“The Polymerase chain reaction is an in vitro method of DNA synthesis in which using the Taq DNA polymerase, the DNA can be amplified.”
The polymerase chain reaction is a routine word used in the genetic laboratory because it is a basic requirement for a genetic or molecular lab.
Replication is a process of DNA synthesis, however, for us mimicking replication in a lab is not possible. But after the discovery of the thermostable DNA polymerase, the dream of synthesising DNA in a lab comes true.
What is the problem with the normal DNA polymerase? well, it can not work at a higher temperature.
A thermostable Taq DNA polymerase, isolated from the hot water bacteria can even synthesise DNA at a higher temperature. Using ingredients such as dNTPs and other enhancers along with Taq, one can synthesise DNA in PCR.
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.
In the present article, we will understand the PCR- polymerase chain reaction, starting from basics to advance. Furthermore, we will also discuss some of the important types of PCR used to enhance PCR results.
Overview of the article,
- History and overview
- Principle of PCR
- Steps in PCR
- PCR machine
- PCR reagents
- PCR procedure/ protocol
- PCR results
- Applications of PCR
- limitations of PCR
History and overview:
In 1983, Kary Mullis described the technique of in vitro gene amplification and called it a polymerase chain reaction. Later on, he was awarded the Nobel Prize for his finding.
However, the story of PCR was begun when the Taq DNA polymerase was isolated from the thermostable bacteria. In 1996, Thomas D Brook discovered the bacteria from the hot spring of water and named it as Thermus aquaticus. Later on, in the year 1976, Chien et al., isolated DNA 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 a single gene fragment cannot be visualized into the gel. So for analysis of particle gene, we required millions of copy of that particular gene.
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. The machine used in the PCR technique is called 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 a thermocycler.
“A common genetic tool- a laboratory technique used to obtain multiple copies of target DNA fragments using Taq DNA polymerase in a temperature-dependent reaction called PCR- a polymerase chain reaction.”
Theoretically, the definition of the PCR can be as stated,
“PCR is a technique in which using the dNTPs, primers, Taq DNA polymerase and template DNA, the DNA can be synthesised artificially.”
Or we can say,
“PCR- a polymerase chain reaction is a cyclic temperature-dependent reaction used to amplify the gene of interest.”
In short, we can define PCR as,
“An in vitro technique of DNA amplification is known as PCR.”
Chemicals: dNTPs, distil water, PCR reaction buffer, enzyme Taq DNA polymerase, primers and template DNA.
Instruments: thermocycler, spinner and agarose gel electrophoresis unit.
Other utilities: PCR tubes, stands, pipettes, tips.
Principle of PCR:
The principle of the PCR is based on the temperature variations, heating and cooling- thermocycling divided the entire reaction into three parts:
Denaturation- in which the template dsDNA becomes single-stranded and provides a template to facilitate amplification.
Annealing- in which the primer binds to the single-stranded complementary DNA.
Extension- Taq DNA polymerase uses the 3’ end of the primer and starts DNA synthesis by adding nucleotides to the growing DNA strand.
These steps are simultaneously repeated for 25 to 40 times in which after each cycle of amplification, the DNA templated doubled.
“Copying DNA using PCR is called amplification.”
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.
The entire PCR reaction is basically governed by the temperature, different temperature zone facilitates different reactions in the reaction. Denaturation, annealing and extension are three PCR steps complete the entire reaction followed by initial denaturation and final extension.
PCR step 1: Denaturation
- Temperature: 90°C to 95°C
- Time 30 sec to 90 sec
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.
PCR step 2: annealing
- Temperature: 55°C to 65°C
- Time: 30 to 60 sec
After the denaturation, The primer binds to the single-stranded DNA at its exact annealing temperature. Each primer has its own annealing temperature, at that particular temperature, the primer binds to its complementary sequence.
Generally, the annealing temperature is ranging from 55ºC to 65ºC. Annealing temperature lower than that 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 can cause non-specific bindings and primer dimers.
