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Polymerase Chain Reaction - PCR PCR - PCR
Table of Contents : Table of Contents:
Introduction to PCR - Polymerase Chain Reaction Uvod u PCR - PCR
Polymerase Chain Reaction (PCR) PCR (PCR)
PCR, a Concept to be Discovered PCR, koncept se Pronalazač
General Principles of the PCR General Principles od PCR
Polymerases/Reaction Specificity and Efficiency Polymerases / Reaction specifičnost i efikasnost
PCR and Molecular Cloning PCR i molekularnu Klon
Misincorporation: Errors of In Vitro Systems Misincorporation: Pogreške u in vitro sustavi
Reaction Specificity Reakcije specifičnost
Major Advantages of PCR as a Cloning Method include its Rapidity, Sensitivity, and Robustness Veći Prednosti PCR kao Klon Metoda obuhvataju brzina, osjetljivost, i Robusnost
Limitations of PCR Ograničenja PCR
Instruments for PCR Uređaji za PCR
Oligonucleotide Synthesis Oligonucleotide Synthesis
Will PCR ever be replaced? PCR će ikada biti zamijenjena? – Helicase-dependent Amplification (HDA) -- Helicase-ovisna pojačanje (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). Više od 30 godina, uvođenje rekombinantnim DNA tehnologija kao alata za biološke znanosti revolutionized proučavanju života. Molekularnu kloniranje dozvoljeno je studija pojedinih gena žive organizme, no ove tehnike je ovisna o dobivanju relativno veliku količinu čistoga DNA. Depended na replikacija DNA, od plazmidi ili drugim vektori tijekom stanične diobe od mikroorganizama (1). Istraživači pronađena je izuzetno marljiv i teško da bi se dobila specifičnu DNA u količini od mase geni prisutni u biološki uzorak (2 ). Rekombinantnim DNA tehnologijom omogućila je prvi molekularna analiza i prenatalna dijagnoza nekoliko ljudskih bolesti. Fetalna DNA dobivenih amniocentesis uzorkovanja mogu biti analizirani uz ograničenje enzim probavu, elektroforeza, hibridizacija i južne transfer do klon gena ili oligonucleotide probe (3). Međutim, južni upijajući dopušteno samo elementarni mapiranje gena u nepovezanih pojedinaca (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, skraćenica za PCR (5,6), dozvoljeno proizvodnju velikih količina specifične DNA iz kompleksa DNK predloška na jednostavan enzimska reakcija. PCR je nedavno razvio postupak za in vitro amplifikacija DNA. PCR je transformirao način na koji gotovo sve studije zahtijevaju manipulacije DNA fragmenata svibanj biti izvedena kao rezultata njezina jednostavnost i korisnost (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). U 1980s, Kary Mullis (Slika 1) i jedan tim istraživača na kit Corporation u kit Corporation zače i način na start i stop jedan polymerase je akcija na specifične točke uzduž jednog obala DNA. Mullis i shvatio da harnessing ovu komponentu molekularnih reprodukcija tehnologija, target DNA mogao biti exponentially pojačavaju. DNA pojačanje Ovaj postupak je na temelju in vitro nego in vivo proces (5,6,8). Cell-free DNA pojačanje by PCR je bio u mogućnosti da se pojednostavio mnoge standardne postupke za kloniranje, analizom, izmjenom i nukleinske kiseline (1). Previous tehnike za isolating određeni dio DNA se oslonio na kloniranje gena - zamoran i spor postupak. PCR, s druge strane Mullis Kerry izjavio "omogućuje vam odabrati komad DNA ste zainteresirani i imate koliko to želite "(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. Kad kit drugih znanstvenika na kraju uspio u tome da je PCR nastupati kao željenu u pouzdani način, oni su jedna od ogromnog moćna tehnika za pružanje bitno neograničena količina precizan genetski materijal molekularnih biologa i ostalih potrebnih za njihov rad (8). Od Prvo izvješće in1985, više od 5000 znanstvenih radova objavljeni su po 1992 (1). Osim toga, veliki broj publikacija, naravno, čini nemoguće pregledati sve važne doprinose