The PCR is a technique that enables the specific amplification and hence detection of target DNA sequences from a mixture of nucleic acid extract. Nowadays, a lot of different types of PCR techniques are using for various purposes.
A combination of short, specific primers and thermo stable DNA polymerases are used to amplify (millions or billions!) the target sequence through repeated cycle of template denaturation, primer annealing and primer extension along the template strand. The operation of PCR cycles for 30-40 times allows an exponential increase in the amount of target DNA strands.
By addition of reverse transcription (RT) step PCR can also be applied to the synthesis of complementary DNA (cDNA) for RNA template.
The technique is widely used in the microbiology laboratories, many biological and medical fields, including molecular biology researches, medical diagnostics and even some ecological branches for the detection of variety of microorganisms including fungi, bacteria, mycoplasmas, viruses and viroids. Nowadays, (in 2020) the name “PCR” is very commonly seen in news due to this covid-19 pandemic.
Principle of PCR
In the PCR experiment, the target DNA is mixed with Taq polymerase, the two oligonucleotide primers, and the supply of nucleotides. The amount of target DNA can be minimal because PCR is extremely sensitive and will work with just a single starting molecule. The reaction is started by heating the mixture to 94°C. Under this temperature, the hydrogen bonds that hold together the two polynucleotides of the double helix are broken, so the target DNA becomes denatured into single-stranded molecules. Then temperature should be reduced to 50–60°C, which results in some rejoining of the single strands of the target DNA. But also, it allows the primers to attach to their annealing positions. After that DNA synthesis can begin, so the temperature is raised to 74°C, just below the optimum for Taq polymerase.
The cycle(chain) of denaturation–annealing–synthesis is now repeated. Usually, PCR machines run for 30 cycles. It means that after 30 cycles, there will be over 130 million short products derived from each starting nucleic acid. In real terms, this equates to several micrograms of PCR products from a few nanograms or less of target DNA.
Types of PCR
Reverse Transcriptase PCR (RT-PCR)
RT PCR is quite famous these days due to its’ tremendous usefulness in identifying Coronavirus patients. Reverse Transcription PCR (RT-PCR) is a traditional PCR modification whereby RNA molecules are first translated into complementary DNA (cDNA) molecules which can then be amplified by PCR. In RT-PCR the RNA template is initially converted using reverse transcriptase to a cDNA. The cDNA also uses PCR to act as a blueprint for exponential amplification. RT-PCR can be carried out either in a single tube, or in different tubes as two steps. The one-step method is more efficient with fewer chances of contamination and variable incorporation.
RT-PCR is used in testing techniques, gene injection, the treatment of genetic disorder and cancer detection.
Reverse-Transcriptase Real-Time PCR (RT-qPCR)
RT-PCR is usually aligned with Reverse Transcriptase Real-Time PCR (RT-qPCR) generating q-PCR; This allows in real-time quantification of DNA after amplification.
Real-Time PCR (Quantitative PCR (qPCR))
Quantitative PCR (qPCR), also known as real-time PCR or quantitative real-time PCR, is a PCR-based technique which combines amplification of a target DNA sequence with quantification of that DNA species concentration in the reaction. Real-time PCR is based on the use of fluorescent dye.
The sample’s nucleic acid concentration is quantified using the fluorescent dye or using the oligonucleotides labeled fluorescent.
q-PCR is used in genotyping and pathogen quantification, microRNA analysis, cancer diagnosis, microbial load monitoring and Genetically Modified Organisms (GMO) diagnosis.
The polymerase chain reaction (PCR) is a test tube system for DNA replication that allows selective amplification of a “target” DNA sequence of several million folds in just a few hours. PCR allows the synthesis of different DNA fragments using a DNA-polymerase enzyme which is involved in the cellular genetic material replication. This enzyme synthesizes a complementary DNA sequence as a small fragment (primer) is connected to one of the DNA strands chosen to start the synthesis at the specific site. Primers restrict the sequence to be repeated, and the effect is the multiplication of billions of copies of a single DNA sequence. Conventional PCR is used in selective DNA isolation, amplification and quantification of DNA, medical and diagnostic approaches, diagnosis of infectious diseases, forensic studies and fields of research.
Amplified fragment length polymorphism (AFLP) PCR
It is a technique based on PCR which uses selective amplification of a portion of digested DNA fragments to produce specific fingerprints for interesting genomes. Without prior knowledge of the genomic sequence this technique can quickly generate large numbers of marker fragments for any organism. AFLP PCR uses restriction enzymes to digest genomic DNA and allows adaptors to be applied to the fragment’s sticky ends. A portion of the restriction fragments is then selected for amplification using primers complementing the adaptor sequence.
