Polymerase chain reaction
The polymerase chain reaction (PCR) is a method widely used to make millions to billions of copies of a specific DNA sample rapidly, allowing scientists to amplify a very small sample of DNA (or a part of it) sufficiently to enable detailed study. PCR was invented in 1983 by American biochemist Kary Mullis at Cetus Corporation. Mullis and biochemist Michael Smith, who had developed other essential ways of manipulating DNA, were jointly awarded the Nobel Prize in Chemistry in 1993.[1]
PCR is fundamental to many of the procedures used in
The majority of PCR methods rely on
Almost all PCR applications employ a heat-stable DNA polymerase, such as Taq polymerase, an enzyme originally isolated from the thermophilic bacterium Thermus aquaticus. If the polymerase used was heat-susceptible, it would denature under the high temperatures of the denaturation step. Before the use of Taq polymerase, DNA polymerase had to be manually added every cycle, which was a tedious and costly process.[4]
Applications of the technique include
Principles
PCR amplifies a specific region of a DNA strand (the DNA target). Most PCR methods amplify DNA fragments of between 0.1 and 10
A basic PCR set-up requires several components and reagents,[8] including:
- a DNA template that contains the DNA target region to amplify
- a DNA polymerase; an enzyme that polymerizes new DNA strands; heat-resistant Taq polymerase is especially common,[9] as it is more likely to remain intact during the high-temperature DNA denaturation process
- two DNA sense and anti-sense strands of the DNA target (DNA polymerase can only bind to and elongate from a double-stranded region of DNA; without primers, there is no double-stranded initiation site at which the polymerase can bind);[10]specific primers that are complementary to the DNA target region are selected beforehand, and are often custom-made in a laboratory or purchased from commercial biochemical suppliers
- deoxynucleoside triphosphates, or dNTPs (sometimes called "deoxynucleotide triphosphates"; nucleotides containing triphosphate groups), the building blocks from which the DNA polymerase synthesizes a new DNA strand
- a buffer solution providing a suitable chemical environment for optimum activity and stability of the DNA polymerase
- better source needed]
The reaction is commonly carried out in a volume of 10–200
Procedure
Typically, PCR consists of a series of 20–40 repeated temperature changes, called thermal cycles, with each cycle commonly consisting of two or three discrete temperature steps (see figure below). The cycling is often preceded by a single temperature step at a very high temperature (>90 °C (194 °F)), and followed by one hold at the end for final product extension or brief storage. The temperatures used and the length of time they are applied in each cycle depend on a variety of parameters, including the enzyme used for DNA synthesis, the concentration of bivalent ions and dNTPs in the reaction, and the
- Initialization: This step is only required for DNA polymerases that require heat activation by hot-start PCR.[13] It consists of heating the reaction chamber to a temperature of 94–96 °C (201–205 °F), or 98 °C (208 °F) if extremely thermostable polymerases are used, which is then held for 1–10 minutes.
- DNA melting, or denaturation, of the double-stranded DNA template by breaking the hydrogen bondsbetween complementary bases, yielding two single-stranded DNA molecules.
- Annealing: In the next step, the reaction temperature is lowered to 50–65 °C (122–149 °F) for 20–40 seconds, allowing annealing of the primers to each of the single-stranded DNA templates. Two different primers are typically included in the reaction mixture: one for each of the two single-stranded complements containing the target region. The primers are single-stranded sequences themselves, but are much shorter than the length of the target region, complementing only very short sequences at the 3' end of each strand.
- It is critical to determine a proper temperature for the annealing step because efficiency and specificity are strongly affected by the annealing temperature. This temperature must be low enough to allow for hybridization of the primer to the strand, but high enough for the hybridization to be specific, i.e., the primer should bind only to a perfectly complementary part of the strand, and nowhere else. If the temperature is too low, the primer may bind imperfectly. If it is too high, the primer may not bind at all. A typical annealing temperature is about 3–5 °C below the Tm of the primers used. Stable hydrogen bonds between complementary bases are formed only when the primer sequence very closely matches the template sequence. During this step, the polymerase binds to the primer-template hybrid and begins DNA formation.