razvoju i primjeni PCR tehnologiji, no Pokušat ćemo pregled ovdje najvažnija zbivanja u praksi osnovnih 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 je mislio da je zamišljen Dr. Kerry Mullis u 1983, dok radimo na kit Corporation u Emeryville, CA. Ipak, neke pionir rad je također radi Gobind Khorana u 1971 koji je opisano osnovno načelo Umnožava dio DNA pomoću dvije početnice. napretku tada bio ograničen upaljač sinteza i polymerase pročišćavanje pitanja (9). U Mullis glavu, izum je rastao iz teorijskih shema za obavljanje ograničene dideoxynucleotide redoslijed jedinstvenih ljudskih gena koriste sintetička oligonucleotides u svrhu dijagnosticiranje zajedničkih ljudskih bolesti mutacije. očigledan prepreka da takva izravni slijed strategije je visoke složenosti ljudskog genoma (3,3 X 109 parova baza). tome, drugi oligonucleotide ili upaljač je dodao da blokira napredovanje u sinteza prvi udžbenik. Kasnije međutim , Ovaj drugi je bio uključen u prvom povezano s drugim DNA Strand, tako da se svaka plaža u mutant alel bi doprinijeti eventualne signal. Ako plan uključuje simultano hibridizacija, početnice za svaku obala je Modified by grijanje u smjesu, a zatim ponavlja žarenje i proširenje korake, onda je primarna signal bi se povećati čak i dalje. ponavljanja koraka će omogućiti proizvodi u prvom krugu da se umnožavati u drugom krugu, na yield dva primjerka. ponavlja ciklus opet bi rezultat u četiri primjerka, et dalje. nekoliko tjedana prije toga prošao velik ideja je pokušala (8). početnice Dva su umjetni biti savršeno komplementarnih na kraju svake od 110 base pair regiji jedan klon segment ljudskog b-globin gene, pojačanje je obavljena, i proizvoda su utvrđene acrylamide gel elektroforeze. Na kraju rezultat je bio predvidljiv 110 base pair fragment DNA i početkom PCR kao osnovnih tehnika molekularne biologije (5,6).
In Mullis's original PCR process(5,6,8), the enzyme was used in vitro (in a controlled environment outside an organism). U Mullis je izvorni postupak PCR (5,6,8), enzim je korišten in vitro (u kontroliranim okoliša izvan jednog organizma). The double-stranded DNA was separated into two single strands by heating it to 96°C. Dvostruka DNA je bila odvojena u dva smjera po jednu grijanje je do 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. U ovom temperatura, međutim, E. coli DNA polymerase je uništeno, tako da je enzim je da se replenished nakon grijanje stage svakog ciklusa. 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 je originalni PCR postupak je vrlo neučinkovit s obzirom da je potrebno mnogo vremena, ogromnu količinu DNA-Polymerase, i trajne pozornost tijekom PCR postupak.
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). Ispit je PCR amplifikacija mehanizam otkriti njegove jednostavnosti ali i svojim eleganciju (Slika 2). Oligonucleotide početnice su prvi osmišljen kako bi se komplementarni do kraja je slijed da se pojačavaju, a zatim mješoviti u molar višak s DNA predložak i deoxyribonucleotides u jednom odgovarajući tampon. Nakon grijanje na denaturirati izvorni strukturi i hlađenje "Cofloor" za promicanje upaljač žarenje, oligonucleotides svaki povezano s različitih obala jedna od target fragment. početnice su smještena tako da kada svaki je produžen do akcija DNA polymerase, novo umjetni strukturi će se preklapaju i obvezujuća site suprotno oligonucleotide. kao proces denaturation, žarenje, i polymerase ekstenzija je nastavila početnice više puta povezano s obje izvorne DNA predložak i komplementarnih stranice u novo umjetni strukturi su proširena i na izradu nove kopije DNA (Slika 3). krajnji rezultat je eksponencijalni porast u ukupnom broju fragmenata DNA sekvenci koje uključuju između PCR početnice, koji su konačno zastupljena na teorijske obilje 2n, gdje je n broj ciklusa (1 , 7,13).
