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Polymerase Chain Reaction - PCR Polymerase Chain Reaction - PCR
Table of Contents : Indholdsfortegnelse:
Introduction to PCR - Polymerase Chain Reaction Introduktion til PCR - Polymerase Chain Reaction
Polymerase Chain Reaction (PCR) Polymerase chain reaction (PCR)
PCR, a Concept to be Discovered PCR, et koncept at blive opdaget
General Principles of the PCR General Principles af PCR
Polymerases/Reaction Specificity and Efficiency Polymeraser / Reaction Specificitet og Effektivitet
Utility of PCR Anvendelighed af PCR
PCR and Molecular Cloning PCR og Molekylær Kloning
Misincorporation: Errors of In Vitro Systems Misincorporation: Fejl af in vitro-Systems
Reaction Specificity Reaktion Specificitet
Major Advantages of PCR as a Cloning Method include its Rapidity, Sensitivity, and Robustness Store fordele ved PCR som et Kloning Metode medtage sin hurtighed, følsomhed, og Robustness
Limitations of PCR Begrænsninger af PCR
Instruments for PCR Instrumenter til PCR
Oligonucleotide Synthesis Oligonucleotide Synthesis
Will PCR ever be replaced? Vil PCR nogensinde blive erstattet? – Helicase-dependent Amplification (HDA) -- Helicase afhængige Amplification (HDA)
More than 30 years ago, the introduction of recombinant DNA technology as a tool for the biological sciences revolutionized the study of life. Molecular cloning allowed the study of individual genes of living organisms; however this technique was dependent on obtaining a relatively large quantity of pure DNA. This depended on the replication of the DNA of plasmids or other vectors during cell division of microorganisms (1). Researchers found it extremely laborious and difficult to obtain a specific DNA in quantity from the mass of genes present in a biological sample (2). Recombinant DNA technology made possible the first molecular analysis and prenatal diagnosis of several human diseases. Fetal DNA obtained by amniocentesis sampling could be analyzed by restriction enzyme digestion, electrophoresis, southern transfer and hybridization to a cloned gene or oligonucleotide probes (3). However, southern blotting permitted only rudimentary mapping of genes in unrelated individuals (4). Mere end 30 år siden indførelsen af rekombinant dna-teknologi som et redskab for de biologiske videnskaber revolutionerede studiet af liv. Molekylær kloning tilladt studiet af enkelte gener af levende organismer, men denne teknik var afhængige af at få en forholdsvis stor mængde ren DNA. Dette afhang af replikation af DNA i plasmider eller andre vektorer ved celledeling af mikroorganismer (1). Forskerne fandt det yderst besværlig og vanskelig at opnå en specifik DNA i mængde fra massen af gener til stede i en biologisk prøve (2 ). Rekombinant dna-teknologi gjort det muligt for første molekylær analyse og prænatal diagnose af en række menneskelige sygdomme. Føtalt DNA udvundet ved amniocentesis prøveudtagning kan analyseres ved restriktionsenzymer fordøjelse, elektroforese, sydlige overførsel og hybridisering til et klonet gen eller oligonukleotide prober (3). Men sydlige blotting kun tilladt rudimentær kortlægning af gener i uafhængige personer (4).
PCR, an acronym for Polymerase Chain Reaction (5,6), allowed the production of large quantities of a specific DNA from a complex DNA template in a simple enzymatic reaction. PCR is a recently developed procedure for the in vitro amplification of DNA. PCR has transformed the way that almost all studies requiring the manipulation of DNA fragments may be performed as a results of its simplicity and usefulness (7). PCR, en forkortelse for Polymerase Chain Reaction (5,6), gjorde det muligt for produktion af store mængder af en specifik DNA fra en kompleks DNA-skabelon på en enkel enzymatisk reaktion. PCR er en nylig udviklet procedure for in vitro opformering af DNA. PCR har forvandlet den måde, at næsten alle de undersøgelser, som kræver manipulation af DNA-fragmenter kan udføres som et resultat af sin enkelthed og nytte (7).
In the 1980s, Kary Mullis (Figure 1) and a team of researchers at Cetus Corporation at Cetus Corporation conceived of a way to start and stop a polymerase's action at specific points along a single strand of DNA. Mullis also realized that by harnessing this component of molecular reproduction technology, the target DNA could be exponentially amplified. This DNA amplification procedure was based on an in vitro rather than an in vivo process (5,6,8). Cell-free DNA amplification by PCR was able to simplify many of the standard procedures for cloning, analyzing, and modifying nucleic acids (1). Previous techniques for isolating a specific piece of DNA relied on gene cloning – a tedious and slow procedure. PCR, on the other hand Kerry Mullis stated “lets you pick the piece of DNA you’re interested in and have as much of it as you want” (2,8). I 1980'erne, Kary Mullis (figur 1) og et hold af forskere på Cetus Corporation på Cetus Corporation udtænkt af en måde at starte og stoppe en polymerase's indsats på specifikke punkter langs en enkelt streng i DNA. Mullis også indset, at ved at styre denne komponent af molekylære reproduktion teknologi, mål-DNA kan være eksponentielt forstærkes. Denne DNA-forstærkning proceduren var baseret på en in vitro snarere end en in vivo-processen (5,6,8). Cell-free DNA-forstærkning ved PCR var i stand til at forenkle mange af faste procedurer for kloning, analysere, og om ændring af nukleinsyrer (1). Forrige teknikker til isolering af et bestemt stykke DNA påberåbes gen kloning - en kedelig og langsommelig procedure. PCR, på den anden side Kerry Mullis erklærede "lader dig vælge de stykke DNA, du er interesseret i, og de har så meget af det som du vil "(2,8). When other Cetus scientists eventually succeeded in making the polymerase chain reaction perform as desired in a reliable fashion, they had an immensely powerful technique for providing essentially unlimited quantities of the precise genetic material molecular biologists and others required for their work (8). Since the first report in1985, more than 5000 scientific papers were published by 1992 (1). Furthermore, the large number of publications of course makes it impossible to review all the important contributions to the development and application of PCR technology; however we will attempt to review here the most important developments in the practice of basic PCR. Når andre Cetus forskerne i sidste ende lykkedes at gøre polymerase chain reaction udføre som ønsket på en pålidelig måde, de havde en uhyre kraftfuld teknik til at give væsentlige ubegrænsede mængder af det præcise genetiske materiale molekylær biologer og andre, der kræves for deres arbejde (8). Da første rapport in1985, mere end 5000 videnskabelige afhandlinger blev offentliggjort i 1992 (1). Endvidere er det store antal af publikationer selvfølgelig gør det umuligt at gennemgå alle de vigtige bidrag til udviklingen og anvendelsen af PCR-teknologien, men vi vil forsøge at gennemgå her den vigtigste udvikling i praksis af grundlæggende PCR.
