PCR-Polymerase Chain reaction (PCR)

Content

• Introduction
• History
• Principle of PCR
• Stages of PCR
• PCR techniques     
• PCR Requirements
• Steps of PCR
• General guidelines for Primers     
•  Analysis Of Products of PCR
• Types of PCR
• Application of PCR
• Disadvantages of PCR
• Conclusion
• Reference

Introduction

The advent of the polymerase chain reaction (PCR) radically transformed biological science from the time it was first discovered (Mullis, 1990). For the first time, it allowed for specific detection and production of large amounts of DNA. PCR-based strategies have propelled huge scientific endeavors such as the Human Genome Project. The technique is currently widely used by clinicians and researchers to diagnose diseases, clone and sequence genes, and carry out sophisticated quantitative and genomic studies in a rapid and very sensitive manner. One of the most important medical applications of the classical PCR method is the detection of pathogens. In addition, the PCR assay is used in forensic medicine to identify criminals. Because of its widespread use, it is important to understand the basic principles of PCR and how its use can be modified to provide for sophisticated analysis of genes and the genome.

Polymerase chain reaction (abbreviated PCR) is a laboratory technique for rapidly producing (amplifying) millions to billions of copies of a specific segment of DNA, which can then be studied in greater detail. PCR involves using short synthetic DNA fragments called primers to select a segment of the genome to be amplified, and then multiple rounds of DNA synthesis to amplify that segment.

The PCR technique is based on the enzymatic replication of DNA. In PCR, a short segment of DNA is amplified using primer mediated enzymes. DNA Polymerase synthesises new strands of DNA complementary to the template DNA. The DNA polymerase can add a nucleotide to the pre-existing 3’-OH group only. Therefore, a primer is required. Thus, more nucleotides are added to the 3’ prime end of the DNA polymerase.

History

The history of modern PCR begins in 1976 with the isolation of Taq DNA polymerase from the thermophilic bacterium Thermus aquaticus. Its isolation meant that molecular biologists now had a thermostable enzyme capable of repeat PCR cycling without adding fresh DNA polymerase after each cycle. For those of us who can remember that far back, resetting the PCR reaction as many as 40 times over a 4–5 hour period was not much fun and did not feel like a great use of time, so the Taq enzyme made our lives better in so many ways!

Taq DNA polymerase was an instant success; in 1989, Science magazine selected the DNA polymerase molecule as the 'Molecule of the year'. Taq enzyme's impact on molecular biology became apparent in 1988 when Kary Mullis and the Cetus Corporation commercialized the enzyme for widespread use. With the introduction of thermal cyclers and commercial Taq enzymes, we could finally say goodbye to those laborious afternoons in front of the water bath to amplify DNA.

´  1983: Kary Mullis, a scientist working for the Cetus Corporation came up with the idea for the polymerase chain reaction.

´  1985: The polymerase chain reaction was introduced to the scientific community.

´  1987: Was awarded with noble prize for this discovery.

Principle Of PCR

PCR makes it possible to obtain, by in vitro replication, multiple copies of a DNA fragment from an extract. Matrix DNA can be genomic DNA as well as complementary DNA obtained by RT-PCR from a messenger RNA extract (poly-A RNA), or even mitochondrial DNA. It is a technique for obtaining large amounts of a specific DNA sequence from a DNA sample. This amplification is based on the replication of a double-stranded DNA template. It is broken down into three phases: a denaturation phase, a hybridization phase with primers, and an elongation phase. The products of each synthesis step serve as a template for the following steps, thus exponential amplification is achieved.

The polymerase chain reaction is carried out in a reaction mixture which comprises the DNA extract (template DNA), Taq polymerase, the primers, and the four deoxyribonucleoside triphosphates (dNTPs) in excess in a buffer solution. The tubes containing the mixture reaction are subjected to repetitive temperature cycles several tens of times in the heating block of a thermal cycler (apparatus which has an enclosure where the sample tubes are deposited and in which the temperature can vary, very quickly and precisely, from 0 to 100°C by Peltier effect). The apparatus allows the programming of the duration and the succession of the cycles of temperature steps. Each cycle includes three periods of a few tens of seconds. The process of the PCR is subdivided into three stages as follows:

                                                             Fig.-Steps of PCR

Stage of PCR

Exponential Amplification: With every cycle, amount of product is doubled.  The reaction is very sensitive.