A DNA polymerase is a naturally occurring enzyme, a biological macromolecule that catalyzes the formation and repair of DNA. A DNA polymerase je prirodno pojavljuje enzim, biološki macromolecule da catalyzes formiranje i popravak DNA. It works by binding to a single DNA strand and creating a complementary strand. It works by binding to jedan obala DNA i stvaranja komplementarnih obala. 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). U točan odgovor od svih živih tvari ovisi o ovoj aktivnosti, gdje je funkcija na dvostruke DNA kada stanice podijele (10,11). Tek nedavno su znanstvenici naučili manipulirati na ovu aktivnost i primjenjuju se na znanstvenim istraživanjima. Najraniji PCR eksperimenti utlilized i Klenow fragment Escherichia coli DNA polymerase I na temperaturi od 37C do proširiti specifične ciljeve iz ljudskih genocid DNA (5,6). Često ove PCR reakcije proizvedene nepotpuno čista ciljani proizvod kao suđeni po gel elektroforeze (1). ove početne PCR amplifications s Klenow fragment nisu bili vrlo specifične (5,6). Iako je jedinstvena DNA fragment mogao biti pojačan ~ 200000 fold iz DNA genocid, samo oko 1% od PCR proizvod je ciljanim slijed (13). Specifično hibridizacije sonda je bio dužan da analizirati pojačan DNA (5,6).
Some PCR conditions were determined to increase the stringency of primer hybridization such as lower MgCl2 concentrations and higher annealing temperatures. Neki PCR uvjeti određeni su povećanje stringency od prvog hibridizacija, kao što su niže koncentracije MgCl2 i žarenje višim temperaturama.
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. Nadalje, koncentracija enzim i početnice, žarenje vrijeme, produljenje vrijeme, i broj PCR ciklusi sve pronađene su na snazi na specifičnost od 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. Također, koncentracija određenu sekvencu u uzorku može također utjecati na relativne homogeneity od PCR products (1,7,13,14,15). Deoxyribonucleotide triphosphates i magnezija u odgovarajući tampon također su važni sastojci za PCR. Uspješnost i specifičnost od PCRs mogu biti pogođeni varijacije u koncentraciji i sistem slobodnog magnezija, deoxyribonucleotide triphosphates, i početnice. 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). Ove Reagensi moraju biti optimiziran kako bi se postigao visok prinos i specifičnost (14). Također je otkriveno da je utjecaj temperature i oligonucleotide fitilj dužine na specifičnost i efikasnost pojačanje koje je PCR (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). The deaktiviranje i Klenow fragment Escherichia coli DNA polymerase I na visoke temperature potrebne za nasukavanje razdvajanja potreban dodatak enzim denaturation korak nakon svakog ciklusa (5,6). Prije nego što se 1988, bilo tko vodi postupak PCR reakcija je bio dužan da posjeda po strpljivo niz vodu kupke ili grijanje blokova i dodati svježe aliquot of E. coli DNA polymerase nakon svakog denaturation korak, koji je obično provodi immersing reakcija u posudu kipuće vode za jedan i pol minute do 3 minute (7 ). Ova vrlo zamorno korak je eliminiran od uvođenja od thermostable DNA polymerase, Taq DNA polymerase (12) jednom, na početku je PCR reakcije. Thermostable properties of DNA polymerase aktivnosti su izolirane iz Thermus aquaticus (Taq) (Slika 4) koja raste u geysers od preko 110C, i uvelike su pridonijeli da je prinos, specifičnost, automatizacija i uslužni program za PCR (1,7,12). Enzim Taq može izdržati ponovio grijanje na 94C i tako svaki put kad je mješavina je hlađen na dopustiti oligonucleotide početnice na sveže za katalizator za proširenje je već prisutan (1,7). No, veći žarenje temperature nisu bile osnovane do jednog "najvažniji razvoj PCR development" (8), pročišćavanje i distribuciju komercijalne od vrućine-otpornih DNA polymerase iz termofilni bakterija 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). Izolacije od vrućine-otpornih DNA polymerase također dozvoljeno upaljač žarenje i proširenje biti provedena na uzvisinama temperature (1,7,12,13), pritom smanjujući