PCR was thought to be conceived by Dr. Kerry Mullis in 1983 while working at the Cetus Corporation in Emeryville, CA. However, some pioneering work was also done by Gobind Khorana in 1971 who described a basic principle of replicating a piece of DNA using two primers. Progress then was limited by primer synthesis and polymerase purification issues (9). In Mullis’s head, the invention grew from a theoretical scheme to perform limited dideoxynucleotide sequencing of unique human genes using synthetic oligonucleotides for the purpose of diagnosing common human disease mutations. An obvious obstacle to such a direct sequencing strategy was the high complexity of the human genome (3.3 X 109 base pairs). Thus, a second oligonucleotide or primer was added to block the progression of the synthesis of the first primer. Later however, this second primer was included to bind to the other DNA strand, so that each strand of the mutant allele would contribute to the eventual signal. If the scheme involving simultaneous hybridization of primers to each strand was modified by heating the mixture and then repeating the annealing and extension steps, then the primary signal would be increased even further. Repeating the steps would enable the products of the first round to be duplicated in the second cycle, to yield two copies. Repeating the cycle again would result in four copies, et cetera . Several weeks passed before this great idea was attempted (8). Two primers were synthesized to be perfectly complementary to each end of the 110 base pair region of a cloned segment of the human b-globin gene, the amplification was performed, and the products were identified by acrylamide gel electrophoresis. The end result was the anticipated 110 base pair DNA fragment and the beginning of PCR as a basic technique in molecular biology (5,6). PCR blev anset for at være udtænkt af Dr. Kerry Mullis i 1983, mens der arbejdes på Cetus Corporation i Emeryville, Californien. Men nogle banebrydende arbejde blev også gjort ved Gobind Khorana i 1971 der er beskrevet et grundlæggende princip for at kopiere et stykke DNA med to primere. Fremskridt derefter blev begrænset af primer syntese og polymerase rensning spørgsmål (9). Mullis's hoved, opfindelsen voksede fra en teoretisk ordningen til at udføre begrænsede dideoxynucleotide sekventering af unikke menneskelige gener ved hjælp af syntetisk oligonucleotides med henblik på at diagnosticere fælles sygdom hos mennesker mutationer. En åbenlys hindring for en sådan direkte sekventering strategi var den store kompleksitet i det menneskelige genom (3,3 X 109 base par). Dermed er en anden oligonukleotide eller primer blev tilføjet for at blokere for udviklingen i en syntese af den første primer. Senere dog Denne anden primer blev medtaget for at binde sig til den anden DNA-streng, så hver del af mutant allel vil bidrage til den endelige signal. Hvis ordningen involverer samtidige krydsning af primere til hvert indsatsområde blev ændret ved opvarmning blandingen og derefter gentage annealing og udvidelse trin, så det primære signal ville være steget endnu mere. repetermekanisme de skridt vil gøre det muligt for varer fra den første runde, der skal gentages i den anden cyklus, at udbyttet to kopier. repetermekanisme cyklen igen vil resultere i fire eksemplarer, et cetera. adskillige uger, før dette store idé blev forsøgt (8). To primere blev syntese at være perfekt supplement til hver ende af de 110 base pair-området i et klonet segment af den menneskelige b-globin gen, forstærkningen blev udført, og produkterne blev identificeret af acrylamid gelelektroforese. Slutresultatet blev de forventede 110 base par DNA-fragment og begyndelsen af PCR som en grundlæggende teknik i molekylær biologi (5,6).
In Mullis's original PCR process(5,6,8), the enzyme was used in vitro (in a controlled environment outside an organism). I Mullis oprindelige PCR-processen (5,6,8), enzymet blev anvendt in vitro (i et kontrolleret miljø uden for en organisme). The double-stranded DNA was separated into two single strands by heating it to 96°C. Den dobbelt-strandede DNA blev opdelt i to individuelle strenge ved at opvarme det til 96 ° C. At this temperature, however, the E.Coli DNA polymerase was destroyed so that the enzyme had to be replenished after the heating stage of each cycle. Ved denne temperatur dog, at E. Coli DNA-polymerase blev ødelagt, så enzymet skulle genopfyldes efter opvarmning etape af hver cyklus. Mullis's original PCR process was very inefficient since it required a great deal of time, vast amounts of DNA-Polymerase, and continual attention throughout the PCR process. Mullis oprindelige PCR-processen var meget ineffektive, da det krævede en hel del tid, enorme mængder af DNA-polymerase og løbende opmærksomhed under PCR-processen.
Examination of the PCR amplification mechanism reveal its simplicity but also its elegance (Figure 2). Oligonucleotide primers are first designed to be complementary to the ends of the sequence to be amplified, and then mixed in molar excess with the DNA template and deoxyribonucleotides in an appropriate buffer. Following heating to denature the original strands and cooling to promote primer annealing, the oligonucleotides each bind to a different strand of the target fragment. The primers are positioned so that when each is extended by the action of a DNA polymerase, the newly synthesized strands will overlap the binding site of the opposite oligonucleotide. As the process of denaturation, annealing, and polymerase extension is continued the primers repeatedly bind to both the original DNA template and complementary sites in the newly synthesized strands and are extended to produce new copies of DNA (Figure 3). The end result is an exponential increase in the total number of DNA fragments that include the sequences between the PCR primers, which are finally represented at a theoretical abundance of 2n, where n is the number of cycles (1,7,13). Undersøgelse af PCR-amplifikation mekanisme afslører sin enkelthed men også dens elegance (figur 2). Oligonukleotide primere er først designet til at være et supplement til de ender af sekvensen skal forstærkes, og derefter blandet i molar overskydende med DNA-skabelon og deoxyribonucleotides i en passende buffer. Efter opvarmning til denaturere den originale dele og afkøling til fremme af primer annealing, oligonucleotides hver binde sig til en anden del af målet fragment. primere er anbragt således, at når hver udvides ved hjælp af en DNA-polymerase, den nyligt syntese strengene vil overlappe den bindende stedet for det modsatte oligonukleotide. Da processen med denaturering, annealing, og polymerase udvidelse er fortsat primere gentagne gange binde sig til både de oprindelige DNA-skabelon og supplerende lokaliteter i de nyligt syntetiserede indsatsområder og udvides til at fremstille nye kopier af DNA (Figur 3). Slutresultatet er en eksponentiel stigning i det samlede antal af DNA-fragmenter, der indeholder sekvenser mellem de PCR primere, som er endeligt repræsenteret på en teoretisk overflod af 2n, hvor n er antallet af cykler (1 , 7,13).
A DNA polymerase is a naturally occurring enzyme, a biological macromolecule that catalyzes the formation and repair of DNA. En DNA-polymerase er en naturligt forekommende enzym, et biologisk makromolekyle at catalyzes dannelse og reparation af DNA. It works by binding to a single DNA strand and creating a complementary strand. Det virker ved at binde sig til en enkelt DNA-streng og skabe en supplerende indsatsområde. The accurate replication of all living matter depends on this activity, where it functions to duplicate DNA when cells divide (10,11). Only recently have scientists learned to manipulate this activity and apply it to scientific research. The earliest PCR experiments utlilized the Klenow fragment of Escherichia coli DNA polymerase I at a temperature of 37C to amplify specific targets from human genomic DNA (5,6). Often these PCR reactions produced incompletely pure target product as judged by gel electrophoresis (1). These initial PCR amplifications with the Klenow fragment were not highly specific (5,6). Although a unique DNA fragment could be amplified ~200,000 fold from genomic DNA, only about 1% of the PCR product was the targeted sequence (13). A specific hybridization probe was required to analyse the amplified DNA (5,6). Den nøjagtige replikering af alle levende materiale afhænger af denne aktivitet, når det fungerer at duplikere DNA, når cellerne kløft (10,11). Først for nylig har videnskabsfolk lært at manipulere denne aktivitet og anvende den til videnskabelig forskning. Tidligst PCR eksperimenter utlilized de Klenow fragment af Escherichia coli DNA-polymerase jeg ved en temperatur på 37c til at uddybe specifikke mål fra menneskelige genom DNA (5,6). Ofte bliver disse PCR-reaktioner produceret ufuldstændigt ren målrette produktet som bedømmes ved gelelektroforese (1). Disse indledende PCR forstærkninger med Klenow fragment ikke var meget specifikke (5,6). Selv om en unik DNA-fragment kan forstærkes ~ 200000 fold fra genomisk DNA, er det kun omkring 1% af PCR-produktet blev de målrettede sekvens (13). En specifik hybridisering sonden var forpligtet til at analysere de forstærkede DNA (5,6).