Levelling of Stage: Reaction slows as DNA polymerase loses activity.

Plateau: No more product accumulate due to exhaustion of reagents and enzymes.

                                                                Fig.- Different Stages of PCR

 

PCR Requirement 

´  Template DNA: A segment of DNA to be amplified. Extracted from the sample.

´  Reaction buffer: (Tris-HCI, ammonium ions, KCI), magnesium ions, bovine serum albumin).

´  This buffer provides the ionic strength and buffering capacity needed during the reaction.

´  Monovalant and Divalant cations: Magnesium Chloride, Potassium. Works as co-factor for enzyme,

 

Fig.-PCR Requirement

´  Primers: small pieces of artificially made DNA strands.

       • Complimentary to 3' end of target DNA.

       • 20-30 nucleotides.

       • Two primers:

       a) A forward primer

       b) A reverse primer

´  DNA polymerase: It combines at the end of the primer and then sequentially adds new nucleotides to the DNA strand at 3' end complementary to the target DNA

• Taq Polymerase- has the unique characteristic to work efficiently in higher temperature. 

extracted from the bacteria Thermus aquaticus.

• Deoxynucleotide Triphosphates (dNTPs): dATP, dCTP, dGTP, and dTTP. raw material or the basic building blocks of the new DNA strands.

´  PCR Machine: A thermal cycler

Heat Stable DNA Polymerase

PCR (Polymerase chain reaction) is a technique that’s used to quickly generate millions of copies of a specific DNA segment to be used for more in-depth analysis. In PCR, a section of the genome to be amplified goes through multiple cycles of DNA synthesis at different temperatures, which allows numerous copies of the target region to be generated. During the process, the temperature can rise to around 60°C, causing denaturation of most enzymes. DNA polymerase is the best enzyme for PCR because it is thermostable and does not denature at higher temperatures.

•           Taq DNA Polymerase: - Isolated from Thermus aquaticus, It lack proofreading thus has low fidelity. (1/9000nt)
•            Pfu DNA Polymerase: - Isolated from Pyrococcus furiosus. It has proofreading activity, thus high the fidelity of the PCR products.
•            Vent DNA Polymerase: - Isolatedfrom Thermococcus litoralis, It has proofreading activity, thus high the fidelity of the PCR product.
•            Pwo DNA Polymerase: - It has isolated from pyrococcus woesei. Fidel Enzyme.

Steps in PCR

Step 1: Denaturing

·         The reaction mixture is heated to 94-95⁰C, for between 15 and 30 seconds.

·         The high temperature causes the hydrogen bonds between the bases in two strands of template DNA to break and the two strands to separate.

·         This results in two single strands of DNA, which will act as templates to produce the new copies of each strand of DNA.

·         It is important that the temperature is maintained at this stage for long enough to ensure that the DNA strands have separated completely.

Fig.-Denaturation of DNA template by disrupting the hydrogen bonds between complementary bases of the DNA strands, yielding single strands of DNA.

Step 2: Annealing

·   The reaction is cooled to enable the primers to attach to a specific location on the single-stranded template DNA by way of hydrogen bonding.

·  The temperature depends on the characteristics of the primer, but is usually between 50 and 65⁰C.

·  The two separated strands of DNA are complementary and run-in opposite directions (from one end – the 5’ end – to the other – the 3’ end). As a result, there are two primers – a forward primer and a reverse primer.

·  This step is essential because the primers serve as the starting point for DNA synthesis, by providing a short region of double stranded DNA for the polymerase enzyme to work with. Only once the primer has bound can the polymerase enzyme attach and start making the new complementary strand of DNA from the loose DNA bases, in the extending step.

·    The annealing step usually takes about 10-30 seconds.

Fig.-Then the temperature is lowered to a temperature between 46-60 °C. The exact temperature depends on the experiment, and is known as the annealing temperature. At this point, the primers will attach, or anneal, to their binding positions on the single strands of the template DNA.

Step 3: Extending

·   The heat is increased to 72⁰C to enable the new DNA to be made by a special Taq DNA polymerase enzyme which adds DNA bases.

·   Taq DNA polymerase is an enzyme taken from the bacteria Thermus aquaticus (“Taq”):

·   This bacterium normally lives in hot springs so can tolerate temperatures above 80⁰C, but its optimum temperature is 72⁰C.