neprilagođeno žarenje na nontarget sekvence (non-specifične pojačanje) ili povećanje specifičnost. taj način, za mnoge amplifications, PCR proizvod mogao biti otkriven kao jedan ethidium bromid-stained band na electrophoretic gel (12). povećao specifičnost i povećana prinosa od DNA target sequence. Štoviše, više PCR proizvoda mogao bi biti pojačan iz genocid DNA, vjerojatno zbog smanjenja u sekundarne strukture predložak smjera na uzvisinama temperatura koristi za upaljač ekstenziju. Gornja granica za veličinu Klenow fragment polymerase pojačanje je samo o 400bp. Taq polymerase i drugih thermostable polymerases su umjetni fragmenti do 10 kb (1,7,12,13). Dostupnost Taq polymerase je uvelike pojednostavljena automatizaciju reakcija kao što je mnogo lakše zadatak da konstruirati jedan stroj koji će ciklus reakcija cijev kroz različite temperature nego u proizvodnju uređaja da će nastupati u obje thermocycling i dodatak enzim aliquots. Trenutno postoji velika raznolikost thermocyclers komercijalno dostupna. razvoj To je značajan čimbenik u brzom primjene ove tehnologije od strane znanstvene zajednice (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). Pored proizvodnje dvostrukim nasukan, tupim-završila DNA fragmenata koji svibanj biti formirana by PCR, dvije druge značajke od PCR shemu uvelike pridonijeti da je uslužni program za PCR. First, poziciju obvezujuća za početnice definira granice fragment je pojačan i stoga je prije molecular klon zahtjev za priznavanje endonuclease ograničenje sučelja nije potrebna za PCR. Kao što samo ograničeni broj DNA sekvence su ograničenje sučelja, PCR uvelike povećava fleksibilnost i izbor fragment veličinu i sastav. Kao drugo, to je nije potrebno za PCR oligonucleotides da se upravo komplementarni na predlošku DNA. "Tails" svibanj biti dodano na 5 'kraju od prvog uvesti sekvence unutar priming web-mjesta koja ovako svibanj biti iskorišten uvesti ograničenje endonuclease prepoznavanje web stranice ili druge korisne sekvence kao što su mutacije u DNA pojačan. Ova pojava dozvoljeno nastanku PCR kao metoda za brzo DNA kloniranje (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) Molekularna klon je imala koristi od nastajanja PCR kao tehnika. Direct klon je prvi put provedena koristeći 110 bp DNA fragment pojačan by oligonucleotide PCR i početnice koje su sadržane ograničenje endonuclease priznavanje sučelja dodano njihovim 5 'kraja. Ove web stranice su korišteni olakšati klon od pojačan DNA u M13 plasmid (17). 110 bp fragment je poredan kako bi potvrdili da je ovaj pristup je brzo, ali pouzdan pristup klon. (Slika 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-based DNA kloniranje uključuje replikacija DNA in vivo, koji je povezan s vrlo visoka vrijednost od kopiranja, jer je lektura mehanizama. Međutim, kada je replicirati DNA in vitro kao s PCR, kopiranje error stopa je znatno veća. Najpoznatiji i naširoko koristi polymerase, Taq DNA polymerase međutim, nema povezane 3'to 5 'exonuclease dati lektura funkcija. Tako je stopa pogreške zbog base misincorporation tijekom replikacija DNA je vrlo visok za Taq: za 1 kb slijed koji je kroz 20 učinkovitih ciklusi za umnožavanje, oko 40% novih DNA strukturi umjetni by PCR koristeći ovaj enzim će sadržavati netočne nukleotidne nastanu iz kopiranje error (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. Stoga, čak i ako je PCR reakcija uključuje pojačanje od jedne DNA sekvence, konačni proizvod će biti spoj gotovo odgovara, ali ne i identične DNA sekvenci. Usprkos pogreške zbog replikaciju in vitro, sekvenciranje DNA od ukupnog PCR proizvod svibanj dati točan slijed s obzirom da je trgovačko društvo u pogrešne baze u osnovi je slučajan i doprinos od jedne pogrešne baze na jednu ili više strukturi se osvaja po doprinosa od ogromne većine strukturi koja će imati ispravan slijed. Međutim, ako je PCR proizvod će biti klon u stanice, nekoliko pojedinačnih klonovi svibanj treba biti poredan kako bi utvrdili ispravnu (konsenzus) slijed, prije provođenja daljnjih eksperimenata.