Some PCR conditions were determined to increase the stringency of primer hybridization such as lower MgCl2 concentrations and higher annealing temperatures. Nogle PCR betingelser var fast besluttet på at øge stramning af primer krydsning såsom lavere MgCl2 koncentrationer og højere annealing temperaturer.
Furthermore, the concentration of enzyme and primers, the annealing time, extension time, and number of PCR cycles all were found to effect the specificity of the PCR. Desuden er koncentrationen af enzym-og primere, annealing tid, udvidelse tid, og antallet af PCR-cyklusser alle blev fundet virkning specificiteten af PCR.
Also, the concentration of a specific sequence in a sample can also influence the relative homogeneity of the PCR products (1,7,13,14,15). Deoxyribonucleotide triphosphates and magnesium in an appropriate buffer are also important ingredients for PCR. The efficiency and specificity of PCRs can be affected by variations in the concentration and ratio of free magnesium, deoxyribonucleotide triphosphates, and primers. Også koncentrationen af en bestemt sekvens i en prøve kan også påvirke den relative homogenitet af PCR-produkter (1,7,13,14,15). Deoxyribonucleotide triphosphater og magnesium i en passende buffer er også vigtige ingredienser til PCR. Effektiviteten og specificitet PCRs kan påvirkes af variationer i koncentration og forholdet til fri magnesium, deoxyribonucleotide triphosphater, og primere. These reagents must be optimized in order to achieve high specificity and yield (14). It was also discovered that the effect of temperature and oligonucleotide primer length on the specificity and efficiency of amplification by the polymerase chain reaction (15). Disse reagenser skal være optimeret med henblik på at opnå en høj specificitet og udbytte (14). Det blev også opdaget, at effekten af temperatur og oligonukleotid primer længde på specificitet og effektivitet forstærkelse af polymerase chain reaction (15).
The inactivation of the Klenow fragment of Escherichia coli DNA polymerase I at the high temperature required for strand separation required the addition of enzyme after the denaturation step of each cycle (5,6). Prior to 1988, anyone conducting a PCR reaction procedure was obliged to sit patiently by a series of water baths or heating blocks and add a fresh aliquot of E.Coli DNA polymerase after each denaturation step, which was typically carried out by immersing the reaction vessel in boiling water for ½ a minute to 3 minutes (7). This rather tedious step was eliminated by the introduction of a thermostable DNA polymerase, the Taq DNA polymerase (12) once, at the beginning of the PCR reaction. The thermostable properties of the DNA polymerase activity were isolated from Thermus aquaticus (Taq) (Figure 4) that grow in geysers of over 110C, and have contributed greatly to the yield, specificity, automation, and utility of the polymerase chain reaction (1,7,12). The Taq enzyme can withstand repeated heating to 94C and so each time the mixture is cooled to allow the oligonucleotide primers to bind the catalyst for the extension is already present (1,7). However, higher annealing temperatures were not established until the single “most important development of PCR development” (8), the purification and commercial distribution of a heat-resistant DNA polymerase from the thermophilic bacterium Thermus aquaticus ( Taq ) (12). Inaktivering af de Klenow fragment af Escherichia coli DNA-polymerase Jeg ved høj temperatur kræves for streng adskillelse kræves tilsætning af enzym efter denaturering trin i hver cyklus (5,6). Forud for 1988, der gennemfører en PCR-reaktion proceduren blev tvunget at sidde tålmodigt ved en række af vand bade eller opvarmning blokke og tilføje en ny prøve af E. Coli DNA-polymerase efter hver denaturering skridt, som var typisk udføres af dyppet reaktionen fartøj i kogende vand i ½ minut til 3 minutter (7 ). Denne ret kedelige skridt blev elimineret ved at indføre en thermostable DNA-polymerase, Taq DNA-polymerase (12) én gang i begyndelsen af PCR-reaktion. Thermostable egenskaber af DNA-polymerase aktivitet blev isoleret fra Thermus aquaticus (Taq) (Figur 4), der vokser i gejsere på over 110c, og har bidraget meget til det udbytte, specificitet, automatisering og nytteværdi af polymerase chain reaction (1,7,12). Taq enzym kan tåle gentagne opvarmning til 94c og så hver gang blandingen er afkølet til at tillade oligonukleotide primere til at forpligte katalysator for udvidelse er allerede til stede (1,7). Dog, højere annealing temperaturer ikke var etableret indtil enkelt "vigtigste udvikling af PCR udvikling" (8), rensning og kommerciel distribution af en varme-resistente DNA-polymerase fra termofile bakterien Thermus aquaticus (Taq) (12).
The isolation of a heat-resistant DNA polymerase also allowed primer annealing and extension to be carried out at elevated temperatures (1,7,12,13), thereby reducing mismatched annealing to nontarget sequences (non-specific amplification) or increasing specificity. In this way, for many amplifications the PCR product could be detected as a single ethidium bromide-stained band on an electrophoretic gel (12). This increased specificity also increased DNA yield of the target sequence. Moreover, longer PCR products could be amplified from genomic DNA, probably due to a reduction in the secondary structure of the template strands at the elevated temperature used for primer extension. The upper size limit for Klenow fragment polymerase amplification was only about 400bp. Taq polymerase and other thermostable polymerases have synthesized fragments up to 10 kb (1,7,12,13). The availability of Taq polymerase has also greatly simplified the automation of the reaction as it is a much easier task to construct an apparatus that will cycle a reaction tube through different temperatures than to manufacture a device that would perform both the thermocycling and the addition of enzyme aliquots. Currently there is a great variety of thermocyclers available commercially. This development has been a significant factor in the rapid application of this technology by the scientific community (7). Isoleringen af et varmeresistent DNA-polymerase også tilladt primer annealing og udvidelse, der skal udføres ved høje temperaturer (1,7,12,13), hvilket reducerer forkerte annealing til nontarget sekvenser (non-specifik amplifikation) eller stigende specificitet. denne måde, for mange forstærkninger PCR-produkt kan opdages som en enkelt ethidiumbromid-farvet bånd på en gel elektroforese (12). Denne øgede specificitet også øget DNA udbytte af målet sekvens. øvrigt længere PCR-produkter kunne blive forstærket fra genomiske DNA, sandsynligvis på grund af en reduktion i den sekundære struktur af skabelonen delområder ved forhøjet temperatur anvendes til primer forlængelse. Den øvre grænse for Klenow fragment polymerase forstærkningen var kun omkring 400bp. Taq Polymerase og andre thermostable polymeraser har syntese fragmenter op til 10 kb (1,7,12,13). Tilgængeligheden af Taq-polymerase er også stærkt forenklet automatisering af reaktionen, da det er en meget nemmere opgave at konstruere et apparat, der vil cyklus en reaktion rør gennem forskellige temperaturer end til fremstilling af en anordning at ville udføre både thermocycling og tilsætning af enzym delprøver. øjeblikket er der en lang række thermocyclers tilgængelige kommercielt. Denne udvikling har været en væsentlig faktor i den hurtige anvendelse af denne teknologi ved det videnskabelige samfund (7).
