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🧳 | Reduced flights by Kushiro bus and fixed-route bus Corona cluster for crew members


Reduced flights by Kushiro bus and fixed-route bus Corona cluster for crew members

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PCR tests conducted by February 2 confirmed infection of 16 bus crew members.

The Kushiro Bus in Kushiro City, Hokkaido, will be on the route until March 3 due to the outbreak of a new coronavirus infection on the crew. → Continue reading


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Polymerase chain reaction

Polymerase chain reaction(Polymerase Rensa Hanno,English: polymerase chain reaction) IsDNAA reaction or technique that amplifies a specific region of a sample by millions to billions of times. Take the initials in EnglishPCR method, Or simplyPCRIt is also called "Polymerase Chain Reaction" in English.

DNA polymeraseと 呼 ば れ る酵素Utilizing the function of, any cycle through a series of temperature changesgeneArea orgenomeCopy spaceExponentialTarget (Mouse arithmeticThe objective is to amplify from a small sample of DNA to a sufficient amount to study its details.[1][2][3].Medical,Molecular biology,Forensic medicineIt is a useful technology that is widely used in fields such asCarrie MarisInvented by (Kary Mullis)[4][5],Nobel PrizeWas awarded.

Since the PCR method was established,Sequencing, Experiments such as gene mutation induction become possible,Molecular genetics,Physiology,TaxonomyIn addition to being utilized in research fields such as, analysis of ancient DNA samples,Forensic medicine,Paternity testUsed inDNA typingIdentification of infectious agentsInfectionTechnology development related to diagnosis (Nucleic acid amplification test), etc. have advanced dramatically. Also, from the PCR methodReverse transcription polymerase chain reaction,Real-time PCR,DNA sequencingEtc. are derived and developed. Therefore, today the PCR methodBiology,Medical scienceIt is the basis of gene analysis in a wide range of fields including[6][7].


The PCR method operates by a series of thermal cycles of raising and lowering the temperature of a DNA solution containing a mixture of reagents. In the repeated cycle of heating and cooling of this DNA sample, dissociation of double-stranded DNA, binding of primer,酵素Three reactions, DNA synthesis by reaction, proceed, and finally a large amount of DNA fragments in a specific region are replicated.

In the PCR method, in addition to the DNA sample to be amplified (template), a large amount ofPrimer(In the target DNA regionComplementaryShort single-stranded DNA with sequence (Oligonucleotide)) and free, a component of DNAnucleotideAndPolymeraseIs a type ofDNA synthase(DNA polymerase) ThreereagentTo use.

  1. The first step is to heat the double-stranded DNA of the DNA double helix at high temperature.DegenerationAnd physically separate into single-stranded DNA. The temperature at which denaturation occurs depends on the base composition and length (number of bases) of DNA, and generally longer DNA requires higher temperature.
  2. Next, the solution containing the single-stranded DNA is cooled, and the primer is bound to the complementary sequence site of the single-stranded DNA to partially form the double strand (annealing). When the cooling is rapid, it is difficult for long DNAs to recombine into double strands, but short DNA fragments (Oligonucleotide) Takes advantage of the fact that they can be easily combined. As a result, a primer is bound to a part of the target long single-stranded DNA. By keeping the primer overwhelmingly more than the DNA, the tendency of the DNA and the primer to bind becomes more predominant than the tendency of the DNA and the DNA to bind.
  3. Next, heat the solution slightly to remove this double-stranded DNA site.templateAsDNA polymeraseBy making the primer act as a starting point and releasenucleotideDNA that is complementary to the single-stranded part酵素Are synthesized. After the DNA is synthesized, the temperature is raised again to return to the first step, and this cycle is repeated from the first DNA denaturation to promote the amplification. As the PCR reaction progresses, the generated DNA itself is used as a template for replication, and the original DNA template isExponentialIs amplified toChain reactionAdvances.

As described above, the PCR method utilizes the difference in the rate of denaturation and annealing depending on the DNA chain length to repeat DNA synthesis simply by repeatedly raising and lowering the temperature of the reaction solution, and amplifying a partial region of any DNA. Is.

If the DNA polymerase used is heat-sensitive, the polymerase will denature along with the DNA at the high temperature of the denaturation step,DeactivationResulting in. Therefore, when the PCR method was first developed, DNA polymerase was added as an enzyme every time DNA was denatured, which was time-consuming and expensive.[8].. Currently,Thermus aquaticusTo sayThermophileIs a thermostable DNA polymerase derived fromTaq polymeraseBy using such as, it is possible to continuously proceed the reaction without adding an enzyme in the middle.