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). Više nedavno, problem nevjerovanje od replikacija DNA tijekom PCR reakcija je znatno smanjena pomoću alternativne heat-stable DNA polymerases koji su povezani 3 'za 5' exonuclease aktivnosti. Pyrococcus furiosus (Pfu) DNA polymerases i Thermococcus Litoralis (izlazni otvor) su postali široko koristiti, jer je lektura koje njihove pridružene 3 'za 5' exonuclease aktivnosti (18). Dobit PCR proizvod Pfu, na primjer, ima mnogo nižoj razini od mutacije uvedena kopiranjem greške: za 1 kb segment DNA koja je kroz 20 učinkovitih cikluse umnožavanje, oko 3,5% u DNA strukturi proizvod nositi u jednoj promijenjenoj 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. Novi pristupi za poboljšanje specifičnost je razvila na temelju prepoznavanja da Taq DNA polymerase zadržava znatnu enzimska aktivnost na temperaturama ispod optimalna za sintezu DNA. Ovako, početnice žarenje non-posebno na djelomično single nasukan predložak regiji može biti produžen pred reakcije dostiže 72 ° C za produženje posebno annealed početnice. Ako DNA polymerase se aktivira tek nakon reakcija je dostigao visoke (> 70 ° C) temperaturama, non-target pojačanje mogu biti minimiziran (19,20). "Hot start "Pristup može biti ostvarivan priručnik Uz jedan od bitnih reagens u selekciji tube temperature na uzvisinama. Dodatak ssDNA binding protein također je izvijestila da povećate određene pojačanje. A više user friendly pristup je koristiti bilo inhibicija ili deaktiviranje od DNA polymerase sama. Two types of inhibition of Taq DNA polymerase have been tried including oligonucleotide inhibition (21) and antibody (22) inhibition. Dvije vrste inhibicije, Taq DNA polymerase su pokušali uključujući oligonucleotide inhibicija (21) i antitijela (22) inhibicija. Highly specific oligonucleotide inhibitors of both Taq DNA polymerases have been produced. Visoko specifične oligonucleotide inhibitori i Taq DNA polymerases su proizvedene. These selectively inhibit DNA polymerase activity at temperatures below 40°C and have been shown to function in Hot Start applications. Ove odabire zabraniti DNA polymerase aktivnosti na temperaturama ispod 40 ° C te su prikazane funkcije u Hot Start aplikacija. Alternatively, one can use an antibody against Taq DNA polymerase. Alternativno, možete koristiti jedan jedan antitijela protiv 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. The antitijela inhibits DNA polymerase do temperature od PCR je takva da antitijelo je denaturirani na temperaturi većoj od 55 ° C, čime je u objavljivanju enzim. However there are disadvantages to this type of Hot Start conditions. Međutim postoje nedostaci na ovu vrstu Hot Start uvjetima. 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. U tom slučaju, treba jednom za svaku antitijela različitih enzim koji se koriste u jednom PCR i za veliki broj PCRs ovo mogu ustati troškovi značajno. 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). Najviše odgovara obliku Hot Start je da se izmjeni DNA polymerase na takav način da ga je neaktivna na sobnoj temperaturi (temperatura-sensitive mutant), te je samo re-aktivirati sljedeći inkubacije na 95 ° C za 6-15 minuta (23 ).