In addition to the production of double-stranded, blunt-ended DNA fragments which may be formed by PCR, two other features of the PCR scheme contribute greatly to the utility of PCR. First, the position of binding of the primers defines the boundaries of the amplified fragment and therefore the prior molecular cloning requirement of restriction endonuclease recognition sites is not required for PCR. As only a limited number of DNA sequences are restriction sites, PCR greatly increases the flexibility of choice of fragment size and composition. Secondly, it is not necessary for PCR oligonucleotides to be exactly complementary to the template DNA. “Tails” may be added to the 5’ end of the primer to introduce sequences within the priming sites which thus may be exploited to introduce restriction endonuclease recognition sites or other useful sequences such as mutations into the amplified DNA. This phenomena allowed the emergence of PCR as a method for rapid DNA cloning (1,7,13). Ud over produktionen af dobbelt-strandede, stumpt-tidsbegrænsede DNA-fragmenter, der kan være dannet ved PCR, to andre elementer af PCR-ordningen i høj grad bidrage til nytten af PCR. Først stilling binding af primere definerer grænserne for det forstærkede fragment og derfor forudgående molekylær kloning krav om begrænsning endonuclease anerkendelse websteder er ikke påkrævet for PCR. Da kun et begrænset antal DNA-sekvenser er begrænsning websteder, PCR øger fleksibiliteten i valget af fragment størrelse og sammensætning. For det andet er det ikke er nødvendigt for PCR oligonucleotides for at være nøjagtig komplementær til den skabelon DNA. "Haler" kan føjes til 5 'ende af primer til at indføre sekvenser inden for priming websteder, der således kan udnyttes til at indføre restriktioner endonuclease anerkendelse websteder eller andre nyttige sekvenser såsom mutationer i det forstærkede DNA. Denne fænomener tilladt fremkomsten af PCR som en metode til hurtig DNA kloning (1,7,13).
Molecular cloning has benefited from the emergence of PCR as a technique. Direct cloning was first conducted using a 110 bp DNA fragment amplified by PCR and oligonucleotide primers which contained restriction endonuclease recognition sites added to their 5’ ends. These sites were used to facilitate cloning of the amplified DNA into an M13 plasmid (17). The 110 bp fragment was also sequenced to confirm that this approach was a rapid yet reliable approach to cloning. (Figure 5) Molekylær kloning har nydt godt af fremkomsten af PCR som en teknik. Direkte kloning blev først gennemført ved hjælp af et 110 bp DNA-fragment forstærkes ved PCR og oligonukleotide primere, som indeholdt begrænsning endonuclease anerkendelse sites tilføjet til deres 5 'slutter. Disse arealer blev brugt til at lette kloning Det forstærkede DNA i en M13 plasmid (17). 110 bp-fragment blev også rækkefølge for at bekræfte, at denne fremgangsmåde var en hurtig endnu pålidelige tilgang til kloning. (fig. 5)
Cell-based DNA cloning involves DNA replication in vivo, which is associated with a very high fidelity of copying because of proofreading mechanisms. However, when DNA is replicated in vitro as with PCR, the copying error rate is considerably greater. The most widely used polymerase, Taq DNA polymerase however, has no associated 3’to 5’ exonuclease to confer a proofreading function. Thus the error rate due to base misincorporation during DNA replication is rather high for Taq : for a 1 kb sequence that has undergone 20 effective cycles of duplication, approximately 40% of the new DNA strands synthesized by PCR using this enzyme will contain an incorrect nucleotide resulting from a copying error (16). Cell-baseret DNA kloning indebærer DNA replikation in vivo, som er forbundet med en meget høj nøjagtighed for kopiering på grund af korrekturlæsning mekanismer. Men når DNA er replikeret in vitro som med PCR, kopieringen fejlprocent er betydeligt større. Den mest udbredte polymerase, Taq DNA-polymerase dog ikke har nogen tilknytning 3'to 5 'exonuclease at indebære en korrekturlæsning funktion. Således fejlprocenten grund til at basere misincorporation under DNA replikation er temmelig høj for Taq: for en 1 kb sekvens, der har undergået 20 effektive cyklusser overlapning, at cirka 40% af de nye DNA-strenge syntese ved PCR ved hjælp af dette enzym vil indeholde en forkert nukleotid skyldes et kopiering fejl (16). Therefore, even if the PCR reaction involves amplification of a single DNA sequence, the final product will be a mixture of almost matching, but not identical DNA sequences. Despite the errors due to replication in vitro , DNA sequencing of the total PCR product may give the correct sequence due to the fact that the incorporation of incorrect bases is essentially random and the contribution of one incorrect base on one or more strands is overwhelmed by the contributions from the huge majority of strands which will have the correct sequence. However, if the PCR product is to be cloned in cells, several individual clones may need to be sequenced in order to determine the correct (consensus) sequence, prior to conducting further experiments. Derfor, selv om PCR-reaktion indebærer forstærkelse af en enkelt DNA-sekvens, det endelige produkt vil være en blanding af næsten matcher, men ikke identiske DNA-sekvenser. På trods af de fejl som følge af replikation in vitro, DNA-sekventering af det samlede PCR-produkt kan give den korrekte sekvens skyldes, at inkorporeringen af ukorrekte baser i det væsentlige tilfældige og bidrag fra en ukorrekt basere på et eller flere områder er overvældet af bidragene fra de enorme flertal af de indsatsområder, som har den korrekte sekvens. Men hvis PCR-produkt skal klonet i cellerne, flere individuelle kloner kan det være nødvendigt at rækkefølge med henblik på at fastslå den korrekte (konsensus) sekvens, forud for udførelse af yderligere eksperimenter.
More recently, the problem of infidelity of DNA replication during the PCR reaction has been considerably reduced by using alternative heat-stable DNA polymerases which have associated 3’ to 5’ exonuclease activity. Pyrococcus furiosus ( Pfu ) DNA polymerases and Thermococcus Litoralis (VENT) are becoming more widely used because of the proofreading conferred by their associated 3’ to 5’ exonuclease activity (18). The resulting PCR product of Pfu for example, has a much lower level of mutations introduced by copying errors: for a 1 kb segment of DNA that has undergone 20 effective cycles of duplication, about 3.5% of the DNA strands in the product carry an altered base (16). På det seneste er problemet med vantro af DNA-replikation under PCR-reaktionen er blevet betydeligt reduceret ved anvendelse af alternative varme-stabile DNA polymeraser, som er knyttet 3 'til 5' exonuclease aktivitet. Pyrococcus furiosus (Pfu) DNA polymeraser og Thermococcus Litoralis (VENT) bliver mere og mere udbredte på grund af den korrekturlæsning tillagt ved deres associerede 3 'til 5' exonuclease aktivitet (18). Den resulterende PCR-produktet af Pfu for eksempel har et meget lavere niveau af mutationer indført ved kopiering fejl: i stedet for en 1 kb segment af DNA, der har undergået 20 effektive cyklusser for dobbeltarbejde, ca 3,5% af det DNA-strenge i produktet foretage en ændret base (16).