The nucleotide sequences at both ends of the DNA region to be amplified are determined, and corresponding primers are artificially synthesized. At this time, the primer is a complementary sequence that binds to the 2'side of each strand of the double-stranded DNA to be amplified, and usually has about 3 bases. They are often custom made in the lab or can be purchased from a commercial biochemical supplier.

Reaction solution preparation

Amplification target DNA, primer, DNA polymerase and DNA synthesis material (Substrate)Deoxynucleotide triphosphate (dNTP), And a buffer solution to create an optimal salt concentration environment in which the enzyme works, and a PCR device (Thermal cycler). For the buffer solution included in the distributed PCR reagent kit,BivalentCationIs often included[9].NormallymagnesiumIon (Mg2+), but PCR-mediated DNA mutagenesis is highmanganeseIon (Mn2+) Can also be used to increase the error rate during DNA synthesis.[10].Taq In the case of DNA polymerase,As a monovalent cationpotassiumIon (K+) May be included[11].. In some casesAmmonium sulfateMay be added.Ammonium ion(NH4+) Has the effect of destabilizing the weak hydrogen bond between the mismatched primer and the template base pair, and can increase the specificity.[11].

PCR cycle

  1. 94 reaction solution° CHeat to about 30 seconds to 1 minute to keep double-stranded DNA into single strands (Fig. 2).
  2. 60° CRapidly cool to a certain degree (slightly different depending on the primer) andPrimerAnneal (Fig. ②).
  3. No separation of primersDNA polymeraseReheat to the optimum temperature range for the activity of. Depending on the experimental purpose, its temperature is 60-72° CIt is set to a degree. This temperature is usually maintained for 1 to 2 minutes depending on the length of time required for DNA synthesis and the length of amplification (Fig. XNUMX).
  4. Up to this point is one cycle, and by repeating the steps from ① to ③, a specific DNA fragment is amplified.

When the PCR treatment is repeated n times, 1 double-stranded DNAn-Amplify 2n times.However, since the maximum of 2 cycles is usually performed, the term of XNUMXn is approximately negligible.If the number of cycles is further increased, the DNA polymerase loses its activity over time and reagents such as dNTPs and primers are consumed, so that the reaction is limited and the series of reactions is finally stopped.

Points to remember

The success or failure of this reaction depends on the base sequences of the DNA to be amplified and the primer, each set temperature and time during the cycle, and the like. If they are inappropriate, they may amplify irrelevant DNA sequences or may show no amplification. In addition, there is a possibility that mutation will occur during the synthesis process, so it is necessary to check the nucleotide sequence of the product depending on the purpose of use.

Application of PCR

Amplification and quantification of DNA

PCR is a technique for dramatically amplifying a region of target DNA, and even a very small amount of DNA sample may be analyzed by passing through PCR. This proves that only trace amounts of DNA are availableForensic medicineIt is especially important in such fields.Or, for example, tens of thousands of years agoAncient DNAPCR is also effective for analysis of[12].

Quantitative PCR(Also called real-time PCR, or simply qPCR.RT-PCRNote that the amount of a specific DNA sequence present in a sample can also be estimated by[13].. this is,Gene expressionIt is used for purposes such as quantitatively determining levels. In quantitative PCR, the abundance of the target DNA region that originally existed is quantified by measuring the concentration of the PCR product that is amplified during the PCR cycle in real time during the PCR cycle process. can do. There are two major methods, one is non-specifically retained between the duplexes.fluorescenceAnother method is to use a dye, and the other method is to use a probe to which a fluorescent label is added in advance and which encodes a specific sequence. In the latter method, fluorescence can be detected only when the probe and its complementary DNA are hybridized.

Combined real-time PCR and reverse transcriptionRT-qPCR(Reverse transcription polymerase chain reaction), the method calledRNAIt is possible to quantify. In this technique, mRNA is the firstcDNAAnd the cDNA is quantified by qPCR. This method is often used to detect genes related to genetic diseases such as cancer and to measure expression levels.[14].

Application to biological research

PCRMolecular biology,GeneticsIt has been applied to various research fields including.