·  The bacteria’s DNA polymerase is very stable at high temperatures, which means it can withstand the temperatures needed to break the strands of DNA apart in the denaturing stage of PCR.

·   DNA polymerase from most other organisms would not be able to withstand these high temperatures. For example, human polymerase works ideally at 37˚C (body temperature).

·  At 72⁰C, the Taq polymerase begins to build the complementary strand. It attaches to the primer and then adds DNA bases to the single strand one-by-one in the 5’ to 3’ direction.

·   The result is a brand-new strand of DNA and a double-stranded molecule of DNA.

·  The duration of this step depends on the length of DNA sequence being amplified. It usually takes around one minute to copy 1,000 DNA bases.

Fig.-optimal temperature for the polymerase enzyme (1), from which PCR takes its name, to start working. The polymerase enzyme builds DNA strands, and it will extend the DNA from the primer along the DNA template, creating a new DNA strand, which combines with the single-stranded template to form a double strand. The polymerase enzyme uses dNTPs (2), free DNA nucleotide bases as the building blocks for the new strand.

General Guidelines for primers

1. Length:

´  Shorter primers have a tendency to go and anneal to the non-target sequence of the DNA template.

´  Short primer may offer sufficient for a simple template such as a small plasmid

´  But a long primer may be required when using eukaryotic genomic DNA as template. In practice, 20-30 nucleotides is generally satisfactory.

2. Mismatches:

´  Do not need to match the template completely.

´  Often beneficial to have C or Gas the 3' terminal nucleotide which makes the binding of the 3' end of the primer to the template more stable.

3. Melting Temperature Tm:

´  Melting temperature is the temperature at which one half of the DNA duplex will dissociated and become single stranded. Typically, the annealing temperature is about 3-5 degrees Celsius bellow the Tm of the primers used.

´  Primers with melting temperatures in the range of 52-58°C generally produce the best results. Primers with melting temperatures above 65°C have a tendency for secondary annealing.

´  Tm can be calculated from the following formula:

´  Tm= (4 x [G+C]) + 2 x [A+T])

4. Internal Secondary Structure:

´  Should be avoided in order to prevent the primer to fold back on itself and not be available to bind to the template.

5. Primer-Primer Annealing:

´  Also, important to avoid the two primers being able to anneal to each other. Extension by DNA polymerase of two self-annealed primers leads to formation of a primer dimer.

6. G/C content:

´  Ideally a primer should have a near random mix of nucleotides, a 50% G/C content.

Analysis of Product in PCR

There are two main methods of visualizing the PCR products: (1) staining of the amplified DNA product with a chemical dye such as ethidium bromide, which intercalates between the two strands of the duplex or (2) labeling the PCR primers or nucleotides with fluorescent dyes (fluorophores) prior to PCR amplification. The latter method allows the labels to be directly incorporated in the PCR product. The most widely used method for analyzing the PCR product is the use of agarose gel electrophoresis, which separates DNA products on the basis of size and charge. Agarose gel electrophoresis is the easiest method of visualizing and analyzing the PCR product. It allows for the determination of the presence and the size of the PCR product (Figure). A predetermined set of DNA products with known sizes are run simultaneously on the gel as standardized molecular markers to help determine the size of the product.

1.Agarose gel electrophoresis: Tells:

´  Any band present in agarose gel electrophoresis.

´  Any other band of different size.

´  Is there a smear pattern

´  Single sharp band of expected size is present or not.

2. Cloning of Product: Done when gene is present in very tiny amount.

3. Sequencing of Product: By automated sequencer machine to analyse sequence of DNA formed as PCR product.

Fig.- Analysis of product in PCR

Types Of PCR 

•          Reverse Transcriptase PCR (RT-PCR)
•         Multiplex PCR
•         Hot Start PCR
•         Nested PCR
•         Cold PCR

1.Reverse Transcriptase PCR (RT-PCR)

Reverse transcription is the enzyme-mediated synthesis of a DNA molecule from an RNA template. The resulting DNA, known as cDNA, can be used as a template for PCR amplification. Reverse transcription followed by PCR is known as RT-PCR (reverse transcription-PCR).