New approaches to improve specificity have been developed based on the recognition that the Taq DNA polymerase retains considerable enzymatic activity at temperatures well below the optimum for DNA synthesis. Thus, primers annealing non-specifically to a partially single stranded template region can be extended before the reaction reaches 72°C for extension of specifically annealed primers. If the DNA polymerase is activated only after the reaction has reached high (>70°C) temperatures, non-target amplification can be minimized (19,20). This “Hot start” approach can be accomplished by manual addition of an essential reagent to the selection tube at elevated temperatures. The addition of ssDNA binding protein has also been reported to increase specific amplification. A more user friendly approach is to use either inhibition or inactivation of the DNA polymerase itself. Nye metoder til at forbedre specificitet er blevet udviklet baseret på erkendelsen af, at de Taq DNA-polymerase bevarer betydelig enzymatiske aktivitet ved temperaturer langt under det optimale for DNA syntese. Således primere annealing ikke-specifikt til en delvis enkelt strandede skabelon regionen kan udvides før reaktion når op på 72 ° C for udvidelse af specifikt udgloedet primere. Hvis DNA-polymerase kun aktiveres efter reaktionen har nået høj (> 70 ° C) temperaturer, uden for målgruppen amplifikation kan minimeres (19,20). Denne "Hot start "Tilgang kan udføres ved manuel tilsætning af et vigtigt reagens til udvælgelsen rør ved høje temperaturer. Tilføjelsen af ssDNA bindende protein er også blevet rapporteret at øge specifik opformering. En mere brugervenlig fremgangsmåde er at bruge enten hæmning eller inaktivering af DNA polymerase selv. Two types of inhibition of Taq DNA polymerase have been tried including oligonucleotide inhibition (21) and antibody (22) inhibition. To typer af hæmning af Taq DNA-polymerase er blevet forsøgt herunder oligonukleotide hæmning (21) og antistof (22) hæmning. Highly specific oligonucleotide inhibitors of both Taq DNA polymerases have been produced. Stærkt oligonukleotide hæmmere af både Taq DNA polymeraser er blevet produceret. These selectively inhibit DNA polymerase activity at temperatures below 40°C and have been shown to function in Hot Start applications. Disse selektivt hæmmer DNA-polymerase aktivitet ved temperaturer under 40 ° C og har vist sig at fungere i Hot Start applikationer. Alternatively, one can use an antibody against Taq DNA polymerase. Alternativt kan man bruge et antistof mod Taq DNA-polymerase. The antibody inhibits the DNA polymerase until the temperature of the PCR is such that the antibody is denatured at a temperature greater than 55°C, thereby releasing the enzyme. Antistoffet hæmmer DNA-polymerase, indtil temperaturen af PCR er sådan, at antistof er denatureret ved en temperatur på over 55 ° C, hvorved der frigives enzymet. However there are disadvantages to this type of Hot Start conditions. Men der er ulemper for denne type Hot Start betingelser. In this case, one needs an antibody for each different enzyme used in a PCR and for a large number of PCRs this can rise costs significantly. I dette tilfælde må man se en antistof for hver enkelt enzym bruges i en PCR og for et stort antal PCRs dette kan medføre omkostninger betydeligt. The most convenient form of Hot Start is to modify the DNA polymerase in such a way that it is inactive at room temperature (temperature-sensitive mutant), and is only re-activated following incubation at 95°C for 6-15 minutes (23). Den mest bekvemme form af varm start er at ændre de DNA-polymerase på en sådan måde, at det er inaktivt ved stuetemperatur (temperatur-følsomme mutante), og er kun aktiveret igen efter inkubation ved 95 ° C i 6-15 minutter (23 ).
Because of its simplicity, PCR is a popular technique with a wide range of applications På grund af sin enkelhed, PCR er en populær teknik med en bred vifte af applikationer
including direct sequencing, genomic cloning, DNA typing, detection of infectious microorganisms, site-directed mutagenesis, prenatal genetic disease research, and analysis of allelic sequence variations (1,7,13,16) which depend on essentially three major advantages of the method: herunder direkte sekventering, genomiske kloning, DNA-typebestemmelse, afsløring af smitsomme mikroorganismer, site-dirigeret mutagenese prænatal genetisk sygdom, forskning og analyse af allelic sekvens varianter (1,7,13,16), som afhænger af hovedsagelig tre store fordele af metoden :
Speed and ease of use: DNA cloning by PCR can be performed in a relatively short amount of time, within a few hours. Usually, a PCR reaction consists of around 30 cycles each cycle containing a denaturation, synthesis and reannealing step, with an individual cycle typically taking 3 Fart og brugervenlighed: DNA kloning af PCR kan udføres inden for en relativt kort tid, inden et par timer. Normalt er en PCR-reaktion består af omkring 30 cykler hver cyklus indeholder en denaturering, syntese og reannealing skridt, med en individuel cyklus typisk under 3 
5 min in an automated thermal cycler. This is clearly quicker than the time required for cell-based DNA cloning, which could take weeks of time. Furthermore, it is quite easy to setup a PCR reaction and the use of a thermocycler machine is also easy. Some time is required for the design and synthesis of oligonucleotide primers, but this has been simplified by the availability of computer software for primer design and rapid commercial or academic synthesis of custom oligonucleotides. Optimization of PCR conditions may be required such as primer annealing temperature, magnesium concentration, and primer concentration. However, the creation of gradient PCR machines which allow a variety of primer annealing temperatures to be tested at the same time has greatly decreased the time required for this step. 5 min på en automatisk termisk cycler. Dette er klart hurtigere end den tid der kræves for celle-baserede DNA kloning, hvilket kan tage uger af gangen. Desuden er det temmelig nemt at setup en PCR-reaktion og anvendelsen af en thermocycler maskine er også let. Nogen tid der kræves til design og syntese af oligonukleotide primere, men dette er blevet forenklet ved tilgængeligheden af computer software til primer design og hurtig kommerciel eller akademiske syntese af brugerdefinerede oligonucleotides. Optimering af PCR betingelser kan kræves såsom primer annealing temperatur, magnesium koncentration, og primer koncentration. Men oprettelsen af gradient PCR-maskiner, som muliggør en vifte af primer annealing temperaturer, der skal testes på samme tid har i høj grad faldt den tid der kræves til dette skridt. Once the optimal conditions for a reaction have been obtained, the reaction can then be simply repeated (1,7,13,16). Når de optimale betingelser for en reaktion er blevet fremstillet, reaktionen kan derefter blot gentages (1,7,13,16).
Sensitivity: PCR is capable of amplifying sequences from minute amounts of target DNA, even the DNA from a single cell (24). Such exquisite sensitivity has afforded new methods of studying molecular pathogenesis and has found numerous applications in forensic science, in diagnosis, in genetic linkage analysis using single-sperm typing and in molecular paleontology studies, where samples may contain minute numbers of cells. Følsomhed: PCR er i stand til at supplere sekvenser fra minut mængder af mål-DNA, selv DNA fra en enkelt celle (24). Sådanne udsøgte følsomhed har givet nye metoder til at studere molekylær patogenese og har fundet talrige anvendelser inden for retsmedicin, i diagnosticering, genetiske sammenkobling analyse vha. single-sperm maskinskrivning og i molekylær paleontology undersøgelser, hvor prøverne kan indeholde minut antal celler. However, the extreme sensitivity of the method means that great care has to be taken to avoid contamination of the sample under investigation by external DNA, such as from minute amounts of cells from the operator (1,7,13,16). Men den ekstreme følsomhed af metoden betyder, at stor omhu har der skal træffes for at undgå forurening af prøven, der efterforskes af eksterne DNA, såsom fra minut mængder af celler fra den erhvervsdrivende (1,7,13,16).
Robustness: A broad range of nucleic acid sources are suitable templates for PCR amplification. Purified DNAs from various species and sources have been amplified. PCR can permit amplification of specific sequences from material in which the DNA is badly degraded or embedded in a medium from which conventional DNA isolation is problematic. Robustness: En bred vifte af nukleinsyre kilder er egnet skabeloner til PCR-amplifikation. Renset udpegede nationale myndigheder fra forskellige arter og kilder er blevet forstærket. PCR kan tillade forstærkning af specifikke sekvenser fra materiale, hvor DNA er stærkt forringet eller indlejret i et medium, hvorfra konventionelle DNA isolation er problematisk. As a result, it is again very suitable for molecular anthropology and paleontology studies, for example the analysis of DNA recovered from archaeological remains. Som et resultat, er det igen meget velegnet til molekylær antropologi og paleontology undersøgelser, for eksempel analyse af dna inddrives fra arkæologiske afdøde. It has also been used successfully to amplify DNA from formalin-fixed or paraffin-embedded tissue samples, which has important applications in molecular pathology and, in some cases, genetic linkage studies. Det har også været brugt med held til at uddybe DNA fra formalin-fast eller paraffin-indstøbte vævsprøver, der har vigtige anvendelser inden for molekylær patologi og i nogle tilfælde, genetiske sammenkobling undersøgelser. Generally, the success of PCR amplification is greatest when target fragments are relatively abundant (1,7,13,16). Generelt succes for PCR-amplifikation er størst, når målet fragmenter er relativt rigelige (1,7,13,16).