  • PCR can be used to selectively amplify and isolate specific DNA regions in the genome. Such use of PCR isSouthern blotting,Northern blottingIs widely used for the production of such hybridization probes and for DNA cloning that requires a large amount of DNA fragments derived from a specific DNA region.
  • PCRDNA sequenceIs often important in doing.. By various PCR, for example, a gene sequence or a DNA region to be analyzed can be extracted from a completely unknown genome and amplified.
  • PCRClassic experiments such as DNA cloningIt is often used in the process. For example, it is used when inserting a specific genomic region from a large genome into a vector. It can also be used to analyze or amplify DNA fragments already inserted in the vector. By partially changing the PCR protocol,mutationCan be artificially induced.
  • PCR is often used for research on ancient DNA. Most of such ancient DNA has been decomposed by ultraviolet rays and hydrolysis, and there is often only a trace amount of double-stranded DNA, so it is possible to analyze only by applying amplification by PCR. .. As an example of research that actually used PCR,NeanderthalsThe bone, 4 years oldMammothFrozen tissue ofEgypt OfmummyThere is a brain, etc.ロシアemperorAnd British KingRichard IIIIs being identified[12].. In some cases, it may be possible to restore the original DNA sequence to some extent by applying PCR amplification even to a DNA sample that has been considerably degraded.
  • Gene expressionPCR is also used to study the pattern. You can analyze body tissues and individual cells at various time-series stages to see which genes were activated/deactivated,Quantitative PCRCan also be used to quantify the actual expression level in detail.
  • PCRGenetic linkageIs also used for research. For example,Amplifies several loci from individual sperm simultaneously,MeiosisAfterChromosome crossoverStudies have been reported[15].. In this study, rare rare crossover events between very close loci were directly observed by analyzing thousands of sperm. Similarly, aberrant deletions, insertions, translocations, or inversions can be analyzed.
  • PCR can be used to induce site-directed mutagenesis of any gene or genomic region. By investigating these mutants, it is possible to elucidate, for example, the function of the protein, or to proceed with research for altering or improving the function of the protein.

Prenatal diagnosis

Whether a PCR is a carrier of a particular inheritance before the child is born, or actuallysickTest whether you are affected byPrenatal diagnosisCan be used for[16].Prenatal examinationDNA sample forAmniocentesisbyChorionic villus sampling, Or can be obtained by analysis of a very small amount of fetal cells circulating in the mother's bloodstream. PCR analysisPre-implantation diagnosisIs also essential and can test mutations in individual cells of the developing embryo.

Organ transplant tissue typing

PCROrgan transplantationEssential toOrganizational typingIt can also be used as a highly sensitive test.Blood TypeA proposal to replace the traditional antibody-based test with a PCR-based test against was also made in 2008[17].

Cancer genetic analysis

Many forms of cancer are associated with various types of cancer development.gene(Oncogene) ArrayWith variations, PCR technology could be used to analyze this mutation to tailor treatment strategies to patients. PCR alsoleukemia,LymphomaSuch asMalignantEnables early diagnosis of disease. It is being developed in the field of cancer research, and PCR is now routinely used. It has been reported that direct PCR assay of genomic DNA samples can detect translocation-specific malignant cells at least 10,000 times more sensitive than other methods[18].. PCR also allows the isolation and amplification of tumor suppressors. For example, it is possible to quantify single cells using quantitative PCR and analyze the abundance and combination of DNA, mRNA, and protein.[19].

Diagnosis of infectious disease

PCR aids in sensitive and rapid diagnosis of infectious diseases caused by bacteria and viruses[20].. In PCR,Mycobacteria ,Anaerobic bacteria, ま た はTissue cultureAssayAnimal modelfromウ イ ル スIt is also possible to quickly identify microorganisms that cannot be cultured or microorganisms that grow slowly. In addition, PCR diagnosis can distinguish between non-pathogenic strains and pathogenic strains by not only detecting infectious pathogens but also determining whether the bacterium has a specific gene[20][21].. On the other hand, various defects have been reported (Later).