RT reactions typically include the following reagents

1.      RNA template: RNA can be purified from a variety of biological sample types. RNA is inherently unstable, so strict precautions must be taken to prevent degradation during the extraction process and subsequent handling steps. The quality and purity of the RNA template are crucial to the success of RT-PCR.

2.      Reverse transcriptase: Reverse transcriptase enzymes are RNA-directed DNA polymerases derived from retroviruses that can synthesize single-stranded cDNA fragments complementary to an RNA template.

3.      Oligonucleotide primers: Three different types of primers can be used in the RT reaction (Figure):

·   Oligo(dT): Primers that selectively anneal to the poly(A) tails found on most messenger RNA (mRNA) molecules. Only polyadenylated RNAs will be reverse transcribed in an oligo(dT)-primed reaction. The resulting cDNA can be used in multiple PCR reactions, allowing for analysis of more than one gene.

·   Random hexamers: A mixture of random hexanucleotide primers that anneal to sequences throughout the target RNA, resulting in reverse transcription of both polyadenylated and nonpolyadenylated RNAs. Random hexamer-primed cDNA can be used in multiple PCR reactions, allowing for analysis of more than one target region.

·    Sequence-specific: Primers that hybridize to a specified gene sequence and result in reverse transcription of a specific mRNA. When using sequence-specific primers, a new RT reaction must be performed for each gene of interest.

4.  Nucleotides: The four dNTPs – dATP, dTTP, dCTP, and dGTP – are the building blocks of the cDNA strands synthesized by reverse transcription.

5.   RNase inhibitor: Addition of an RNase inhibitor helps protect the RNA template from the activity of RNA-degrading ribonucleases present in the laboratory environment.

6.  Reaction buffer: RT reaction buffer provides optimal conditions for enzyme activity. An appropriate buffer is usually supplied with the reverse transcriptase enzyme.

                                                                      Fig- RT-PCR

´  RNA molecules are first converted to complementary DNA (cDNA) using reverse transcriptase (RNA dependent DNA polymerase).

´  cDNA then acts as a template.

´  Can be conducted in a single tube or as two steps in different tubes.

´   USES:

1.      Detection of infectious agent.
2.       Genetic disease diagnosis.
3.       Gene insertion.
4.     Diagnosis of cancer.

2.Multiplex PCR

In multiplex PCR, two or more primer sets designed for amplification of different targets are included in the same PCR reaction. Using this technique, more than one target sequence in a clinical specimen can be amplified in a single tube. As an extension to the practical use of PCR, this technique can save time and effort. The primers used in multiplex reactions must be selected carefully to have similar annealing temperatures and must be not complementary to each other. The amplicon sizes should be different enough to form distinct bands when visualized by gel electrophoresis. Multiplex PCR can be designed in either single-template PCR reaction that uses several sets of primers to amplify specific regions within a template, or multiple-template PCR reaction, which uses multiple, templates and several primers sets in the same reaction tube (Fig.). Although the use of multiplex PCR can reduce costs and time to simultaneously detect two, three, or more pathogens in a specimen, multiplex PCR is more complicated to develop and often is less sensitive than single-primer-set PCR. The advantage of multiplex PCR is that a set of primers can be used as internal control, so that we can eliminate the possibility of false positives or negatives. Furthermore, multiplex PCR can save costly polymerase and template in short supply.

 

Fig.- Multiplex PCR

 

Principle

M-PCR is the simultaneous amplification of more than one target sequence in a single reaction tube using more than one primer pair. This co-amplification of two or more targets in a single reaction is dependent on the compatibility of the PCR primers used in the reaction. All primers in the reaction must have similar melting temperatures (Tm) so they anneal to and dissociate from complementary DNA sequences at approximately the same temperatures, allowing each amplification to proceed at the selected temperature. This procedure could not be done if one primer set was annealing at the time that another primer set was dissociating from its target. Therefore, all primers must be selected so their TmS are within a few degrees (°C) of each other. Each amplification proceeds independently of the others (as long as none of the reagents is present at rate-limiting concentrations) and each specific amplification product or amplicon is synthesized in an unencumbered way. Primers should also be chosen that define amplicons of approximately the same size range (100–500 bp), so each is synthesized efficiently and at equal rates. Each M-PCR assay must also have a detection step capable of identifying each amplicon. This can be done by gel electrophoresis with visual identification of separate amplicons of different size or by hybridization with specific DNA probes and detection using spectrophotometry, fluorometry, autoradiography, or chemiluminescence.

Types of Multiplex PCR

Multiplexing reactions can be broadly divided in two categories:

1. Single Template PCR Reaction

This technique uses a single template which can be a genomic DNA along with several pairs of forward and reverse primers to amplify specific regions within a template.

2. Multiple Template PCR Reaction

It uses multiple templates, and several primers sets in the same reaction tube. Presence of multiple primers may lead to cross hybridization with each other and the possibility of mis-priming with other templates.

ADVANTAGES:

·    This technique has the potential to produce considerable savings in time and effort within the laboratory.

·   Without compromising on the utility of the experiment.

DISADVANTAGES:

·    Optimization is difficult; since many sets of forward and reverse primers are to be designed for use.

·    Increases cost. Presence of multiple primer may lead to cross hybridization with each other and the possibility of miss-priming with other templates.

3.Hot Start PCR

As soon as the PCR reagents have all been mixed together, it is possible for the DNA polymerase to start synthesis.

This may happen while the reaction mixture is being heated for the first time, and is at a temperature low enough to allow non-specific annealing of primer to template, generating a range of non-specific products. 

This problem would be prevented if DNA synthesis could not take place until the first cycle had reached its maximum temperature. This is the basis of hot-start PCR. In the simplest form, the DNA polymerase is not added to the reaction tubes until they have reached the DNA melting temperature of the first cycle.

 

Figure- Diagrammatic Representation of Hot-Start PCR

4.Nested PCR

Nested polymerase chain reaction (Nested PCR) is a modification of polymerase chain reaction intended to reduce non-specific binding in products due to the amplification of unexpected primer binding sites. Nested polymerase chain reaction involves two sets of primers, used in two successive runs of polymerase chain reaction, the second set intended to amplify a secondary target within the first run product.

Figure- Diagrammatic Representation of Nested PCR

 

5. Cold PCR

´  Based on modification of the critical temperature at which mutation-containing DNA is preferentially denatured over wild (non-mutated) type.

     • APPLICATION:

1.      ✓ Detection of mutation in oncology specimens.

2.      ✓ Assessment of residual disease after surgery or chemotherapy Tailoring the therapy for individual patients

3.      ✓ Disease staging and molecular profiling for prognosis.

Fig.- Diagrammatic Representation of Cold PCR

 

Application Of PCR

•       Research
•       Clinical

Research Application

•       DNA Sequencing
•       Bioinformatics
•       Classification of Organisms
•       Gene Expression studies
•       Drug Discovery

Clinical Application

•        Diagnosis of Infection- Viral, Bacterial & Parasitic.
•        Cancer- Diagnosis & Prognosis

1.       Mutation Detection - Oncogenes, Tumour Suppressor genes 
2.      Chromosomal Changes Translocation, Rearrangements
3.       Monoclonality detection-B & T cell Lymphoma
4.      Minimal Residual Disease follow up cases.

•      Genetic diseases - Down's Syndrome, Cystic Fibrosis
•      Forensic Pathology-

1.       Paternity of child
2.       Identify corpse or mutilated body.
3.       Identify criminal

·         Gene Therapy

DISADVANTAGES OF PCR

•        Setting up and running requires high technical skills.
•        Not easy to quantitate results
•        High 'equipment cost.
•        High sterile environment should be provided

Conclusion

PCR is one vital process to amplify specific DNA fragments from small amounts of DNA sample. The speed and ease of use, sensitivity, specificity and robustness of PCR has revolutionized molecular biology and made PCR the most widely used technique with great spectrum of research and diagnostic applications.

 

Reference

•     https://old.abmgood.com/marketing/knowledge_base/polymerase_chain_variation_system.php
•      https://www.researchgate.net/figure/Schematic-workflow-for-co-amplification-at-lower-denaturation-temperature-COLD-PCR_fig1_343071799
•      https://www.sigmaaldrich.com/IN/en/technical-documents/technical-article/genomics/pcr/what-is-hot-start-pcr
•      https://bento.bio/
•      https://bento.bio/protocol/biotechnology-101/introduction-to-pcr/
•      https://www.genome.gov/genetics-glossary/Polymerase-Chain-Reaction