- Extension/elongation: The temperature at this step depends on the DNA polymerase used; the optimum phosphate group of the dNTPs with the 3'-hydroxy groupat the end of the nascent (elongating) DNA strand. The precise time required for elongation depends both on the DNA polymerase used and on the length of the DNA target region to amplify. As a rule of thumb, at their optimal temperature, most DNA polymerases polymerize a thousand bases per minute. Under optimal conditions (i.e., if there are no limitations due to limiting substrates or reagents), at each extension/elongation step, the number of DNA target sequences is doubled. With each successive cycle, the original template strands plus all newly generated strands become template strands for the next round of elongation, leading to exponential (geometric) amplification of the specific DNA target region.
- The processes of denaturation, annealing and elongation constitute a single cycle. Multiple cycles are required to amplify the DNA target to millions of copies. The formula used to calculate the number of DNA copies formed after a given number of cycles is 2n, where n is the number of cycles. Thus, a reaction set for 30 cycles results in 230, or 1,073,741,824, copies of the original double-stranded DNA target region.
- Final elongation: This single step is optional, but is performed at a temperature of 70–74 °C (158–165 °F) (the temperature range required for optimal activity of most polymerases used in PCR) for 5–15 minutes after the last PCR cycle to ensure that any remaining single-stranded DNA is fully elongated.
- Final hold: The final step cools the reaction chamber to 4–15 °C (39–59 °F) for an indefinite time, and may be employed for short-term storage of the PCR products.
To check whether the PCR successfully generated the anticipated DNA target region (also sometimes referred to as the amplimer or
Stages
As with other chemical reactions, the reaction rate and efficiency of PCR are affected by limiting factors. Thus, the entire PCR process can further be divided into three stages based on reaction progress:
- Exponential amplification: At every cycle, the amount of product is doubled (assuming 100% reaction efficiency). After 30 cycles, a single copy of DNA can be increased up to 1,000,000,000 (one billion) copies. In a sense, then, the replication of a discrete strand of DNA is being manipulated in a tube under controlled conditions.[16] The reaction is very sensitive: only minute quantities of DNA must be present.
- Leveling off stage: The reaction slows as the DNA polymerase loses activity and as consumption of reagents, such as dNTPs and primers, causes them to become more limited.
- Plateau: No more product accumulates due to exhaustion of reagents and enzyme.
Optimization
In practice, PCR can fail for various reasons, such as sensitivity or contamination.[17][18] Contamination with extraneous DNA can lead to spurious products and is addressed with lab protocols and procedures that separate pre-PCR mixtures from potential DNA contaminants.[8] For instance, if DNA from a crime scene is analyzed, a single DNA molecule from lab personnel could be amplified and misguide the investigation. Hence the PCR-setup areas is separated from the analysis or purification of other PCR products, disposable plasticware used, and the work surface between reaction setups needs to be thoroughly cleaned.