  • Human immunodeficiency virus (HIV) Is a difficult target to discover and eradicate. The initial diagnosis of infection is the virus circulating in the bloodstream.antibodyHowever, the antibody does not appear until weeks after infection, the maternal antibody masks the infection of the newborn, and the amount of antibody does not change when treated with an HIV therapeutic agent. was there. Therefore, a high-sensitivity PCR method that can detect only one viral genome from over 50,000 cellular DNA samples was developed.[22].. This method enables early detection of infectious diseases, virus testing of donated blood, rapid infection testing of newborns, and quantification of antiviral treatment effects.
  • tuberculosis OfSome disease-causing microbes such as these are known to be difficult to sample from patients and grow slowly in the laboratory,cultureA lot of time and effort was spent in the base diagnosis. Tests by PCR can detect disease-causing microorganisms in samples, and from genetic analysisAntibiotic resistanceIt is possible to detect the presence or absence, etc., which may lead to the setting of an effective treatment policy and the evaluation of the treatment effect.
  • 家畜orwildThrough a group of animalsdiseaseBiology Diffusion and emergence of new toxic subtypes can be monitored by PCR testing.
  • By using a primer specific to the target sequence of viral DNA, viral DNA can be detected by PCR and also used for DNA sequencing. Due to the high sensitivity of PCR, virus detection may be possible immediately after infection and before the onset of disease[23].. Early detection may give the physician a significant lead time for treatment. The viral load contained within the patient can also be quantified by PCR-based DNA quantification techniques.
  • Social Services LizardIs caused by a bacterium called B. pertussis. This bacterium is characterized by a serious acute respiratory tract infection affecting a wide variety of animals and humans, leading to the death of many young children.Communicate and LizardBinds to cell receptors by two dimers and plays a role in cell immunityT lymphocyteIs a protein toxin that reacts with[24].. Since the sequence in the pertussis toxin gene can be detected by PCR, pertussis can be diagnosed very efficiently compared to the culture method.[25].

Application to forensics

PCR-basedDNA typing(Fingerprinting)Forensic medicineWidely applied in the field.

  • DNA typing is世界It is a technology that can uniquely distinguish one person from the entire population. Applying this technology, a small amount of DNA sampleCrime sceneIn some cases, suspects can be identified by collecting, analyzing, and comparing prisoners. A simpler method of use is to quickly exclude suspects during criminal investigations. Alternatively, test crime evidence decades ago to confirm the perpetration of convicted people, orImmunityIt also leads to doing.
  • Forensic DNA typing (forensic DNA typing) is an effective way to identify or exclude suspected crimes from the analysis of evidence found at crime scenes. The human genome has many repeat regions found within gene sequences or in noncoding regions of the genome. Specifically, up to 40% of human DNA is known to be repetitive[26].. There are two types of these repetitive non-coding regions in the genome, one called variable length tandem repeat (VNTR), which is 2-10 base pairs long, while the other is short tandem repeat (STR). It is called a repeat section of 100-2 base pairs. Then, PCR amplification can be performed using the primers flanking each repeat region. A statistically high probability of uniquely identifying each individual can be obtained by examining the size distribution of several STR fragments obtained from each individual.[26].. Furthermore, the complete sequence of the human genome has already been determined, and since this sequence information can be easily accessed from the NCBI website, various applications have been made. For example, the FBI has compiled a set of DNA marker sites used for identification, which are called the Combined DNA Index System (CODIS) DNA database.[26].. This database can be used to statistically determine the probability that a DNA sample will match. PCR is a very powerful and important analytical tool for forensic DNA typing because it requires very small amounts of target DNA for analysis. For example,Hair follicleHuman hair to which is attached contains enough DNA, if any, to perform the analysis. Similarly, severalspermA skin sample from under the fingernails, or a small amountbloodCan provide enough DNA for definitive analysis[26].
  • On the other hand, on the contrary, due to the format with low discriminationDNA fingerprintingIt is,Paternity testIs utilized for.. In this case, the subject, such as the body of an unidentified human, is compared to the DNA of the expected relatives, ie parents, siblings, children, etc. To identify the biological parents of the adopted (kidnapped) child,NewbornOf the actual biological father ofConfirm (Or excluded) is also used.
  • AmelogeninGene-based PCR enables real-time male and female sex determination from forensic bone samples. This allows you to determine the sex of ancient specimens and suspected criminals.[27].

PCR technical limitations

PCR has many advantages: the principle and the actual work are very simple, the results can be obtained quickly, and they are very sensitive.Quantitative PCR(qPCR, quantitative PCR) also has the advantage of being able to quantify the targeted DNA region.On the other hand, PCR is also known to have various technical restrictions and limitations.