Despite its huge popularity, PCR has certain limitations as a method for selectively cloning specific DNA sequences. Trods sin enorme popularitet, PCR har visse begrænsninger som en metode til selektivt kloning specifikke DNA-sekvenser.
In order to construct specific oligonucleotide primers that permit selective amplification of a particular DNA sequence, some prior sequence information is usually necessary. For at konstruere oligonukleotide primere at tillade selektiv opformering af en bestemt DNA-sekvens, nogle forudgående sekvens oplysninger er normalt nødvendigt. This normally means that the DNA region of interest has been partly characterized previously, often following prior cell-based DNA cloning. Dette normalt betyder, at DNA-regionen interesse er delvis karakteriseret tidligere, ofte efter forudgående celle-baseret DNA kloning. However, a variety of approaches have been developed that reduce or even exclude the need for prior DNA sequence information concerning the target DNA. Previously uncharacterized DNA sequences can sometimes be cloned using PCR with degenerate oligonucleotides if they are members of a gene or repetitive DNA family at least one of whose members has previously been characterized. Men en række forskellige tilgange er blevet udviklet, at nedsætte eller endog udelukke behovet for forudgående DNA-sekvens oplysninger om mål-DNA. Tidligere uncharacterized DNA-sekvenser kan undertiden være klonet ved hjælp af PCR med degenerere oligonucleotides hvis de er medlemmer af et gen eller gentagne DNA-familien mindst et af medlemmerne har tidligere været præget. In some cases, PCR can be used effectively without any prior sequence information concerning the target DNA to permit indiscriminateamplification of DNA sequences from a source of DNA that is present in extemely limited quantities. I nogle tilfælde, PCR kan anvendes effektivt uden nogen forudgående sekvens oplysninger om mål-DNA til at tillade indiscriminateamplification af DNA-sekvenser fra en kilde af DNA, der er til stede i extemely begrænsede mængder. Therefore, although PCR can be applied to ensure whole genome amplification, it does not have the advantage of cell-based DNA cloning in offering a way of separating the individual DNA clones comprising a genomic DNA library. Derfor, selv om PCR kan anvendes til at sikre hele genamplifikationsteknik, det ikke har den fordel, celle-baseret DNA kloning i at tilbyde en måde at adskille de enkelte DNA-kloner, der omfatter en genomiske DNA-biblioteket.
The amount of PCR product obtained in a single reaction is also much more limited than the amount that can be obtained using cell-based cloning where scale-up of the volumes of cell cultures is possible. Størrelsen af PCR-produkt, der fremstilles i en enkelt reaktion er også langt mere begrænset end det beløb, der kan opnås ved hjælp af celle-baseret kloning, hvor skala-up af de mængder af cellekulturer er muligt. The efficiency of a PCR reaction will vary from template to template and according to various factors that are required to optimize the reaction but typically only comparatively small amounts of product are achieved. Effektiviteten af en PCR-reaktion vil variere fra skabelon til skabelon og i henhold til forskellige faktorer, der kræves for at optimere den reaktion, men typisk kun relativt små mængder af produktet er nået.
Although the theoretical yield of PCR is exponential, the actual yield of a PCR is much less indicating that the scheme is operating with less than its maximum potential. For example, the amount of product at each cycle eventually levels off. This plateau may be explained by the following phenomena. First, some of the template may never be available due to strand breaks or failure of the DNA to dissociated from other macromolecules during purification and the initial thermocycles. Secondly, the amount of enzyme is finite and eventually activity may decrease. Thirdly, as the concentration of the double-stranded product reaches high levels, competition increases between annealing of template (PCR product) to primer and reannealing of the complementary template strands (1,7,13). Selvom det teoretiske udbytte af PCR er eksponentiel, det faktiske afkast af en PCR er langt mindre angiver, at ordningen fungerer med færre end dens maksimale potentiale. For eksempel er mængden af produktet i hver cyklus sidst niveauer off. Dette plateau kan forklares med følgende fænomener. For det første er nogle af den skabelon mai aldrig være til rådighed på grund af streng pauser eller fiasko af DNA til at skille fra andre makromolekyler under rensningen og den indledende thermocycles. For det andet er den mængde enzym er afgrænset og i sidste ende aktivitet mai falde. Tredje, som den koncentration af det dobbelte strandede vare når høje niveauer, konkurrencen øges mellem annealing af skabelon (PCR-produkt) til primer og reannealing af komplementære skabelon indsatsområder (1,7,13).
An obvious and many times great disadvantage of PCR as a DNA cloning method has been the size range of the DNA sequences that can be cloned. Et indlysende og mange gange stor ulempe ved PCR som en DNA kloning metode har været den størrelse vifte af DNA-sekvenser, der kan klones. Unlike cell-based DNA cloning where the size of cloned DNA sequences can approach 2 Mb, reported DNA sequences cloned by PCR have typically been in the 0.1 I modsætning til celle-baseret DNA kloning, hvor størrelsen af klonede DNA-sekvenser kan henvende sig til 2 Mb, rapporterede DNA-sekvenser klonet ved PCR har typisk været på 0,1 
5 kb size range, often at the lower end of this scale. 5 kb størrelsesorden, ofte i den lavere ende af denne skala. Small fragments of DNA can usually be amplified easily by PCR, however it becomes increasingly more difficult to obtain efficient amplification as the desired product length increases. Små fragmenter af DNA kan normalt blive forstærket let ved PCR, men det bliver stadig vanskeligere at opnå en effektiv forstærkning som den ønskede vare længde øges. Barnes (25) recognized a target length limitation to PCR amplification of DNA. He used a combination of a high level of an exonuclease-free, N-terminal deletion mutant of Taq DNA polymerase, Klentaq1, with a very low level of a thermostable DNA polymerase exhibiting a 3'-exonuclease activity (Pfu, Vent, or Deep Vent) to conduct high fidelity long PCR. Barnes (25) anerkendt en målsætning længde begrænsning til PCR-amplifikation af DNA. Han brugte en kombination af et højt niveau af en exonuclease-fri, N-terminal sletning mutant af Taq DNA-polymerase, Klentaq1, med et meget lavt niveau af et thermostable DNA polymerase udstille en 3'-exonuclease aktivitet (Pfu, Vent, eller Deep Vent) at foretage high fidelity lang PCR. At least 35 kb of bacteriophage lambda can be amplified to high yields from 1 ng of lambda DNA template. Use of this method yielded increased base-pair fidelity, the ability to use PCR products as primers, and the maximum yield of target fragment. Other conditions have been identified for effective amplification of longer targets, including amplification of up to 22 kb of the beta-globin gene cluster from human genomic DNA and up to 42 kb from phaga lambda DNA (26). Mindst 35 kb af bakteriofag lambda kan forstærkes med høje udbytter fra 1 ng af lambda DNA-skabelon. Brug af denne metode gav øget base-pair troskab, evnen til at anvende PCR-produkter som primere, og det maksimale udbytte af mål-fragment. Andre betingelser er blevet identificeret for en effektiv forstærkning af længere mål, herunder forstærkning på op til 22 kb af beta-globin gen klynge fra menneskelige genom DNA og op til 42 kb fra phaga lambda DNA (26). The conditions for these long PCRs included increased pH, addition of glycerol and dimethyl sulfoxide, decreased denaturation times, increased extension times, and the use of a secondary thermostable DNA polymerase that possesses a 3'-to 5'-exonuclease, or "proofreading," activity. Betingelserne for disse lange PCRs medtaget øget pH, tilsætning af glycerol og dimethylsulfoxid, faldt denaturering gange, øget udvidelse gange, og brugen af en sekundær thermostable DNA-polymerase, der besidder en 3'-5'-exonuclease eller "korrekturlæsning, "Aktivitet. The "long PCR" protocol maintained the specificity required for targets in genomic DNA by using lower levels of polymerase and temperature and salt conditions for specific primer annealing. The ability to amplify DNA sequences of 10-40 kb will bring the speed and simplicity of PCR to genomic mapping and sequencing and facilitate studies in molecular genetics (26). Generally, the conditions for long range PCR involve a combination of modifications to standard conditions with a two-polymerase system. Den "lange PCR" protokol opretholdes specificitet kræves til mål i genomisk DNA ved hjælp af lavere polymerase og temperatur og salt betingelser for specifikke primer annealing. Muligheden for at supplere DNA-sekvenser af 10-40 kb vil bringe den hastighed og enkelhed af PCR til genomiske kortlægning og sekventering og lette studier i molekylær genetik (26). Sædvanligvis er betingelserne for lange række PCR involvere en kombination af ændringer til standardbetingelser med en to-polymerase system. This provides optimal levels of DNA polymerase and 3’to 5’ exonuclease activity which serves as a proofreading mechanism (16). Dette giver optimale niveauer af DNA-polymerase og 3'to 5 'exonuclease aktivitet, der tjener som et korrekturlæsning mekanisme (16).