Specificity can be adjusted by experimental conditions so that no spurious products are generated. Primer-design techniques are important in improving PCR product yield and in avoiding the formation of unspecific products. The usage of alternate buffer components or polymerase enzymes can help with amplification of long or otherwise problematic regions of DNA. For instance, Q5 polymerase is said to be ~280 times less error-prone than Taq polymerase.[19][20] Both the running parameters (e.g. temperature and duration of cycles), or the addition of reagents, such as formamide, may increase the specificity and yield of PCR.[21] Computer simulations of theoretical PCR results (Electronic PCR) may be performed to assist in primer design.[22]
Applications
Selective DNA isolation
PCR allows isolation of DNA fragments from genomic DNA by selective amplification of a specific region of DNA. This use of PCR augments many ways, such as generating
Other applications of PCR include
Some PCR fingerprint methods have high discriminative power and can be used to identify genetic relationships between individuals, such as parent-child or between siblings, and are used in paternity testing (Fig. 4). This technique may also be used to determine evolutionary relationships among organisms when certain
Amplification and quantification of DNA
Because PCR amplifies the regions of DNA that it targets, PCR can be used to analyze extremely small amounts of sample. This is often critical for
qPCR allows the quantification and detection of a specific DNA sequence in real time since it measures concentration while the synthesis process is taking place. There are two methods for simultaneous detection and quantification. The first method consists of using fluorescent dyes that are retained nonspecifically in between the double strands. The second method involves probes that code for specific sequences and are fluorescently labeled. Detection of DNA using these methods can only be seen after the hybridization of probes with its complementary DNA (cDNA) takes place. An interesting technique combination is real-time PCR and reverse transcription. This sophisticated technique, called RT-qPCR, allows for the quantification of a small quantity of RNA. Through this combined technique, mRNA is converted to cDNA, which is further quantified using qPCR. This technique lowers the possibility of error at the end point of PCR,[27] increasing chances for detection of genes associated with genetic diseases such as cancer.[5] Laboratories use RT-qPCR for the purpose of sensitively measuring gene regulation. The mathematical foundations for the reliable quantification of the PCR[28] and RT-qPCR[29] facilitate the implementation of accurate fitting procedures of experimental data in research, medical, diagnostic and infectious disease applications.[30][31][32][33]
Medical and diagnostic applications
Prospective parents can be tested for being
- PCR can also be used as part of a sensitive test for tissue typing, vital to organ transplantation. As of 2008,[update] there is even a proposal to replace the traditional antibody-based tests for blood type with PCR-based tests.[34]
- Many forms of cancer involve alterations to malignant diseases such as leukemia and lymphomas, which is currently the highest-developed in cancer research and is already being used routinely. PCR assays can be performed directly on genomic DNA samples to detect translocation-specific malignant cells at a sensitivity that is at least 10,000 fold higher than that of other methods.[35] PCR is very useful in the medical field since it allows for the isolation and amplification of tumor suppressors. Quantitative PCR for example, can be used to quantify and analyze single cells, as well as recognize DNA, mRNA and protein confirmations and combinations.[27]
Infectious disease applications
PCR allows for rapid and highly specific diagnosis of infectious diseases, including those caused by bacteria or viruses.
Characterization and detection of infectious disease organisms have been revolutionized by PCR in the following ways:
- The human immunodeficiency virus (or tests have been developed that can detect as little as one viral genome among the DNA of over 50,000 host cells.[38] Infections can be detected earlier, donated blood can be screened directly for the virus, newborns can be immediately tested for infection, and the effects of antiviral treatments can be quantified.
- Some disease organisms, such as that for tuberculosis, are difficult to sample from patients and slow to be grown in the laboratory. PCR-based tests have allowed detection of small numbers of disease organisms (both live or dead), in convenient samples. Detailed genetic analysis can also be used to detect antibiotic resistance, allowing immediate and effective therapy. The effects of therapy can also be immediately evaluated.
- The spread of a earlier epidemicscan also be determined by PCR analysis.