One of the technical limitations of PCR is that it requires a priori knowledge of the sequence of the target region in order to generate primers that allow selective amplification.[28]..That is, the PCR practitioner usually needs to know the sequence information before and after the target DNA region in advance so that the primer and the template bind properly.Therefore, in principle, it is impossible to perform PCR on a target whose sequence information is completely unknown.Also, like any other enzyme, DNA polymerase itself is prone to errors during DNA synthesis and may mutate the sequence of the PCR amplification product produced.[29].. Furthermore, since PCR can amplify even a small amount of DNA, amplification may occur based on erroneously mixed DNA, resulting in ambiguous or erroneous results.

Many techniques and procedures have been developed to avoid these problems and optimize PCR conditions.[30][31].. For example, in order to minimize the possibility that the sample will be contaminated by the contamination of foreign DNA, by using separate rooms for each step of reagent preparation and PCR processing/analysis, they can be spatially separated. Separation is effective[32]..In addition, it is effective to always use disposable new tubes and pipette tips with filters to operate samples and reagents, thoroughly clean the workbench and equipment, and always work in a clean space.[33].. Reconsidering primer design and investigating the types of buffers and polymerase enzymes is also important in improving the yield of PCR products and avoiding the formation of spurious products. To the buffer systemFormamideAddition of reagents such as may increase PCR specificity and yield[34].. Computer simulation of theoretical PCR results (Electronic PCR) has also been developed to support primer design.[35].

Characteristics of PCR in diagnosis of infectious diseases

PCR is a very powerful and practical research tool, and indeed, in many infectious diseases, the etiology sequencing has been elucidated using PCR. This method is knownウ イ ル スIt also helps identify unknown viruses and contributes greatly to understanding the disease itself. If the procedure can be further simplified and a highly sensitive detection system can be developed, PCR will becomeClinical examinationIt is believed that it will take an important position in the room[36].. However, the use of PCR in the diagnosis of infectious diseases has been pointed out to have various disadvantages as well as advantages.

利 点

  • Human capital OfgenomeOnly specific DNA fragments (hundreds to thousands of base pairs) can be selectively amplified from a sample containing a long DNA molecule such as (30 billion base pairs).[1].
  • The purpose can be achieved with a very small amount of DNA.
  • Amplification reaction can be completed in a short time[2]..The time required for amplification is as short as 2 hours or less.
  • Testing is possible by using primers specific for each pathogen while keeping the equipment (PCR machine) used in common.[2].
  • Even if the pathogen is dead (even if it loses its infectivity), it can be amplified if the target nucleic acid is preserved, and even dangerous pathogens can be inactivated before testing.[2].
  • Sensitivity can be increased by using Nested PCR, real-time PCR, etc.[2].


  • When biomaterials such as organs and tissues are used as samples, the detection rate differs depending on the sampling site and pretreatment of the material, and even if the result is negative, the presence of pathogens in the living body cannot always be denied.[2].
  • There is a limit to the amplification of nucleic acid due to the decrease in reaction efficiency due to generation of reaction inhibitors, etc., and the result will be negative if the nucleic acid cannot be amplified to a detectable amount. Cannot deny the existence of pathogens[2].
  • Testing of biomaterials such as blood and feces may be affected by the inhibitory substances in the test materials, so purification work is required, but it is not said to be complete.[2].. In addition, the storage condition of test materials, the number of freeze-thaw cycles, and the methods and conditions for nucleic acid purification can have a large impact on test efficiency and test results.[2].
  • Contamination from positive controls during testing, contamination from previous tests and experiments, and contamination of reagents with nucleic acids can lead to false positives (contamination)[2].

History and background

With Kjell KleppeH. Gobind KhoranaEt al. used an enzyme assay with primers and a short DNA template.vitroIn 1971Journal of Molecular BiologyFirst published in (Molecular Biology Journal)[37].. This was to explain the basic principle of PCR, but it did not receive much attention at that time, and the invention of the polymerase chain reaction was generally found in 1983.Mullis penaltiesIs considered to be due to[38].

When Mullis developed PCR in 1983, heCaliforniaEmeryvilleSo the firstBiotechnologyA company that is one of the companies (Cetus Corporation) Was working. Mullis said, "One night,Pacific coast highwayWhile driving in the car, I came up with the idea of ​​PCR."[39].. When he was thinking about a new method to analyze changes (mutations) in DNA, he tried to amplify a partial region of nucleic acid by repeating the DNA synthesis reaction using the already known oligonucleotide and DNA polymerase at that time. Came up with[39].

