural and numerical chromosomal anomalies through PCR.
The polymerase chain reaction is highly sensitive biological technique. The chance of cross contamination is always high in case of the PCR. Always perform PCR reaction in a sterile area otherwise the chance of the false positive result will increase if any of the ingredients are contaminated.
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