- Viral DNA can be detected by PCR. The primers used must be specific to the targeted sequences in the DNA of a virus, and PCR can be used for diagnostic analyses or DNA sequencing of the viral genome. The high sensitivity of PCR permits virus detection soon after infection and even before the onset of disease.[36] Such early detection may give physicians a significant lead time in treatment. The amount of virus ("viral load") in a patient can also be quantified by PCR-based DNA quantitation techniques (see below). A variant of PCR (RT-PCR) is used for detecting viral RNA rather than DNA: in this test the enzyme reverse transcriptase is used to generate a DNA sequence which matches the viral RNA; this DNA is then amplified as per the usual PCR method. RT-PCR is widely used to detect the SARS-CoV-2 viral genome.[39]
- Diseases such as pertussis (or dimers and reacts with different cell types such as T lymphocytes which play a role in cell immunity.[40] PCR is an important testing tool that can detect sequences within the gene for the pertussis toxin. Because PCR has a high sensitivity for the toxin and a rapid turnaround time, it is very efficient for diagnosing pertussis when compared to culture.[41]
Forensic applications
The development of PCR-based
- Forensic DNA typing has been an effective way of identifying or exonerating criminal suspects due to analysis of evidence discovered at a crime scene. The human genome has many repetitive regions that can be found within gene sequences or in non-coding regions of the genome. Specifically, up to 40% of human DNA is repetitive.[5] There are two distinct categories for these repetitive, non-coding regions in the genome. The first category is called variable number tandem repeats (VNTR), which are 10–100 base pairs long and the second category is called short tandem repeats (STR) and these consist of repeated 2–10 base pair sections. PCR is used to amplify several well-known VNTRs and STRs using primers that flank each of the repetitive regions. The sizes of the fragments obtained from any individual for each of the STRs will indicate which alleles are present. By analyzing several STRs for an individual, a set of alleles for each person will be found that statistically is likely to be unique.[5] Researchers have identified the complete sequence of the human genome. This sequence can be easily accessed through the NCBI website and is used in many real-life applications. For example, the FBI has compiled a set of DNA marker sites used for identification, and these are called the Combined DNA Index System (CODIS) DNA database.[5] Using this database enables statistical analysis to be used to determine the probability that a DNA sample will match. PCR is a very powerful and significant analytical tool to use for forensic DNA typing because researchers only need a very small amount of the target DNA to be used for analysis. For example, a single human hair with attached hair follicle has enough DNA to conduct the analysis. Similarly, a few sperm, skin samples from under the fingernails, or a small amount of blood can provide enough DNA for conclusive analysis.[5]
- Less discriminating forms of DNA fingerprinting can help in DNA paternity testing, where an individual is matched with their close relatives. DNA from unidentified human remains can be tested, and compared with that from possible parents, siblings, or children. Similar testing can be used to confirm the biological parents of an adopted (or kidnapped) child. The actual biological father of a newborn can also be confirmed(or ruled out).
- The PCR AMGX/AMGY design has been shown to not only[clarification needed] facilitate in amplifying DNA sequences from a very minuscule amount of genome. However it can also be used for real-time sex determination from forensic bone samples. This provides a powerful and effective way to determine gender in forensic cases and ancient specimens.[42]
Research applications
PCR has been applied to many areas of research in molecular genetics:
- PCR allows rapid production of short pieces of DNA, even when not more than the sequence of the two primers is known. This ability of PCR augments many methods, such as generating hybridization probes for Southern or northern blot hybridization. PCR supplies these techniques with large amounts of pure DNA, sometimes as a single strand, enabling analysis even from very small amounts of starting material.
- The task of DNA sequencing can also be assisted by PCR. Known segments of DNA can easily be produced from a patient with a genetic disease mutation. Modifications to the amplification technique can extract segments from a completely unknown genome, or can generate just a single strand of an area of interest.
- PCR has numerous applications to the more traditional process of DNA cloning. It can extract segments for insertion into a vector from a larger genome, which may be only available in small quantities. Using a single set of 'vector primers', it can also analyze or extract fragments that have already been inserted into vectors. Some alterations to the PCR protocol can generate mutations (general or site-directed) of an inserted fragment.
- Sequence-tagged sites is a process where PCR is used as an indicator that a particular segment of a genome is present in a particular clone. The Human Genome Project found this application vital to mapping the cosmid clones they were sequencing, and to coordinating the results from different laboratories.
- An application of PCR is the phylogenic analysis of DNA from ancient sources, such as that found in the recovered bones of Neanderthals, from frozen tissues of mammoths, or from the brain of Egyptian mummies.[16]In some cases the highly degraded DNA from these sources might be reassembled during the early stages of amplification.