Thermocyclers which automatically regulate temperatures for PCR cycling were introduced in 1986 (Figure 6). In addition to the advances in PCR reagents, new instruments for automated thermal cycling and for analyzing PCR products have been developed. New thermal cyclers have increased rates of heating, cooling, and heat transfer to modified reaction vessels. The reaction vessels accommodated by the first generation thermal cyclers (or even water baths and heating blocks) were standard plastic microfuge tubes. PCR amplification in thin capillary tubes allowed rapid thermal cycling, and DNA synthesis to 20s. The speed of the temperature changes achieved in these systems has allowed the precise definition of temperature optima for each individual step in the PCR cycle. The new generation thermal cyclers also accommodate more samples, have more precise thermal profiles, and are programmable (13). Thermocyclers som automatisk regulere temperaturen for PCR cykling blev indført i 1986 (Figur 6). Ud over de fremskridt inden for PCR-reagenser, nye instrumenter til automatiseret termisk cykling og for at analysere PCR-produkter er blevet udviklet. Ny termisk cyclers steg satserne for opvarmning, køling og varme overførsel til modificerede reaktion fartøjer. Reaktionen fartøjer huses af den første generation termisk cyclers (eller endda vand bade og varme blokke) var standard plast mikrofugeglas rør. PCR-amplifikation i tynde kapillar rør tilladt hurtig termisk cykling, og DNA syntese til 20s. Hastighed temperaturen ændringer opnået i disse systemer har gjort det muligt for den præcise definition af temperatur Optima for hvert enkelt skridt i den PCR cyklus. Den nye generation termisk cyclers også tilgodese flere prøver, har mere præcise termiske profiler, og er programmerbar (13 ).
One of the least appreciated contributions to the widespread application of PCR has been the development of reliable automated chemistry for oligonucleotide synthesis. Until recently, the construction of a single oligonucleotide was a substantial task that could only be performed by a skilled organic chemist. Now it is possible to purchase either an oligonucleotide synthesizer that can be operated by a technician or the oligonucleotides themselves from a commercial or academic source. Multiplex oligonucleotide synthesis machines have been constructed with the aim of reducing the overall cost of synthesis (27,28). En af de mindst værdsat bidrag til den udbredte anvendelse af PCR har været udviklingen af pålidelige automatiseret kemi for oligonucleotid syntese. Indtil for nylig, opførelse af et enkelt oligonukleotide var en væsentlig opgave, som kun kunne udføres af en uddannet organisk kemiker. Nu er det er muligt at købe enten en oligonukleotide synthesizer, der kan drives af en tekniker eller oligonucleotides sig fra en kommerciel eller akademisk kilde. Multiplex oligonucleotid syntese maskiner er blevet konstrueret med det formål at reducere de samlede omkostninger ved syntese (27,28). As the oligonucleotides define the eventual PCR products, there is little doubt that in the absence of their ready supply, PCR would not have enjoyed the wide acceptance that it has gained today (13). Som oligonucleotides definere eventuelle PCR-produkter, er der ikke meget tvivl om, at i mangel af deres store udbud, PCR ville ikke have haft den brede accept af, at den har vundet i dag (13).
Researchers agreed early on that the design of PCR primers was difficult and unreliable. Computer programs were devised to take all of the design criteria into account. Forskerne enedes tidligt, at udformningen af PCR primere var vanskelige og upålidelige. Edb-programmer blev udtænkt til at træffe alle de design kriterier i betragtning. One of the first programs written for primer design was Olga which made use of the implementation of Digital Research GEM (Graphics Environment Manager) on the Atari ST (29). En af de første programmer skrevet til primer design var Olga, der gjorde brug af gennemførelsen af Digital Research GEM (Graphics Environment Manager) på Atari ST (29). Olga was specifically suited to the polymerase chain reaction (PCR) allowing simultaneous analysis of two primer sequences. Olga var specielt velegnet til polymerase chain reaction (PCR) giver mulighed for samtidig analyse af to primer sekvenser. The advantage of Olga was that it provided in one program analyses for direct repeats, secondary structures and primer dimerization as well as several useful 'finishing' tools for workers engaged in PCR optimization and oligonucleotide syntheses. The Primer3 program at the Whitehead Institute is now thought to be the most reliable and versatile tool currently available (30). Fordelen ved Olga var, at det er fastsat i ét program analyser til direkte gentager, sekundære strukturer og primer dimerization samt flere nyttige 'finish' værktøjer til arbejderbeskyttelse i PCR-optimering og oligonukleotide synteser. Primer3 program ved Whitehead Institute er nu troede at være den mest pålidelige og alsidigt værktøj for øjeblikket er til rådighed (30).
PCRs can now be performed enabling the amplification of DNA fragments up to several kilobases in length by more than one million times their initial abundance. The procedure is highly automatable and requires just a few hours from beginning the thermocyling to product analysis. This was not the case previously, and the practical requirements for performing a PCR have been greatly simplified since the first manuscripts of the method (13). Today, most of the initial hitches or inefficiencies of the PCR have been worked out (8). Furthermore, PCR has expanded to include more than 270,000 articles (31). PCRs kan nu udføres der gør det muligt for opformering af DNA-fragmenter op til flere kilobases i længden med mere end én million gange deres oprindelige overflod. Proceduren er yderst automatiske og kræver kun et par timer fra begyndelsen af thermocyling til produkt-analyse. Dette var ikke tilfældet tidligere, og de praktiske krav til udførelsen af en PCR blevet stærkt forenklet, da de første manuskripter af metoden (13). I dag er de fleste af de indledende problemer eller ineffektivitet af PCR er blevet udarbejdet (8). Endvidere PCR har udvides til at omfatte mere end 270000 artikler (31).