Because of its simplicity, PCR is a popular technique with a wide range of applications Zbog jednostavnosti, PCR je popularna tehnika sa širokim rasponom aplikacija
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: uključujući izravan sekvenciranje, kloniranje genocid, DNA tipizacija, otkrivanje zaraznih mikroorganizama, site-usmjeren mutagenesis, prenatalna genetička bolest istraživanja i analiza allelic slijed varijacije (1,7,13,16) koja bitno ovise o tri glavne prednosti metode :
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 Brzina i jednostavnost korištenja: DNA kloniranje by PCR može biti izvedena u relativno kratko vrijeme, u roku od nekoliko sati. Usually, PCR reakcija sastoji se od oko 30 ciklusa svaki ciklus sadrži denaturation, sinteza i reannealing korak, s jedne pojedinačne ciklus obično traje 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 u automatiziranoj termalne cycler. To je jasno brže nego vrijeme potrebno za cell-based DNA klon, koji bi mogao potrajati tjednima. Nadalje, sasvim je jednostavno postaviti PCR reakcija i korištenje jedne thermocycler stroj je također lako. nekog vremena je potrebno za dizajn i sinteza oligonucleotide početnice, ali ovo je pojednostavljena je dostupnost računala softver za upaljač dizajn i brzo komercijalnih ili akademskih sinteza prilagođeni oligonucleotides. Optimiranje PCR uvjeti svibanj biti potrebna, kao što su boja žarenje temperatura, koncentracija magnezija, udžbenik i koncentracija. Međutim, stvaranju gradijenta PCR strojevima koji dopušta različite upaljač žarenje temperature biti testirani istovremeno je uvelike smanjen je vrijeme potrebno za ovaj korak. Once the optimal conditions for a reaction have been obtained, the reaction can then be simply repeated (1,7,13,16). Nakon što je optimalno uvjete za reakcije su dobiveni, reakcije mogu biti jednostavno ponavlja (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. Osjetljivost: PCR je sposoban pojaccavanjem sekvence iz minute količine target DNA, čak i DNA iz jednog stanica (24). Takva je izuzetna osjetljivost afforded nove metode proučavanje molekularne patogeneze i našao brojne aplikacije u forenzičke znanosti, u dijagnozi, u genetski povezivanja analiza pomoću jedno-sperma i upisivanjem u molecular paleontology studija, gdje se uzorci svibanj sadržavati minute broj stanica. 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). No, ekstremna osjetljivost od metoda znači da ima veliku brigu kako bi se izbjeći onečišćenje uzorku pod istragom od strane vanjskih DNA, kao što je iz minute količine stanica od operatera (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. Robusnost: A broad range of nucleic acid izvori su pogodne za predloške PCR amplifikacija. DNAs očistili od raznih vrsta i izvora su se pojačavaju. PCR može dozvola pojačanje određenih sekvenci od materijala u kojem se DNA je jako uništen ili ugrađen u srednje iz kojih konvencionalnog izolaciju DNA je problematičan. As a result, it is again very suitable for molecular anthropology and paleontology studies, for example the analysis of DNA recovered from archaeological remains. Kao rezultat toga, to je opet vrlo pogodna za molekularne antropologije i paleontology studije, na primjer, analizu DNA oporavila od arheološki ostaci. 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. Ona je također bio korišten za uspješno povećati DNA iz formalin-fixed ili parafin-ugrađen uzoraka tkiva, što je važno u aplikacijama molekularna patologija i, u nekim slučajevima, genetski povezivanja studija. Generally, the success of PCR amplification is greatest when target fragments are relatively abundant (1,7,13,16). Općenito, uspjeh PCR je najveće pojačanje kad target fragmenti su relativno bogata (1,7,13,16).
Despite its huge popularity, PCR has certain limitations as a method for selectively cloning specific DNA sequences. Unatoč ogromnoj popularnosti, PCR je određena ograničenja kao metoda za selektivno klon specifične DNA sekvenci.
In order to construct specific oligonucleotide primers that permit selective amplification of a particular DNA sequence, some prior sequence information is usually necessary. Da bi konstruirali oligonucleotide specifične početnice koje dopuštaju selektivno pojačanje u određenom DNA sekvence, neke prije slijed informacija je obično potrebno. This normally means that the DNA region of interest has been partly characterized previously, often following prior cell-based DNA cloning. To obično znači da je DNA područje interesa je dijelom karakterizira ranije, često prije sljedećih cell-based DNA kloniranje. 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. Ipak, različitim pristupima Razvijene su da smanje ili čak isključiti potreba za prije DNA sekvence informacije o DNA target. Ranije uncharacterized DNA sekvence mogu ponekad biti klon pomoću PCR s izrod oligonucleotides ako su članovi jednog gena ili ponavljajući DNA obitelji barem jedan od članova čiji je prethodno bio karakterizira. 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. U nekim slučajevima, PCR može se koristiti na učinkovit način, bez ikakve prethodne slijed informacija o target DNA da dozvola indiscriminateamplification DNA sekvence iz jednog izvora DNA koja je prisutna u extemely ograničena količina. 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. Stoga, iako PCR se može primijeniti kako bi se osiguralo cijeli genom pojačanje, to ne imati prednost cell-based DNA kloniranje u nudeći način odvajanje pojedinih DNA klonovi genocid se sastoji od jedne DNA biblioteke.