- A common application of PCR is the study of patterns of quantitative PCRto quantitate the actual levels of expression
- The ability of PCR to simultaneously amplify several loci from individual sperm[43] has greatly enhanced the more traditional task of genetic mapping by studying chromosomal crossovers after meiosis. Rare crossover events between very close loci have been directly observed by analyzing thousands of individual sperms. Similarly, unusual deletions, insertions, translocations, or inversions can be analyzed, all without having to wait (or pay) for the long and laborious processes of fertilization, embryogenesis, etc.
- Site-directed mutagenesis: PCR can be used to create mutant genes with mutations chosen by scientists at will. These mutations can be chosen in order to understand how proteins accomplish their functions, and to change or improve protein function.
Advantages
PCR has a number of advantages. It is fairly simple to understand and to use, and produces results rapidly. The technique is highly sensitive with the potential to produce millions to billions of copies of a specific product for sequencing, cloning, and analysis. qRT-PCR shares the same advantages as the PCR, with an added advantage of quantification of the synthesized product. Therefore, it has its uses to analyze alterations of gene expression levels in tumors, microbes, or other disease states.[27]
PCR is a very powerful and practical research tool. The sequencing of unknown etiologies of many diseases are being figured out by the PCR. The technique can help identify the sequence of previously unknown viruses related to those already known and thus give us a better understanding of the disease itself. If the procedure can be further simplified and sensitive non-radiometric detection systems can be developed, the PCR will assume a prominent place in the clinical laboratory for years to come.[16]
Limitations
One major limitation of PCR is that prior information about the target sequence is necessary in order to generate the primers that will allow its selective amplification.[27] This means that, typically, PCR users must know the precise sequence(s) upstream of the target region on each of the two single-stranded templates in order to ensure that the DNA polymerase properly binds to the primer-template hybrids and subsequently generates the entire target region during DNA synthesis.
Like all enzymes, DNA polymerases are also prone to error, which in turn causes mutations in the PCR fragments that are generated.[44]
Another limitation of PCR is that even the smallest amount of contaminating DNA can be amplified, resulting in misleading or ambiguous results. To minimize the chance of contamination, investigators should reserve separate rooms for reagent preparation, the PCR, and analysis of product. Reagents should be dispensed into single-use aliquots. Pipettors with disposable plungers and extra-long pipette tips should be routinely used.[16] It is moreover recommended to ensure that the lab set-up follows a unidirectional workflow. No materials or reagents used in the PCR and analysis rooms should ever be taken into the PCR preparation room without thorough decontamination.[45]
Environmental samples that contain humic acids may inhibit PCR amplification and lead to inaccurate results.
Variations
- Allele-specific PCR or The amplification refractory mutation system (ARMS): a diagnostic or cloning technique based on single-nucleotide variations (SNVs not to be confused with SNPs) (single-base differences in a patient). Any mutation involving single base change can be detected by this system. It requires prior knowledge of a DNA sequence, including differences between alleles, and uses primers whose 3' ends encompass the SNV (base pair buffer around SNV usually incorporated).[46] PCR amplification under stringent conditions is much less efficient in the presence of a mismatch between template and primer, so successful amplification with an SNP-specific primer signals presence of the specific SNP or small deletions in a sequence.[47] See SNP genotyping for more information.
- Assembly PCR or Polymerase Cycling Assembly (PCA): artificial synthesis of long DNA sequences by performing PCR on a pool of long oligonucleotides with short overlapping segments. The oligonucleotides alternate between sense and antisense directions, and the overlapping segments determine the order of the PCR fragments, thereby selectively producing the final long DNA product.[48]
- Tm) than the excess primer to maintain reaction efficiency as the limiting primer concentration decreases mid-reaction.[50]
- Convective PCR: a pseudo-isothermal way of performing PCR. Instead of repeatedly heating and cooling the PCR mixture, the solution is subjected to a thermal gradient. The resulting thermal instability driven convective flow automatically shuffles the PCR reagents from the hot and cold regions repeatedly enabling PCR.[51] Parameters such as thermal boundary conditions and geometry of the PCR enclosure can be optimized to yield robust and rapid PCR by harnessing the emergence of chaotic flow fields.[52] Such convective flow PCR setup significantly reduces device power requirement and operation time.
