PCR is used in different branches of science for different purposes. Starting from microbiology to food industry PCR is a must needed technique used in the identification of pathogens.
PCR is commonly used in biotechnology, microbiology, environmental science, medical science, dentistry, anthropology, food industry, animal and plant research.
PCR is a technique used to amplify and quantify DNA present in a biological sample.
In the present article, we have tried to enlist 50 application of PCR in various fields of science.
Before that read our article on PCR: The polymerase chain reaction.
One of the groundbreaking applications of PCR is in the diagnosis and screening of human diseases. An inherited disease, non-inherited disease and infectious diseases can be screened using the PCR.
Either only PCR or associating it with other techniques such as restriction digestion, sequencing and blotting, genetic disorders as well as other non-inherited and infectious disease can be diagnosed.
Inherited genetic disease:
Inherited diseases led by the mutation in a gene. The genes are located on a chromosome and inherited to the offsprings.
Thus, the mutation occurred in a gene also inherited to the consecutive generations, results in genetic abnormalities.
The condition might be autosomal dominant, autosomal recessive or X linked. Depending upon that the individuals may be a carrier of the disease (heterozygous) or affected with the disease (homozygous), Using other methods available, only PCR has the power to distinguish the homozygous from the heterozygous.
For example, thalassemia is an autosomal recessive disorder, only 25% of progenies can be effected with the disease with two recessive alleles.
Using the PCR method called ARMS-PCR, two different sequence-specific primers amplify one mutant allele and one normal allele respectively.
Thus homozygous dominant without the disease, heterozygous carrier and homozygous recessive with two disease genes can be distinguished on an agarose gel.
Nowadays PCR assays for Huntington’s disease, cystic fibrosis, thalassemia, sickle cell anaemia, Haemophilia, Myotonic dystrophy and Duchenne muscular dystrophy are available.
non-inherited genetic disorders:
Cancer is a genetic abnormality arisen due to mutation or group of mutations in some genes results in oncogenes.
Mutations in genes related to cell differentiation and cell death commonly cause cancer. Though cancer origins due to the genetic mutations, it is non-inherited, originated only due to the interaction of genes with the environment.
Using realtime PCR assay, the amount of the oncogene or the expression of gene causing-cancer can be determined.
(Some of the cancer are inherited).
PCR is an accurate, easy and rapid method for identification of microbes. Any pathogens can be identified and estimated in a single real-time assay.
PCR is used in the diagnosis of infectious disease enlisted below,
PCR is a gold standard method for diagnosis of tuberculosis called TB-PCR. the amount of the TB pathogen can be detected within an hour using PCR and it is accurate than other methods available.
HIV1 a causative agent of AIDS, Hepatitis B and C causative pathogen of liver malignancy, cytomegalovirus causative pathogen of the immune inflammation and human papillomavirus a causative agent of cervical cancer are detected using the PCR.
Furthermore, the amount of infection is also measured by mRNA expression analysis.
Selective DNA fragment isolation:
One of the basic primitive applications of the PCR is isolating the DNA fragment of our interest from the rest of genomic DNA.
The total genomic DNA might be contaminated with chemicals and other agents used during DNA extraction.
A low quantity DNA restrict the use of it for a wide array of experiments thus it is important to amplify DNA.
Selective amplification allows one to amplify DNA fragments of interest and used in for downstream applications.
The amplification also allows an analysis of the DNA sample from the very small amount of starting material.
Microbiology: Amplification of microbial DNA
Using the traditional microbiology techniques, identification and characterization of microorganism is a tedious and time-consuming job.
Further, the chance of contamination and infection is also very high in those methods. Instead, the PCR amplification allows to reduce time and increase the accuracy of results.
By examining the DNA sequence through the DNA sequencing, one can characterise and identify any microorganism in rapid time for that the DNA must be amplified first.
Further, the results are more accurate than the conventional microbiology technique.
In addition to this, even, by only amplifying the group of microorganism in a simple PCR experiment using the sequence-specific primer, the microbes can be identified.
PCR revolutionalized the microbiology filed totally by removing the traditional microbial culturing.
The PCR is even used in the identification of different strains of one particular microorganism, for example, different strains of HPV commonly found in cervical cancer, using a different set of primers each strain of HPV can be detected.
Using the PCR based genetic markers such as STRs ( short tandem repeats) and VNTR (variable numbers of tandem repeats) scientists can create a unique DNA fingerprint between different individuals.
STRs and VNTRs are unique DNA sequence present in a genome with different repeat numbers with the difference in a length.
(STR with 6 to 10 nucleotides long and VNTR with 5 to 50 nucleotides long).
No two individuals have the same VNTR or STR markers in the world. Thus by amplifying either STR or VNTR or even both scientists can create a unique DNA pattern which is different from other individuals.
Hence by using the PCR based markers two individuals can be distinguished. Because it has the power to discriminated a person from the population, it is used in the criminal verification and crime scene investigation.
Criminal identification using the PCR based method accepted widely in every country for investigating a criminal case.
Read our article on DNA fingerprinting.
Checking contamination with PCR!! Sounds unrealistic at first glance. But PCR is one of the trusted technique used in the checking of contamination and cross-contamination.
Let’s take an example,
Suppose we have a sample of HPV patient and it is supposed to be contaminated with some other microorganism.
This sample can not be used for HPV culture, interestingly, using the sequence-specific primer of HPV Only HPV DNA is amplified in the PCR, any of the other DNA can not be amplified.
And thus, in the end, we have the pure amplicons of only the HPV DNA only. These PCR amplicons are now can be used in DNA sequencing for further confirmation of results.
Even, If we know the possible source of the contamination, contaminant strains of microbes can also be identified using the sequence-specific primers.
In recent microbial practises, for maintaining the strains, the strains are routinely checked for detection of contamination using the PCR.
One of the fascinating application of PCR is in the checking of maternal cell contamination in the fetal sample.
The fetal samples of chorionic villi or amniotic fluid are taken for prenatal analysis of disorders like thalassemia, sickle cell anaemia and Down syndrome.
During the fetal sample collection, the maternal cells or blood might contaminate the CV or AF sample leads to false-positive results.
As we discussed above, STR and VNTR patterns are different between individuals, thus by doing the PCR based DNA fingerprinting, maternal cell contamination can be detected.
Sex determination has been easy and accurate using the PCR. A Y chromosome-specific marker is selected for it and amplified using a routine PCR protocol.
If amplification is observed, the fetus is male and if amplification is not observed the fetus is female.
PCR in Genotyping:
A genotype is a genetic constitution related to a phenotype. Genotyping using the PCR facilitate identification and characterization of normal as well as the mutant alleles.
Using methods such as nested PCR or allelic specific PCR variations in a genotype, two genotypes can be distinguished
Using two different sets of primers for two different genotypes homozygous normal, heterozygous and homozygous mutant alleles can be identified.
This method is used in knock-out and knock-in mice genotyping studies in which the affected organism of different genotypes can be evaluated.
However, sequencing of PCR amplicons is highly recommended to confirm the results of PCR genotyping.
SNP (single nucleotide polymorphism) an SNV (single nucleotide variations) can be detected using the PCR genotyping methods.
See the image
(Note: if DNA band is observed in NP and MP the individual is heterozygous for this particular SNP).
Read our article on SNP: An Introduction To Single Nucleotide Polymorphism (SNP).
Quantification of nucleic acids
In the case of infectious disease, it is very important to measure the amount of infection, because the amount of infection is directly proportional to the intensity of the infection.
It measures each and every template DNA from every microbe present in a sample, ultimately we can determine the amount of infection.
Quantitative PCR (aslo called Realtime PCR) facilitates quantification of template DNA or RNA in a sample.
If the sample is of retrovirus (having RNA as genetic material), by reverse transcribe it into DNA in a reverse transcription PCR, the amount of mRNA can be measured.
After each round of amplification, the amount of the accumulated DNA amplicons are measured by the real-time PCR machine.
A fluorescently labelled probe or dye is used to hybridize with template DNA, which emits fluorescent, detected by the detector.
The amount of the fluorescent emitted is directly proportional to the amount of the nucleic acid present in a sample.
We have explained both the dye-based method and fluorescent-probe based method is our previous article: Real-time PCR: Principle, Procedure, Advantages, Limitations and Applications.
Estimation of gene expression:
The difference in the gene expression between cells, tissues or organism can be examined using the PCR.
Realtime RT-PCR is a gold standard method for routine estimation of cytokines and chemokines gene expression.
A cytokine is a component of the immune system plays an important role in during organ transplantation. Also involved in autoimmune disorders and inflammatory disorders.
By measuring the amount of mRNA or gene expression regulating cytokines scientists can evaluate the effect of it and monitor its therapeutic effects in patients.
The gene expression PCR assay is now a gold standard method several disease diagnosis used in the clinical microbiology and clinical oncology.
The quantitative PCR is further employed in the gene expression studies in gene therapy experiments and RNA interference studies.
In the RNA interference experiments, the amount of gene suppression or gene silencing is measured using the gene expression assays.
Moreover, in the gene therapy experiments it is used to measure the gene insert into plasmid as well as its expressions in cell lines.
For the gene expression studies instead of DNA the RNA is isolated and it is reverse transcribed into the cDNA. This complementary DNA is amplified and measured in a PCR called reverse transcription PCR.
Validation of cell lines:
Cell lines are developed for different in vitro gene therapy and gene transfer experiments. Those cell lines once cultured, can be validated using the polymerase chain reaction.
The cell from the cell line (possibly contains the insert DNA or DNA of our interest) are taken and in situ PCR is performed.
In the in situ PCR, the amplification is directly performed on the slide containing the cell lines.
Instead of in situ PCR normal PCR can be performed by using the DNA extracted from the cell line.
PCR cloning is another mindblowing application of PCR technique allows DNA amplification and ligation in a single experiment, without doing restriction digestion. A rapid cloning method called direct PCR cloning allows researchers to clone DNA which is not available in larger amount.
We will discuss the PCR cloning in some other article.
Plant research and agriculture
PCR in GM verification:
The genetically modified plant species is created by altering its genetic composition for specific economical benefit.
Once the GM plant is developed, it is very necessary to verify it.
The PCR is used for GM identification as well as quantification of the insert. Even, the method is widely accepted by consumers as well as developers who cross-check the quality of GM using the PCR.
Furthermore, the PCR is a gold standard method for grain handling and grain processing industries for certification and verification of grains.
Marker assistance selection in plants:
Using the RAPD (Rapid amplification of polymorphic DNA) like markers the plant species and members of plant species can be identified and characterised.
Thereupon, the phylogeny of plant species can be created using the PCR-RAPD based marker assistant selection method.
RAPD is a method in which the random universal primers are used for amplification in PCR. based on the polymorphism between different plants, the primers amplify different DNA fragments which are separated on the agarose gel electrophoresis.
Alike us the plant also suffers from pathogenic infections. The plant may die with the infection and if it is economically important, one has to suffer from economical burden.
Viruses, bacteria and fungus are the groups of common pathogens infect plants.
Using PCR detection method, infectious pathogens (for example Xanthomonas, pseudomonas and mycoplasmas) for economically important plants are identified and subsequently, management steps are taken to prevent it.
For developing a novel economical important plant, scientists have to insert a desired gene into the plant genome.
For that, a bacterial vector is used as a carrier of genetic material.
Using the PCR, the insertion of a gene into the vector is verified, in addition to this, the expression of that foreign gene in the plant genome can also be measured by the real-time PCR.
Apart from this, PCR and its variations have anonymous applications in plant research; some of them are:
- Sequence comparison of the marker gene, thus marker gene comparison studies
- RAPD fingerprinting of different plant species
- DNA barcoding for different plant species
- Analysis of economically important proteins and its sequence analysis.
- Analysis and comparisons of allozymes in plants.
PCR in animal research:
Alike humans, the PCR and its variations are commonly used in animal research and disease studies.
Some of the common applications of PCR in animal genetics are:
- In the identification of MTM mutated gene in dogs, responsible for X-linked Myotubular myopathy.
- For Bursal disease virus in avian samples.
- Identification of canino parvo-virus in dogs.
- Deletion study of Meq gene in chickens
PCR in antiviral therapies:
Antiviral therapies are now in trending, albeit, it is under pre-clinical trial phase, not available fully for human subjects.
Scientists are now trying to cure AIDS, TB and cervical cancer like infectious conditions using the antiviral therapies using the HIV-1, HBV, HPV-1 and HSV-2.
The amount of viral nucleic acid reduced after the antiviral therapy dose is measured for checking the success rate of the therapy using the quantitative PCR.
PCR in dentistry:
In recent days, due to the great power and precision of PCR in the identification of pathogens in rapid time, it is also used in the dentistry and related infection diagnosis. s. Ratti, s mutants, s. Sobrinus and s ferus are common pathogens damages dental health.
Taking a sample from the oral cavity and isolating DNA, the amount of pathogen present in the dental cavity can be measured.
Furthermore, the realtime PCR is applied in the periodontal disease and endodontic infections.
PCR in artificial mutagenesis or site-directed mutagenesis:
Inserting a mutation in a DNA sequence (called artificial mutagenesis or site-directed mutagenesis) can be useful in removing restriction sites and gene transfer experiments.
The artificial mutagenesis can be done using different PCR based methods. two of the examples are given into the figure below,
PCR in development of markers:
PCR is one of the basic technique which replaced traditional cloning and blotting methods for the analysis of genes.
Henceforth, instead of blotting, PCR is nowadays used in several genetic markers.
In the RFLP, PCR is now used for amplifying the varients of different lengthed DNA.
PCR is employed in the marker enlisted below,
- STS (short tandem repeat)
- VNTR (variable number of tandem repeat)
- SNP (single nucleotide polymorphism)
- STS ( sequence tag sites)
- SSCP (simple sequence conformation polymorphism)
- RAPD (random amplified polymorphic DNA)
- AFLP (amplified fragment length polymorphism).
PCR is DNA sequencing:
DNA sequencing is a method in which the nucleotide sequence is determined.
In fact, DNA sequencing is used in the conformation of PCR results, once any of the amplicons is identified in the PCR-agarose gel electrophoresis, confirmed using DNA sequencing in the final step if required.
In the DNA sequencing, the PCR technique is one of the important step, which provides the template DNA for final sequencing procedure.
The template provided by the PCR is only the DNA fragment of our interest (which we wish to sequence) and contaminated free.
Through the bridge amplification during the next-generation sequencing, the entire library of the DNA amplicons are being constructed.
Instead of using the whole genomic DNA, using the PCR amplified fragment of DNA, it increases the efficiency, accuracy and surety of sequencing.
Food pathogen detection using PCR:
Detection, identification and confirmation of food-borne pathogens and toxic fungus facilitates by PCR.
Commercialization of food industries increased the risk of food contamination. Food processing, food packaging and food distributing industries are booming fastly. The packaged food reaches to the consumer between 3 to 4 days after cocking.
Although it is packaged under extreme sterile conditions, there might be some chance that the food product may be contaminated or infected by some pathogens.
That pathogen creates hazardous effects on once’s health.
PCR is now widely used at different levels of the food distribution chain to detect the possible contamination in it.
The sequence-specific primers are used for most common food contaminating pathogens is amplified using PCR.
If amplification obtained, the food product suspected to be contaminated with the pathogen.
The PCR based detection method is widely used in meat and meat-related industries.
Furthermore, it is used in the dairy and poultry industries too.
PCR in the methylation assay:
DNA methylation is directly related to gene expression. The methylated sequences can not be processed for translation and thus methylation has great impact on gene expression.
PCR has great utility in the detection of methylation in a sequence using methylation-specific PCR.
However, the method for methylation assay is slightly different having an additional step of bisulfite treatment.
The bisulfite treatment converts the unmethylated cytosine into uracil but the methylated cytosine remains unaffected.
The set of primers are designed, one specific for the methylated cytosine reach sequence with guanine.
Another set of primer is designed specifically to the bisulfite-treated sequence in which the unmethylated cytosines converted into uracil.
This primer set has adenine in place of guanine which binds to uracil and then with the thymine in consequent PCR cycles.
The methylated and unmethylated sequence can be distinguished and quantified in the quantitative PCR.
PCR in cancer:
The PCR is used in the diagnosis, prognosis and monitoring of cancer using the realtime PCR assay. Even the MRD (minimal residual disease) can also be determined by the quantitative PCR.
Breast cancer, cervical cancer, chronic myeloid leukaemia and other related cancer is detected using different PCR assays.
Further, the amount of the mutant gene or oncogene can also be measured.
Interestingly, PCR is also used in cancer therapy monitoring. The success rate of the therapy, thus the expression of oncogene or mutant gene is calculated each time after the therapy using the quantitative PCR.
PCR in gene therapy:
Gene therapies are now a brighter field of genetics in which faulty gene is replaced by the healthy gene using vectors.
The viral-vector mediated gene therapies have more success rate in comparison with the non-viral vector-mediated gene therapies.
PCR is used in the gene therapy for constructing a library for a gene of interest, to confirm the insert in a plasmid, checking transformation of a plasmid in cell lines and expression of GOI in a cell.
Tracking animal migration using PCR:
Now, this is something very innovative.
Environmental scientist used PCR very differently not for disease diagnosis, not for microbial identification but for tracking the migration pattern of animals.
They collect samples from the migration route on an animal, the sample might be a fecus sample, urine sample, hair sample, blood sample or any other body sample.
In the next step, DNA extraction is performed from every sample.
Using the species specif primer for that particular animal, the PCR is performed from the DNA sample collected from every sample.
In the final step, by doing the agarose gel electrophoresis, they conform the amplification and creates the migration map of that animal based on the sample collected.
(if the sample belongs to that animal it will amplify in the PCR).
PCR in organ transplantation:
Bone marrow transplantation is one of the therapeutic options for curing thalassemia and sickle cell like disorders.
Kidney transplant and liver transplant practises are now common in medical science but in the very first step of organ transplantation checking graft-acceptance and graph-rejection is very necessary.
If the graft is rejected, the transplanted organ can induce a strong immune response.
hence, it is crucial to check the compatibility between donor and acceptor.
PCR is the most-trusted method for checking the acceptance and rejection of the graft.
Human leukocyte antigen locus present in our genome is highly variable regions used for the organ transplantation called HLA typing.
As like the DNA fingerprinting, the HLA typing is a unique genetic property of every individual. Different persons have different HLA pattern.
If the HLA pattern matches between acceptors and donor than and then the organ transplantation can be done.
A simple HLA locus-specific primers are used for doing the HLP typing PCR.
If the locus is present, the primer will amplify otherwise remain unamplified. The same type of locus-specific primers are used for the donor as well as acceptor.
We will update this article from time to time by studying other applications of PCR.
PCR is one of the important tools in research as well as in the diagnosis, further, the accuracy of PCR is very high. PCR also save time in some critical experiment otherwise which takes two to three days for completion.