PCR step 3: extension:
- Temperature: 70°C to 72°C
- TIme: 45 Sec
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 the 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:
Time-duration for PCR:
- 1 hour to 4 hours
Template DNA, PCR 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 single 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 an 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.
Concentration: 30 to 50ng.
Another important PCR ingredient is PCR primers. Generally, the primer 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.
Concentration: 10 to 12pMol.
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
Concentration: 200-250μM each.
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 a 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.
Concentration: 1 to1.5 unit.
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:
Concentration: 1X or as per requirement.
The PCR machine
The PCR machine is called 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.
PCR procedure/ protocol:
For any molecular genetic experiment, pre-preparation plays an important role in getting good results.
Before starting the reaction, one must have to be ready for doing the lab work, for that wear a lab coat, gloves, mouth cap and head cap.
Clean the PCR reaction preparation area and arrange all other utilities nearby the reaction preparation.
Now take reagents from the deep freeze and thaw all the reagents properly.
Take a sterile PCR tube and start adding reagents as shown into the table.
Starts adding reagents in a sequential manner to reduce the chance of error.
If you have a ready to use mastermix, you can add it directly, this will save time and increases the efficiency of the reaction.
After the completion of reaction preparation, close all the cap of the tubes and spin it properly, so that all the reagents mix well.
Now put the tubes in the PCR machine one by one in the pre-set PCR protocol. Remember: don’t waste time in setting protocol during the PCR, set it before the reaction preparation and immediately run the PCR.
Meanwhile start preparing the gel for agarose gel electrophoresis, because it will also take time for around 60 to 90 minutes.
Key to success:
“I suggest you get good results is that, weigh and prepare all the reagents accurately. Accuracy results in uniform results.”
After completion of the PCR reaction, turn off the machine and collect all the tubes in “orderly manner”.
Rest tubes for some time in a freeze before doing agarose gel electrophoresis.
You can also rest it for the next day also, no problem in it.
We have covered an amazing article on analysing and interpreting agarose gel electrophoresis results, that portion will master you on this.
Anyway, we will explain to you how to interpret the results of PCR in brief,
Run a DNA ladder along with the PCR amplicons so that we can analyse the results.
Based on the migration of DNA fragment in the gel and our in silico PCR or primer 3 results we can assume what size our PCR amplicons are.
See the image below,
Application of PCR
We have covered an amazing in-depth article on applications of PCR, you can read it here: 50 Powerful applications of PCR.
Here I am only enlisting some of the important applications 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 the 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 from 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.
The PCR is used in;
- Gene editing
- Gene manipulation
- Genetic engineering
- RNAi research
- DNA and RNA quantification
- cDNA and gDNA library preparation
- Developing new assays
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.
Types of PCR:
The simplest version or the original PCR technique utilises only a simple Taq DNA polymerase and no modifications called a conventional PCR.
Gradient PCR is one of the widely used modification of native PCR in which for optimising the PCR reaction, different temperature gradients are created in a machine.
Using these different temperature gradients, the template DNA amplification efficiency can be checked. The best annealing temperature can be selected for further consecutive reactions.
Besides this, the efficiency of different PCR enhancers can also be checked at different temperature using the gradient PCR.
Read more: Gradient PCR
The benefit of using the Taq DNA polymerase in the PCR reaction is its stability at a higher temperature, however, it is also its limitations.
As it is also able to synthesise the DNA at a lower temperature too, using the hot start modifications, the Taq is inserted only at the time of denaturation.
For doing that, different strategies of inactivating Taq DNA polymerase at early in the reaction are available. One of them is the use of enzyme liked antibody.
At the higher temperature, the antibody released the enzyme in the reaction. However, the main objective of the hot start is to activate Taq only when the reaction starts.
Read more: Hot start PCR
Realtime RT PCR:
Yet, another amazing modification of the native PCR is the realtime PCR in which using the fluorochrome chemistry, the template DNA can be estimated.
A probe attached with the fluorochrome emits fluorescence once it is hydrolysed from the template and the template is measured.