- Dial-out PCR: a highly parallel method for retrieving accurate DNA molecules for gene synthesis. A complex library of DNA molecules is modified with unique flanking tags before massively parallel sequencing. Tag-directed primers then enable the retrieval of molecules with desired sequences by PCR.[53]
- Digital PCR(dPCR): used to measure the quantity of a target DNA sequence in a DNA sample. The DNA sample is highly diluted so that after running many PCRs in parallel, some of them do not receive a single molecule of the target DNA. The target DNA concentration is calculated using the proportion of negative outcomes. Hence the name 'digital PCR'.
- DNA helicase, an enzyme that unwinds DNA, is used in place of thermal denaturation.[54]
- Hot start PCR: a technique that reduces non-specific amplification during the initial set up stages of the PCR. It may be performed manually by heating the reaction components to the denaturation temperature (e.g., 95 °C) before adding the polymerase.[55] Specialized enzyme systems have been developed that inhibit the polymerase's activity at ambient temperature, either by the binding of an antibody[13][56] or by the presence of covalently bound inhibitors that dissociate only after a high-temperature activation step. Hot-start/cold-finish PCR is achieved with new hybrid polymerases that are inactive at ambient temperature and are instantly activated at elongation temperature.
- In silico PCR (digital PCR, virtual PCR, electronic PCR, e-PCR) refers to computational tools used to calculate theoretical polymerase chain reaction results using a given set of primers (probes) to amplify DNA sequences from a sequenced genome or transcriptome. In silico PCR was proposed as an educational tool for molecular biology.[57]
- Intersequence-specific PCR (ISSR): a PCR method for DNA fingerprinting that amplifies regions between simple sequence repeats to produce a unique fingerprint of amplified fragment lengths.[58]
- self ligation, resulting in known sequences at either end of the unknown sequence.[59]
- Ligation-mediated PCR: uses small DNA linkers ligated to the DNA of interest and multiple primers annealing to the DNA linkers; it has been used for genome walking, and DNA footprinting.[60]
- Stephen Baylin and James G. Herman at the Johns Hopkins School of Medicine,[61]and is used to detect methylation of CpG islands in genomic DNA. DNA is first treated with sodium bisulfite, which converts unmethylated cytosine bases to uracil, which is recognized by PCR primers as thymine. Two PCRs are then carried out on the modified DNA, using primer sets identical except at any CpG islands within the primer sequences. At these points, one primer set recognizes DNA with cytosines to amplify methylated DNA, and one set recognizes DNA with uracil or thymine to amplify unmethylated DNA. MSP using qPCR can also be performed to obtain quantitative rather than qualitative information about methylation.
- Miniprimer PCR: uses a thermostable polymerase (S-Tbr) that can extend from short primers ("smalligos") as short as 9 or 10 nucleotides. This method permits PCR targeting to smaller primer binding regions, and is used to amplify conserved DNA sequences, such as the 16S (or eukaryotic 18S) rRNA gene.[62]
- Multiplex ligation-dependent probe amplification (MLPA): permits amplifying multiple targets with a single primer pair, thus avoiding the resolution limitations of multiplex PCR (see below).
- Multiplex-PCR: consists of multiple primer sets within a single PCR mixture to produce amplicons of varying sizes that are specific to different DNA sequences. By targeting multiple genes at once, additional information may be gained from a single test-run that otherwise would require several times the reagents and more time to perform. Annealing temperatures for each of the primer sets must be optimized to work correctly within a single reaction, and amplicon sizes. That is, their base pair length should be different enough to form distinct bands when visualized by gel electrophoresis.