Polymerase chain reaction is the most widely used method for in vitro DNA amplification however it requires thermal denaturation or thermocycling to separate the two DNA strands. In vivo , DNA is replicated by DNA polymerases with various accessory proteins. DNA helicase, a DNA polymerase accessory proteins acts to separate duplex DNA inside cells. Vincent et al. (32) have devised a new in vitro isothermal DNA amplification method by mimicking the in vivo replication mechanism. Helicase-dependent amplification (HDA) utilizes a DNA helicase to generate single-stranded templates for primer hybridization. Subsequent primer extension is then catalyzed by a DNA polymerase. HDA does not require an expensive thermocycler and thus PCR may be performed practically anywhere. In addition, it offers several advantages over other isothermal DNA amplification methods by having a simple reaction scheme and being a true isothermal reaction that can be performed at one temperature for the entire process. HDA offers great promise in the development of simple portable DNA diagnostic devices to be used in the field and at the point-of-care (32).
It is said the simplest and most convenient way to define PCR is as a technique . However, such a categorization eliminates the history of PCR's development as many individuals over the years contributed to the ideas behind the theory of PCR and the fine-tuning of the technique. The next simplest answer is to name an individual as the inventor of the polymerase chain reaction. Karry Mullis was awarded the Nobel Prize for Chemistry in 1993 for his discovery of PCR. However, this discovery is contested amongst many scientists, all of which may have contributed to unlocking this puzzle.
It has also been said that PCR did not exist until it was made to work in an experimental system. With this in mind, merely the thought of a concept is not sufficient; a concept must have been successfully been put into practice (33).
Although there is doubt as to the ultimate creator of PCR, and doubt as to the possibility that PCR may somehow or sometime be replaced, there is little doubt the impact that PCR has created over a short time span on the study of molecular biology and life.
References
1. Arnheim, N; Erlich, H; Polymerase Chain Reaction Strategy. ANNUAL REVIEW OF BIOCHEMISTRY, VOL. 61. XIV+1359P. , 1992. p. 131-156.
2. Appenzeller T. Democratizing the DNA sequence., Science , 1990 Mar 2, 247(4946).
3. Saiki R, K.; Scharf S; Faloona F; Mullis K. B; Horn G. T; Erlich HA; Arnheim N., Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia, Science , 1985 Dec 20, 230(4732):1350-4.
4. Southern, EM (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98:503-518.
5. Mullis K. B; Faloona F. A; Scharf S; Saiki R. K; Horn G; Erlich HA, Specific enzymatic amplification of DNA in vitro: the polymerase chain reaction. Cold Spring Harbor Symposia on Quantitative Biology , 1986
6. Mullis K. B; Faloona FA Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction. Methods in Enzymology , 1987, 155:335-50.
7. Gibbs, RA; DNA Amplification by the Polymerase Chain Reaction. Analytical Chemistry, 1990, 62:1202-1214.
8. Mullis, KB; The Unusual Origin of the Polymerase Chain Reaction, Scientific American, April 1990.
9. Kleppe, KE; Khorana , HG; (1971) J. Mol. Biol. 56, 341-346.
10. Keir, H; DNA polymerases from mammalian cells. Prog Nucleic Acid Res Mol Biol. 1965;4:81-128.
11. Fansler, BS; Eukaryotic DNA polymerases: their association with the nucleus and relationship to DNA replication. Int Rev Cytol. 1974;Suppl 4:363-415.
12. Saiki R. K; Gelfand D. H; Stoffel S; Scharf S. J; Higuchi R; Horn G. T; Mullis K. B; Erlich HA. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science , 1988 Jan 29, 239(4839):487-91.
13. Erlich, H. A; Gelfand, D; Sninsky, JJ Recent Advances in the Polymerase Chain Reaction., Science , 1991, v.252, n.5013, 1643-1651.
14. Williams, JF; Optimization strategies for the polymerase chain reaction. Biotechniques. 1989 Jul-Aug;7(7):762-9.
15. Wu DY, Ugozzoli L, Pal BK, Qian J, Wallace RB. The effect of temperature and oligonucleotide primer length on the specificity and efficiency of amplification by the polymerase chain reaction. DNA Cell Biol. 1991 Apr;10(3):233-8.
16. Strachan, T; Read AP; Human Molecular Genetics 2 . 1999. John Wiley & Sons Inc. Chapter 6 Section 1.
17. Scharf S. J; Horn G. T; Erlich HA Direct cloning and sequence analysis of enzymatically amplified genomic sequences. Science , 1986 Sep 5, 233(4768):1076-8.
18. Cline J, Braman JC, Hogrefe HH; PCR fidelity of pfu DNA polymerase and other thermostable DNA polymerases. Nucleic Acids Res. 1996 Sep 15;24(18):3546-51.
19. Falooea, F., Weiss, S., Ferre, F., Mullis, K. 1990 6th Int.Conf . AIDS . Abstr.
20. D’Aquila, RT, Bechtel, LJ, Videler, JA, Eron, JJ, Goeczyca, P., Kaplan, JC 1991. Nucleic Acids Res. 19:3749.
21. Dang C, Jayasena SD. Oligonucleotide inhibitors of Taq DNA polymerase facilitate detection of low copy number targets by PCR. J Mol Biol. 1996 Nov 29;264(2):268-78.
22. Kellogg DE, Rybalkin I, Chen S, Mukhamedova N, Vlasik T, Siebert PD, Chenchik A. TaqStart Antibody: "hot start" PCR facilitated by a neutralizing monoclonal antibody directed against Taq DNA polymerase. Biotechniques. 1994 Jun;16(6):1134-7.
23. Kermekchiev MB, Tzekov A, Barnes WM. Cold-sensitive mutants of Taq DNA polymerase provide a hot start for PCR. Nucleic Acids Res. 2003 Nov 1;31(21):6139-47.
24. H. Li, UB Gyllenstein, X. Cui, RK Saiki, H. Ehrlich, and N. Arnheim. (1988). Amplification and analysis of DNA sequences in single human sperm and diploid cells Nature 335: 414-417.
25. Barnes WM.; PCR amplification of up to 35-kb DNA with high fidelity and high yield from lambda bacteriophage templates. Proc Natl Acad Sci US A. 1994 Mar 15;91(6):2216-20.
26. Cheng S, Fockler C, Barnes WM, Higuchi R. Effective amplification of long targets from cloned inserts and human genomic DNA. Proc Natl Acad Sci US A. 1994 Jun 7;91(12):5695-9.
27. Caruthers MH, Barone AD, Beaucage SL, Dodds DR, Fisher EF, McBride LJ, Matteucci M, Stabinsky Z, Tang JY. Chemical synthesis of deoxyoligonucleotides by the phosphoramidite method. Methods Enzymol. 1987;154:287-313.
28. Beattie KL, Logsdon NJ, Anderson RS, Espinosa-Lara JM, Maldonado-Rodriguez R, Frost JD 3rd. Gene synthesis technology: recent developments and future prospects. Biotechnol Appl Biochem. 1988 Dec;10(6):510-21.
29. Bridges CG. Olga--oligonucleotide primer design program for the Atari ST. Comput Appl Biosci. 1990 Apr;6(2):124-5.
30. Rozen S, Skaletsky H. Primer3 on the WWW for general users and for biologist programmers. Methods Mol Biol. 2000;132:365-86.
31. Location world wide web: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed Search: “PCR”
32. Vincent M, Xu Y, Kong H. Helicase-dependent isothermal DNA amplification. EMBO Rep. 2004 Aug;5(8):795-800. Epub 2004 Jul 09.
33. Paul Rabinow. Making PCR, A Story of Biotechnology , University of Chicago Press, 1996
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