Mullis named this method "polymerase-catalyzed chain reaction",Nature,ScienceI submitted it as a paper to a famous scientific journal such as, but it was not published. On the other hand, the PCR method itself was made by a colleague of Cetus.Sickle cell diseaseIt was applied to a rapid diagnostic method for inherited diseases. It was reported as "Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia" in Science magazine, and it attracted the attention of scientists around the world before the original paper.[5].. Finally in 1987, Mullis's paper Methods in Enzymology Published as "Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction."[40].. Later MullisScientific Americanso,"PCR can start with a single molecule of genetic material DNA and produce 1,000 billion similar molecules in the afternoon. The reaction is easy to perform. It requires a test tube, some simple reagents, and a heat source. It is only"[41].. DNA fingerprinting in 1988Paternity testFirst used in[42].

In recognition of this achievement, Mullis worked with his colleagues at Cetus in 7, seven years after demonstrating PCR technology.Nobel Prize in ChemistryWon[43].. In addition, “Enzymatic Amplification of β-globin Genomic Sequences and Restriction Site Analysis for Diagnosis of Sickle Cell Anemia” by RK Saiki and HA Erlich in 1985 (“Enzymatic amplification of β-globin genomic sequence for diagnosis of sickle cell anemia”). And restriction site analysis'')American Chemical SocietyReceived Chemistry Breakthrough Award in the History of Chemistry category[44][45].. However, some controversy remains regarding the contributions of other scientists to Mullis' work and whether he was the only inventor of the PCR principle, as described below.

PCR was initiallyE. coli5'-3' exonuclease activity was removed by subtilisin treatment of DNA polymerase IKlenow FragmentMost of them used to cause a reaction. However, this enzymecopyThis enzyme had to be added manually after each thermal cycle because it could not withstand the high temperatures required to separate post-cycling DNA double helices and deactivate the DNA polymerase.[46].. As a result, the initial steps of DNA replication were very inefficient and time consuming, requiring large amounts of DNA polymerase and continuous treatment throughout the process. The Cetus research group lives in a high temperature environment (hot spring) of 50 to 80°C to solve this drawback.Thermophilic bacteriumIsThermus aquaticus[47]As a thermostable DNA polymeraseTaq polymeraseWas purified and the PCR method using this was published in Science magazine in 1976.[6].T. aquaticusDNA polymerase isolated from ° C (194 ° F) Stable at high temperature, and maintains activity even after DNA denaturation[48]Eliminates the need to add new DNA polymerase after each cycle[49].. This has opened the way to the simplification and automation of PCR reactions, and has led to the development of a widely applicable method.

In this way, the Cetus Group (including initially Maris) played a large role in the application and development of the PCR method.

However, it was Carey Maris who first conceived this method and showed direction, so MarisNobel Prize in ChemistryWas awarded in 1993. PCR technologyMullis penalties PatentWas acquired and was assigned to work in 1983 when Mullis invented the technology.TaqThe polymerase enzyme is also patent protected.DupontThere were several well-known lawsuits related to this technology, including the unsuccessful lawsuits filed. Swiss pharmaceutical companyEf Hoffman La RochePurchased a patent right in 1992, but the patent right now expires[50].

PCR types and application methods

Conventional PCR
Normal PCR that repeats the reaction for 1 to 25 cycles with one set of primers[2].
Real-time polymerase chain reaction (Real-Time PCR,Real-time PCR
A method of drawing an amplification curve in real time by emitting light from a nucleic acid fragment and detecting it using a dedicated optical instrument.It is used when quantitative rather than qualitative is required.[2].
Reverse transcription polymerase chain reaction (RT-PCR,Reverse transcription polymerase chain reaction
A method in which RNA is converted to cDNA by reverse transcriptase and then PCR is performed.[2].
Nested polymerase chain reaction(Nested PCR)
A method in which the PCR product amplified by PCR is used as a template for the next reaction and another PCR is repeated using another primer pair.[2].
Multiplex polymerase chain reaction (Multiplex PCR,Multiplex PCR
A method to simultaneously perform PCR reactions for multiple target nucleic acids (DNA) in a single reaction tube[2].
Amplified fragment length polymorphism(AFLP)
A method in which genomic DNA containing target nucleic acid (DNA) is cleaved with a restriction enzyme, a short double-stranded DNA (adapter) is bound to the cleaved end, and PCR is performed using its complementary primer.[2].
Loop-Mediated Isothermal Amplification (LAMP method
A method based on a completely different principle from conventional PCR[2][51].


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