- Nanoparticle-assisted PCR (nanoPCR): some nanoparticles (NPs) can enhance the efficiency of PCR (thus being called nanoPCR), and some can even outperform the original PCR enhancers. It was reported that quantum dots (QDs) can improve PCR specificity and efficiency. Single-walled carbon nanotubes (SWCNTs) and multi-walled carbon nanotubes (MWCNTs) are efficient in enhancing the amplification of long PCR. Carbon nanopowder (CNP) can improve the efficiency of repeated PCR and long PCR, while zinc oxide, titanium dioxide and Ag NPs were found to increase the PCR yield. Previous data indicated that non-metallic NPs retained acceptable amplification fidelity. Given that many NPs are capable of enhancing PCR efficiency, it is clear that there is likely to be great potential for nanoPCR technology improvements and product development.[63][64]
- Nested PCR: increases the specificity of DNA amplification, by reducing background due to non-specific amplification of DNA. Two sets of primers are used in two successive PCRs. In the first reaction, one pair of primers is used to generate DNA products, which besides the intended target, may still consist of non-specifically amplified DNA fragments. The product(s) are then used in a second PCR with a set of primers whose binding sites are completely or partially different from and located 3' of each of the primers used in the first reaction. Nested PCR is often more successful in specifically amplifying long DNA fragments than conventional PCR, but it requires more detailed knowledge of the target sequences.
- Overlap-extension PCR or Splicing by overlap extension (SOEing) : a genetic engineering technique that is used to splice together two or more DNA fragments that contain complementary sequences. It is used to join DNA pieces containing genes, regulatory sequences, or mutations; the technique enables creation of specific and long DNA constructs. It can also introduce deletions, insertions or point mutations into a DNA sequence.[65][66]
- PAN-AC: uses isothermal conditions for amplification, and may be used in living cells.[67][68]
- PAN-PCR: A computational method for designing bacterium typing assays based on whole genome sequence data.[69]
- quantitative PCR(real-time PCR).
- Reverse Complement PCR(RC-PCR): Allows the addition of functional domains or sequences of choice to be appended independently to either end of the generated amplicon in a single closed tube reaction. This method generates target specific primers within the reaction by the interaction of universal primers (which contain the desired sequences or domains to be appended) and RC probes.
- Reverse Transcription PCR (RACE-PCR(Rapid Amplification of cDNA Ends).
- RNase H-dependent PCR (rhPCR): a modification of PCR that utilizes primers with a 3' extension block that can be removed by a thermostable RNase HII enzyme. This system reduces primer-dimers and allows for multiplexed reactions to be performed with higher numbers of primers.[70]
- Single specific primer-PCR (SSP-PCR): allows the amplification of double-stranded DNA even when the sequence information is available at one end only. This method permits amplification of genes for which only a partial sequence information is available, and allows unidirectional genome walking from known into unknown regions of the chromosome.[71]
- Solid Phase PCR: encompasses multiple meanings, including Polony Amplification (where PCR colonies are derived in a gel matrix, for example), Bridge PCR[72] (primers are covalently linked to a solid-support surface), conventional Solid Phase PCR (where Asymmetric PCR is applied in the presence of solid support bearing primer with sequence matching one of the aqueous primers) and Enhanced Solid Phase PCR[73] (where conventional Solid Phase PCR can be improved by employing high Tm and nested solid support primer with optional application of a thermal 'step' to favour solid support priming).
- Suicide PCR: typically used in paleogenetics or other studies where avoiding false positives and ensuring the specificity of the amplified fragment is the highest priority. It was originally described in a study to verify the presence of the microbe Yersinia pestis in dental samples obtained from 14th Century graves of people supposedly killed by the plague during the medieval Black Death epidemic.[74] The method prescribes the use of any primer combination only once in a PCR (hence the term "suicide"), which should never have been used in any positive control PCR reaction, and the primers should always target a genomic region never amplified before in the lab using this or any other set of primers. This ensures that no contaminating DNA from previous PCR reactions is present in the lab, which could otherwise generate false positives.