The amplified sequences on the electrophoresis of the agarose gel are isolated and visualized for denaturing. AFLP PCR is used for a variety of applications, such as the assessment of genetic diversity within species or among closely related species, the inferment of population-level phylogenies and biogeographic patterns, the generation of genetic maps and the determination of linkage between cultivars.
Single Specific Primer-PCR (SSP-PCR)
This allows the amplification of double-stranded DNA even when the information about the sequence is only available at one end. This method, the Single Specific Primer-PCR (SSP-PCR), allows for the amplification of genes for which only partial sequence information is available, and permits unidirectional genome walking from known to unknown chromosome regions.
A new PCR method is called Miniprimer PCR using “miniprimers” of engineered polymerase and 10-nucleotides. This approach is used to show sequences of novel 16S rRNA genes which would not have been identified with normal primers. Miniprimer PCR uses a polymerase enzyme that is thermostable and can extend from short primers (9 or 10 nucleotides). This method allows PCR targeting to smaller binding regions, and is used to amplify highly conserved sequences of DNA, such as the rRNA gene 16S (or eukaryotic 18S).
All-specific polymerase chain reactions (AS-PCR) are techniques based on allelele-specific primers that can be used to analyze polymorphism of single nucleotides. Also called the (amplification refractory mutation system) ARMS-PCR is the allele-specific PCR, corresponding to the use of two different primers for two different alleles. One is the mutant set of refractory (resistant) primers to the normal PCR, and the other is the normal set of primers that are refractory to the mutant PCR reaction.
The 3 ‘ends of these primers are modified so that the normal allele can be amplified by one set of the primers while others amplify the mutant allele. This mismatch allows for amplification of a single allele by the primer.
It is commonly used in the diagnosis of single point gene defects, such as sickle cell anemia and thalassemia. It is also used for the precise identification of genotypes from the ABO blood stream.
PCR assembly is a method of assembling large oligonucleotides from multiple shorter fragments of DNA. The size of the oligonuleotides used in PCR is 18 base pairs, while PCR lengths of up to 50bp are used in the assembly to ensure correct hybridization. The oligonucleotides bind to complementary fragments during the PCR processes, and are then loaded with polymerase enzyme. Thus, each cycle of this PCR decreases arbitrarily the length of various fragments, depending on which oligonucleotides locate each other.
Assembly PCR is used to improve the yield of the desired protein and can also be used to make large quantities of RNA for structural or biochemical studies.
Alu PCR is a simple and easy technique of DNA fingerprinting, based on simultaneous examination of multiple genomic loci accompanied by repetitive elements of Alu. Alu components are small stretches of DNA initially distinguished by the activity of the endonuclease limiting Arthrobacter luteus (Alu). Alu elements are one of the most abundant transposable elements found throughout the human genome and have been used as genetic markers and play a role in evolution. In Alu PCR, two complementary fluorochrome-labeled primers are used to perform the PCR and the PCR products are then analyzed.
Several genetically inherited human diseases and various forms of cancer have been used to insert alu. This PCR thus plays a crucial role in the detection of these diseases and mutations.
Co-amplification at lower denaturation temperature-based polymerase chain reaction (COLD-PCR) is a novel form of PCR that selectively amplifies variants of low-abundance DNA from mixtures of wild-type and mutant-containing (or variant-containing) sequences, regardless of the mutation type or position on the amplicon. This method is based on the critical temperature modification at which mutation-containing DNA is preferentially melted over wild type.
After denaturation there is an intermediate annealing process that allows for the hybridization of wild-type and mutant allele. This misalignment significantly affects the fusion temperature of the ds DNA. These heteroduplexes combine and are used as a base. As a result, a greater percentage of small variant DNA will be amplified and will be available for subsequent PCR rounds. PCR plays a vital role in detecting mutations in oncology specimens, especially in heterogeneous tumors and corporal fluids.
This PCR also assists in the determination of latent illness following surgery or chemotherapy and the staging of illness and molecular profiling for prognosis or specific patient tailoring.
Asymmetric PCR is a variation of PCR which is used to amplify one strand of the original DNA more preferably than the other. Asymmetric PCR differs from regular PCR because of the excessive amount of primers used for a selected strand.
If the asymmetric PCR advances, the lower limiting concentration prim is quantitatively integrated into, and used up, newly synthesized double-stranded DNA. Consequently, after depletion of the limiting primer, linear synthesis of the targeted single DNA strand from the excess primary is produced.
It is useful when only one of the two complementary strands, such as in sequencing and hybridization probing, is required for amplification.
Colony PCR is a method in which the identification of the DNA of interest inserted into the plasmid is obtained by designing specific primers of the inserted DNA. Two sets of primers can be used to directly amplify the bacterial colony which contains the plasmid. A bacterial colony which contains all other PCR reagents is taken and inserted directly into the master mix.
The main application of colony PCR is to identify the correct ligation and insert the inserted DNA into bacteria as well as the plasmid yeast.
Digital PCR (dPCR)
Digital PCR (dPCR) is a quantitative PCR technology which provides a sensible and efficient way to measure the amount of DNA or RNA present in a sample.
For dPCR, before the amplification step, the initial sample mix is divided into a large number of individual wells, resulting in either target sequence present in each well or not. Based on the presence or absence of fluorescence the calculation of the absolute number of targets present in the original sample is done in the amplified reaction wells.
Wells with a fluorescent signal are considered positive and are scored as “1” while wells with no such signal are negative and scored as “0.” Through Poisson statistical analysis the concentration of the target sequence present in the initial sample is then determined.
DPCR is used to determine the total number of DNA and RNA viruses, bacteria and parasites in a variety of clinical specimens, particularly where there is no well-calibrated standard.
Fast cycling PCR
Fast cycling PCR is a technology based on PCR which enables the amplification of specific PCR products with significantly reduced cycling time. In this method the concept is the same as traditional PCR, the only difference is when amplifying.
The buffer used in this PCR increases the affinity of Taq DNA polymerases to short single-stranded DNA fragments, reducing the time needed for successful annealing of the primers to just 5 seconds.
Fast cycling PCR is essential for processes requiring rapid cycles, and it also helps to diagnose diseases and mutations quickly.
In-Situ Polymerase Chain Reaction (In-situ PCR) is an effective method for detecting tiny amounts of rare nucleic acid sequences in frozen or paraffin-embedded cells or tissue sections to compartmentalize those sequences within the cells. This approach involves tissue fixation that maintains the cell morphology, which is then accompanied by proteolytic enzyme treatment to provide the PCR reagents with an entry to operate on the target DNA. The reagents amplify the target sequences and are then detected using standard immunocytochemical protocols.
In-situ PCR is applicable for infectious disease diagnosis, DNA quantification, detection of even small amounts of DNA, and is widely used in organogenesis and embryogenesis study.
High Fidelity PCR
High-fidelity PCR is a modified PCR method that uses a low error rate DNA polymerase and results in a high degree of accuracy in interest-bearing DNA replication.
Such enzymes have a significant binding affinity during amplification to get the right nucleoside triphosphate. In the case of an incorrect binding in the active site of polymerase, the incorporation is slowed because of the active site complex architecture.
For experiments whose outcome depends on the correct DNA sequence, such as cloning, SNP analysis, NGS applications, high-fidelity amplification is key.
High-Resolution Melt (HRM) PCR
High-Resolution Melt (HRM) PCR is an incredibly effective tool for mutation identification, polymorphism and epigenetic variations in double stranded DNA Samples.
It is enormously cost-effective over other methods for genotyping, such as sequencing and typing of Taqman SNP. This makes it ideal for projects to genotyping on a large scale. It is quick and strong, allowing huge quantities of samples to be accurately genotyped in rapid time.
It’s fast. With a good quality HRM assay, non-geneticists can perform powerful genotyping in any laboratory that has access to an HRM-capable real-time PCR machine.
Hot start PCR
Hot start PCR is a novel type of traditional polymerase chain reaction (PCR) which reduces the occurrence of unwanted products and primary dimers due to non-specific DNA amplification at room temperatures. The fundamental principle of hot-start PCR is to separate one or more reagents from the reaction mix until the mixture reaches the temperature of denaturation upon heating.
Hot-start PCR significantly reduces non-specific binding, primer-dimer formation, and often increases product yield. It also calls for less effort and lowers the risk of contamination.
Inverse PCR is especially useful for identifying insert positions of various transposons and retroviruses in the host DNA. Inverse polymerase chain reaction (Inverse PCR) is one of the variations of the polymerase chain reaction used when only one sequence is understood to amplify DNA.
Conventional PCR requires primers complementary to both target DNA terminals, but Inverse PCR allows amplification, even if only one sequence from which primers can be designed is available. The inverse PCR involves a series of restriction digestion followed by ligation, resulting in a looped fragment that can then be primed for PCR through a single known sequence section.
Then, as with other processes of reaction in the polymerase chain, the temperature-sensitive DNA polymerase amplifies the DNA.
Methylation-specific PCR (MSP)
Methylation-specific PCR (MSP) is a method used to detect and analyze methylation patterns of DNA in CpG islands. DNA is modified to perform MSP, and PCR is performed with two primer pairs, respectively, which are detectable methylated and nonmethylated DNA.
The DNA undergoes bisulfite treatment for the conversion of cytosine to uracil, and the methylated sequences are then selectively amplified with specific primers
Methylated sequence identification is important as excessive methylation of CpG dinucleotides in the promoter suppresses gene expression.
Multiplex PCR is a popular technique in molecular biology used to amplify multiple targets in a single PCR test cycle. In Multiplex PCR multiple primers and temperature-mediated DNA polymerase are used in a thermal cycler to amplify DNA. All the primers pairs designed for Multiplex PCR must be optimized so that the same temperature of the annealing is optimal for all pairs during PCR.
When multiple sequences are targeted at once, additional information can be generated from a single test run, which would otherwise require a larger amount of reagents and a considerable amount of time and effort. Its technique has been used in a variety of fields, including genotyping, mutation and polymorphism analysis, microsatellite STR analysis, pathogens or genetically modified organisms identification, etc.
Multiplex PCR is useful in diagnostic laboratories to detect various micro-organisms which cause the same types of diseases.
Repetitive sequence-based PCR
Repeated sequence-based PCR (rep-PCR) is a modified PCR technique that uses primers that target non-coding, interspersed repetitive sequences throughout the bacterial genome.
These non-coding, repeated sequence blocks can serve as multiple genetic targets for oligonucleotide organisms, allowing individual bacterial strains to produce specific DNA profiles or fingerprints. The main application of rep-PCR is in the typing of various bacteria by molecular strain. It is also used to discriminate epidemiologically against diverse pathogens.
Solid Phase PCR
Solid-phase PCR (SP-PCR) is a unique PCR technique that enables target nucleic acids to be amplified on a solid support in which one or both primers are immobilized on the surface.
The spatial isolation of the primers minimizes dramatically unfavorable priming interactions, thereby avoiding the creation of priming-dimers and allowing for greater amplification of multiplexing. The fundamental principle of this innovative approach is to add the 5′-end of the primers to the surface, rather than allowing the primers to disperse freely in a bulk solution. A DNA target which is freely diffused can be caught on the surface and then copied by polymerase.
The copy remains attached to the surface whereas after the annealing step, the initial DNA molecule returns to solution. The free end of the attached copy complements its sequence by hybridizing to the primer (attached to the surface), and the amplification process can begin.
Suicide PCR is widely used in experiments where the highest priority is to prevent false positives and to guarantee the accuracy of the amplified sample.
The method requires the use of any combination of primers only once in a PCR which should not have been used in any positive control PCR reaction. These primers should always target a genomic region that has never been amplified before this particular primer or any other set of primers was applied.
This arrangement ensures that there is no contaminating DNA present in the laboratory from previous PCR reactions which could otherwise generate false positives. Suicide PCR is used in paleogenetic studies involving an examination of preserved genetic material from ancient organisms remains.
Thermal asymmetric interlaced PCR (TAIL-PCR)
TAIL PCR is a powerful recuperation tool for DNA fragments adjacent to known sequences.
TAIL – PCR uses three nested primers in sequential reactions along with a low melting temperature random degenerate primer, such that the relative amplification frequencies of similar and non-specific products may be thermally regulated. This approach is highly reliable, such that the unpurified components of TAIL-PCR can be sequenced directly. It also allows full-length functional gene cloning.
Variable Number of Tandem Repeats (VNTR) PCR
In forensic science, they are important markers for the individualization.
Fragments are amplified in VNTR PCR which showed little variation within a species but showed differences between species. Thanks to the polymerase chain reaction (PCR), it can successfully amplify from a very small amount of genomic deoxyribonucleic acid (DNA).
aun, O., & Schönswetter, P. (2012). Amplified fragment length polymorphism: an invaluable fingerprinting technique for genomic, transcriptomic, and epigenetic studies. Methods in molecular biology (Clifton, N.J.), 862, 75–87. https://doi.org/10.1007/978-1-61779-609-8_7
Cardelli, Maurizio. (2011). Alu PCR. Methods in molecular biology (Clifton, N.J.). 687. 221-9. 10.1007/978-1-60761-944-4_15.
T.A.BROWN. (2010). GENE CLONING & DNA ANALYSIS (6th ed.). A John Wiley & Sons, Ltd,Publication.
Erandi Ranasinghe – Chemist