The amount of fluorescence emitted is directly proportional to the amount of DNA present in the sample.
This is the principle of the realtime PCR which is now widely used in the diagnostic and microbial identification.
Here the catch is the use of the coloured molecule, although, different types of probes are used for different applications.
Read more: Real-time PCR
Reverse transcription PCR:
Reverse transcription PCR is actually a variant of the real-time PCR in which instead of DNA the amount of RNA can be measured.
An enzyme called reverse transcriptase converts the total mRNA into the cDNA which is measured using the same chemistry of the real-time PCR.
Thus the amount of the mRNA present in a sample can be estimated using this type of PCR.
As we know, the total mRNA translates into protein, therefore the gene expression can be measured using the reverse transcription PCR.
Read more: Reverse transcription PCR
One of the major limitations of the PCR reaction is that only a single template can be amplified in a single reaction.
Multiplex PCR is a modification using which multiple templates can be amplified using a single set of primers or a single template can be amplified using the multiple sets of primers.
Multiplex PCR is widely applied in the realtime PCR assay for quantification of multiple templates or screening of multiple mutations in a single assay.
Though the method is similar, the optimization must be required for developing different multiplex protocols.
Read more: multiplex PCR
Amplified refractory mutation system is a unique type of PCR reaction set up in which different allele of the same gene can be amplified using ARMS PCR and therefore it is also called as allelic PCR.
Generally, two pairs of primers- one for wild type allele and one for a mutant allele are used to amplify two different alleles.
A primer set for the wild type allele can not amplify the mutant allele and thus a single DNA band for homozygous allele is obtained.
A primer set for mutant type allele can not amplify the wild type allele and thus a single DNA band for a mutant homozygous is obtained.
However, two different DNA bands one for wild type allele and one for mutant type allele is obtained in heterozygous DNA.
Read more: ARMS or allele-specific PCR
Touch down PCR:
Now, this modification is my favourite one! Because it always gives positive results in all assays.
In the touchdown PCR, by gradually decreasing the annealing temperature, the specificity in a PCR reaction can be increased.
First, set up the annealing temperature 10C above the real annealing temperature and then set up each PCR cycle with a decrease in temperature 1C per each cycle until 50C or 55C.
The main objective of doing this is to increase the specificity of the PCR amplification without compromising the specificity.
Read more: Touchdown PCR
Using one of the nested PCR along with the flanking primers, the efficiency of the PCR reaction can be increased by employing the nested PCR methods.
Read more: nested PCR
A rapid, high throughput PCR method in which the insert or the plasmid DNA is amplified directly from the bacterial colony.
For that, the bacterial colonies are taken and PCR is performed directly on it which helps in amplifying insert directly without extracting plasmid DNA.
However, two sets of primers are used for that, one for plasmid specific and one for amplifying rest of the DNA.
Read more: Colony PCR
In situ PCR:
In situ-PCR is yet another excellent method for rapid amplification of a sample DNA. in this method the amplification of target DNA is done directly on the side or in situ. All the reagents such as dNTPs, primers and PCR buffers are added directly on the slide to do PCR.
This method is widely used for paraffin-embedded tissues or for formalin-fixed tissues.
Read more: in situ PCR
Immuno PCR is a combination of real-time PCR and ELISA method.
Using the sensitivity of the ELISA method in the quantification, the specificity of the PCR reaction can be increased using the Immuno PCR.
Read more: Immuno PCR
In silico PCR:
The in silico PCR is a computational tool used to estimate or predict the results of actual PCR reaction.
We have covered an amazing article on step-wise guide on how to do in silico PCR.
Read more: in silico PCR
The droplet PCR is further, an amazing enhancement of the PCR quantification in which using the droplet; the amount of the template DNA is estimated
Droplet PCR is an assay used to estimate the amount of the template, especially, for sensitive assays such as quantification of pathogens.
Read more: Droplet PCR
The polymerase chain reaction is highly sensitive biological technique. The chance of cross-contamination is always high in the 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.
Read further on agarose gel electrophoresis: