Polymerase Chain Reaction Method
Polymerase chain reaction (PCR) in biology and chemistry is a laboratory method or technique used for making millions to billions of copies of a specific DNA molecule. PCR is a cell-free amplification technique used for synthesizing multiple copies of any DNA molecule for medical, forensic, and biological research. The polymerase chain reaction method can help scientists amplify a very small DNA sample to a sufficient amount. PCR technique was developed in 1983 by American biochemist Karry Mullis and received a Nobel prize in 1993 for his discovery.
Today, PCR is a basic tool in molecular biology to make many copies of DNA from small samples.
Large amounts of nucleic acid (DNA and RNA) samples are necessary for molecular and genetic analysis. Scientists used polymerase chain reaction (PCR) to amplify trace amounts of DNA and RNA from a sample. The PCR process has a wide variety of laboratory and clinical applications.
- Forensic labs use PCR for analyzing DNA samples from a crime scene.
- Clinical healthcare labs use PCR to identify infected viruses in patients.
- Pharmaceutical research labs use PCR for analyzing and duplicating DNA and RNA samples and using it in the manufacturing of drugs and vaccines.
Principle of Polymerase Chain Reaction
The PCR principle is based on the enzymatic replication of DNA molecules. It is used for amplification of a specific region of DNA strands. In most cases, polymerase chain reaction (PCR) is used to amplify DNA fragments of between 0.1 and 10 kilo base pairs (kbp) in length but some techniques also allow to amplify DNA fragments up to 40 kbp.
According to the PCR principle, DNA amplification is carried out in three main steps.
- The double-stranded DNA of interest is denatured to separate into two individual DNA strands.
- Each strand in PCR steps is then allowed to hybridize with a primer (renaturation).
- The premier template duplex is used for DNA synthesis by the enzyme DNA polymerase.
These three steps- denaturation, renaturation, and synthesis are repeated again and again to generate multiple forms of targeted DNA.
Present-day, PCR is a common and indispensable technique used by researchers for researching biomedical and forensic samples. The essential requirements of the PCR technique are:
- DNA Template: A target DNA (100 − 35000 bp in length).
- Oligonucleotide Primers: Two primers (synthetic oligonucleotides of 17 − 30 nucleotide length) that are complimentary to regions flanking the target DNA.
- Deoxyribonucleotide triphosphate: Four deoxyribonucleotides (dATP, dCTP, dGTP, dTTP).
- DNA Polymerase: A DNA polymerase that can withstand a temperature of up to 95 ℃.
- Buffer System: A buffer solution that contains magnesium ions or potassium ions provides a suitable chemical environment for DNA denaturation and renaturation, polymerase activity, and stability.
Technique of Polymerase Chain Reaction
The actual technique of polymerase chain reaction involves repeated cycles for amplification of target DNA. Commonly, the polymerase chain reaction is carried out in a thermal cycler and small reaction tubes (0.2–0.5 mL volumes).
The thermal cycler in PCR can heat and cool the reaction tubes to achieve the temperatures required at each step. Typically, thermal cycles in PCR are carried out by a series of 20–40 repeated temperature changes where each cycle commonly consists of two or three discrete temperature steps.
Polymerase Chain Reaction Steps
The actual PCR technique involves repeated cycles to amplify target DNA. Each cycle in the PCR technique has three steps,
- Denaturation
- Renaturation or annealing
- Elongation
The three steps of PCR are related to temperature and time. Each cycle in PCR takes about 3−5 minutes. In normal practice, the PCR is carried out in an automatic machine.
Denaturation
On raising the temperature to about 95 ℃ for about 0.5 to 2 minutes, the DNA gets denatured and two strands are separated by breaking hydrogen bonds between the two strands of DNA. Such types of single strands now act as a template for the production of new DNA strands.
The temperature in the denaturation of the polymerase chain reaction should be provided for a longer time to ensure the separation of the two strands.
Renaturation or Annealing
When the temperature of the mixture is slowly cooled to about 55 ℃, the primer bases pair with the complimentary reagins flanking target DNA strands. Such a process is called renaturation or annealing. In this step, the primers bind to their complementary sequences on the template DNA.
A high concentration of premier ensures annealing between each DNA strand and the primer rather than the two stands of DNA. The two separated strands in the polymerase chain reaction run in the opposite direction. Therefore, two primers such as forward primer and reverse primer used in renaturation or annealing.
Elongation
At this step, the temperature is raised to 72-80℃ and initiation of DNA extension occurs at the 3´ − hydroxyl end of each primer. The premiers are extended by joining the bases complementary to DNA strands by the Taq polymerase enzyme. The synthetic process in PCR is quite comparable to the DNA replication of the leading strand.
Almost all PCR machines contain a heat-stable DNA polymerase, such as Taq polymerase. It is an enzyme that is isolated from the thermophilic bacterium Thermus aquaticus.
The enzyme taq polymerase can tolerate very high temperatures and adds DNA bases to the single strand. Therefore, a double-stranded DNA molecule is obtained during elongation.
Applications of Polymerase Chain Reaction
The polymerase chain reaction (PCR) is a laboratory technique that is used for rapidly producing or amplifying millions to billions of copies of a specific segment of DNA for medical, forensic, and biological research. Therefore, PCR is a common tool used in medical, forensic, and biological research labs. It has a vast number of applications in clinical diagnosis, genetic engineering, and forensic analysis.
Applications of PCR in Clinical Diagnosis
The specificity and sensitivity of PCR is highly useful for the clinical diagnosis of various diseases in humans. It may include genetic diseases, viral diseases, bacterial diseases, etc.
- Diagnosis of inherited diseases: Sickle anemia, β−thalassemia, and phenylketonuria.
- Diagnosis of retroviral infections: PCR test from cDNA is a variable tool for diagnosis and monitoring HIV infection.
- Diagnosis of bacterial infections: PCR test is used for the detection of tuberculosis, a bacterial infection caused by Mycobacterium tuberculosis.
- PCR in sex determination: Sex in humans and other animals can be determined by PCR machine by using primers and DNA probes specific for sex chromosomes. It is also useful for the detection of sex-related disorders in humans.
- Diagnosis of cancers: Several virtually induced cancers in humans can be detected by PCR machine.
- PCR in the diagnosis of COVID−19: The real-time PCR technique used for diagnosis of Covid−19.
PCR in Genetic Engineering
The differences in the genomes of the two organisms can be measured by PCR with random primers. The products are separated by electrophoresis or other chromatographic techniques for comparative identification.
Two genomes found from closely related organisms are expected to yield more similar bands. In genetic engineering, PCR can rapidly produce short pieces of DNA. It can produce large amounts of pure DNA.
PCR in Forensic Analysis
A single molecule of deoxyribonucleic acid (DNA) from any source (blood strains, hair, semen, etc) of an individual is adequate for amplification by polymerase chain reaction (PCR). Therefore, PCR is very important for the identification of criminal activity. Genetic fingerprinting can uniquely identify any person from the entire world population.
- Minute samples of DNA that can be isolated from a crime scene amplified by polymerase chain reaction (PCR) method. Therefore, these DNA samples can be compared with a DNA database of earlier evidence or convicts.
- The PCR method is used for amplifying DNA sequences from a very minuscule amount of genome that is used in DNA paternity testing. Therefore, DNA from unidentified humans obtained from crime spots can be tested, and compared with their possible parents, siblings, or children.
- Modern PCR machines amplify DNA sequences from a very minuscule amount of genome. Therefore, it provides a powerful and effective way to determine gender in forensic samples.
Different Types of PCR
Being a versatile technique, polymerase chain reaction (PCR) can be modified into various types according to the demands of the situation. Some types of PCR machines are:
- Real-time PCR
- Nested PCR
- Inverse PCR
- Multiplex PCR
Real-time Polymerase Chain Reaction (Real-time PCR)
Real-time polymerase chain reaction (Real-time PCR) contains a thermal cycler that can illuminate each nucleic acid sample with a beam of light with one specific wavelength and detect by fluorescence emitter excited by the fluorophore. The thermal cycler has the ability to heat and chill samples rapidly and DNA amplification is detected in real-time with the help of a fluorescent reporter.
Nested Polymerase Chain Reaction (Nested PCR)
Nested polymerase chain reaction (Nested PCR) method is usually designed to improve the sensitivity and specificity of DNA amplification. Two sequential amplification reactions take place in Nested PCR. Each of these uses a different set of primers.
Therefore, it may reduce the non-specific binding of products due to the amplification of unexpected primer binding sites.
Inverse Polymerase Chain Reaction (Inverse PCR)
Inverse polymerase chain reaction (Inverse PCR) is used to amplify DNA with only one known sequence. It is a variant of the polymerase chain reaction that is carried out even if only one sequence is available.
Multiplex Polymerase Chain Reaction (Multiplex PCR)
Multiplex polymerase chain reaction (Multiplex PCR) is a type of polymerase chain reaction method that is used for the amplification of multiple samples in a single PCR experiment. It contains multiple primer sets within a single PCR mixture and amplifies many different DNA sequences simultaneously. The multiple primer sets are designed to work at the same annealing temperature during PCR analysis.
Advantages of Polymerase Chain Reaction
The polymerase chain reaction (PCR) is a quick, simple, and the most accurate technique used to create unlimited copies of DNA from just one original strand. Therefore, PCR technology can play an important role in genetic testing and research.
The most common advantages of polymerase chain reaction method are
- PCR is a highly specific technique that distinguishes DNA sequences by just one nucleotide.
- It is a very useful technique when the amount of DNA sample is limited.
- Polymerase chain reaction (PCR) has various applications in genetic testing and research.
- PCR is an important tool in forensic laboratories because it is used widely in criminal investigations and paternity tests.
- PCR is a rapid and efficient technique because it can amplify a small amount of DNA sample to a million copies in just a few hours.
Frequently Asked Questions (FAQs)
What are the advantages of PCR?
The polymerase chain reaction (PCR) is a quick, simple, and the most accurate method used to create unlimited copies of DNA from just one original strand. Therefore, PCR is a rapid and efficient technique for amplification of a small amount of DNA sample to a million copies in just a few hours. It is a faster and less tedious technique than the traditional methods of gene cloning.
What is the principle of PCR?
Polymerase chain reaction (PCR) in biology is based on the principle of enzymatic replication of the nucleic acids. The double-stranded deoxyribonucleic acid (DNA) of interest is denatured to separate into two individual strands. Each strand of DNA is then allowed to hybridize with a primer. The primer-template duplex is used for DNA synthesis by the enzyme DNA polymerase.
These three steps in PCR, denaturation, renaturation, and synthesis are repeated again and again to generate multiple forms of the target DNA molecule.
What is PCR used for in biology?
The polymerase chain reaction (PCR) method is used in molecular biology to amplify many copies of small sections of DNA or a gene. It is a common tool used in medical and biological research labs to generate thousands to millions of copies of a particular section of DNA from a very small amount of DNA.