- Thermal asymmetric interlaced PCR (TAIL-PCR): for isolation of an unknown sequence flanking a known sequence. Within the known sequence, TAIL-PCR uses a nested pair of primers with differing annealing temperatures; a degenerate primer is used to amplify in the other direction from the unknown sequence.[75]
- Touchdown PCR (Step-down PCR): a variant of PCR that aims to reduce nonspecific background by gradually lowering the annealing temperature as PCR cycling progresses. The annealing temperature at the initial cycles is usually a few degrees (3–5 °C) above the Tm of the primers used, while at the later cycles, it is a few degrees (3–5 °C) below the primer Tm. The higher temperatures give greater specificity for primer binding, and the lower temperatures permit more efficient amplification from the specific products formed during the initial cycles.[76]
- Universal Fast Walking: for genome walking and genetic fingerprinting using a more specific 'two-sided' PCR than conventional 'one-sided' approaches (using only one gene-specific primer and one general primer—which can lead to artefactual 'noise')[77] by virtue of a mechanism involving lariat structure formation. Streamlined derivatives of UFW are LaNe RAGE (lariat-dependent nested PCR for rapid amplification of genomic DNA ends),[78] 5'RACE LaNe[79] and 3'RACE LaNe.[80]
History
The heat-resistant enzymes that are a key component in polymerase chain reaction were discovered in the 1960s as a product of a microbial life form that lived in the superheated waters of Yellowstone's Mushroom Spring.[81]
A 1971 paper in the Journal of Molecular Biology by Kjell Kleppe and co-workers in the laboratory of H. Gobind Khorana first described a method of using an enzymatic assay to replicate a short DNA template with primers in vitro.[82] However, this early manifestation of the basic PCR principle did not receive much attention at the time and the invention of the polymerase chain reaction in 1983 is generally credited to Kary Mullis.[83][page needed]
When Mullis developed the PCR in 1983, he was working in
Mullis has credited his use of LSD as integral to his development of PCR: "Would I have invented PCR if I hadn't taken LSD? I seriously doubt it. I could sit on a DNA molecule and watch the polymers go by. I learnt that partly on psychedelic drugs."[87]
Mullis and biochemist Michael Smith, who had developed other essential ways of manipulating DNA,[1] were jointly awarded the Nobel Prize in Chemistry in 1993, seven years after Mullis and his colleagues at Cetus first put his proposal to practice.[88] Mullis's 1985 paper with R. K. Saiki and H. A. Erlich, "Enzymatic Amplification of β-globin Genomic Sequences and Restriction Site Analysis for Diagnosis of Sickle Cell Anemia"—the polymerase chain reaction invention (PCR)—was honored by a Citation for Chemical Breakthrough Award from the Division of History of Chemistry of the American Chemical Society in 2017.[89][2]
At the core of the PCR method is the use of a suitable
The discovery in 1976 of Taq polymerase—a DNA polymerase purified from the thermophilic bacterium, Thermus aquaticus, which naturally lives in hot (50 to 80 °C (122 to 176 °F)) environments[14] such as hot springs—paved the way for dramatic improvements of the PCR method. The DNA polymerase isolated from T. aquaticus is stable at high temperatures remaining active even after DNA denaturation,[15] thus obviating the need to add new DNA polymerase after each cycle.[3] This allowed an automated thermocycler-based process for DNA amplification.
Patent disputes
The PCR technique was patented by
A related patent battle over the Taq polymerase enzyme is still ongoing[as of?] in several jurisdictions around the world between Roche and Promega. The legal arguments have extended beyond the lives of the original PCR and Taq polymerase patents, which expired on 28 March 2005.[92]
See also
- COVID-19 testing
- DNA spiking
- Loop-mediated isothermal amplification
- Selector technique
- Thermus thermophilus
- Pfu DNA polymerase
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External links
- US Patent for PCR Archived 16 October 2011 at the Wayback Machine
- What is PCR plateau effect? YouTube tutorial video
- History of the Polymerase Chain Reaction from the Smithsonian Institution Archives