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. Iznos PCR proizvod dobiven u jednoj reakcija je i mnogo više nego ograničen iznos koji se može dobiti koristeći cell-based klon gdje scale-up od količine kulture stanica je moguće. 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. Uspješnost jedne PCR reakcija će se razlikovati od predložaka na predlošku i prema različitim faktorima koji su potrebni kako bi optimizirali reakcije, ali obično samo usporedno male količine proizvoda su postignuti.
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). Iako je teorijski prinos od PCR je eksponencijalna, stvarna stopa dobiti od PCR je puno manje ukazuju da je operativni plan s manje od njegove maksimalne potencijal. Na primjer, u iznosu od proizvoda na kraju svakog ciklusa razinama off. Visoravni svibanj To se, objasnio je od sljedećih pojava. Prvo, neke od predloška svibanj nikada biti dostupni zbog nasukavanje pauze ili neuspjeh u DNA na dissociated iz drugih makromolekula za vrijeme čišćenja i početno thermocycles. Drugo, iznos enzim je konačna i na kraju aktivnosti svibanj smanjiti. Treće, kao što je koncentracija u dvostrukim nasukan proizvod doseže visoke razine, povećanje konkurencije između žarenje predloška (PCR proizvoda) u prvom i reannealing od komplementarnih predložak strukturi (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. Očigledan i mnogo puta veliki nedostatak PCR kao kloniranje DNA metoda je u rasponu veličina od DNA sekvenci koje mogu biti klon. 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 Za razliku od cell-based DNA kloniranje gdje veličini klon DNA sekvence mogu pristup 2 MB, izvijestila DNA sekvence klon by PCR su obično je u 0,1 
5 kb size range, often at the lower end of this scale. 5 kB veličine raspona, često na donjem kraju ove skale. 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. Malih fragmenata DNA se obično moľe biti pojačan jednostavno by PCR, međutim, postaje sve više teško da bi se dobila efikasna pojačanje kao željeni proizvod duljina povećava. 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) priznata je cilj ograničenja na dužinu PCR amplifikacija DNA. On je koristiti kombinaciju visoke razine jednog exonuclease-free, N-terminal brisanje mutant od Taq DNA polymerase, Klentaq1, s vrlo niskoj razini a thermostable DNA polymerase izlaganja jednog 3'-exonuclease aktivnosti (Pfu, Vent, ili Deep Vent) provoditi visoka kakvoća long 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). Najmanje 35 KB, bakteriofag lambda može biti pojačan, pa do high donosi od 1 od lambda DNA u predložak. Korištenja ove metode yielded povećao base-pair vjernost, sposobnost da koriste PCR proizvoda kao početnice, a maksimalni urod od ciljnih fragment. Other uvjeti su identificirani za učinkovito pojačanje od više ciljeva, uključujući i pojačanje do 22 kb, beta-globin gene cluster iz ljudske DNA genocid i do 42 kb iz 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. Uvjete za ove duge PCRs uključena povećao pH, dodatak glicerola i dimethyl sulfoxide, denaturation puta je smanjen, povećana proširenje puta, i korištenje sekundarni thermostable DNA polymerase da posjeduje 3'-u 5'-exonuclease, ili "korektura, "Aktivnost. 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. The "long PCR" protokol zadržala specifičnosti potrebne za genocid ciljeva u DNA pomoću nižim razinama polymerase i temperatura i soli uvjete za određene upaljač žarenje. Mogućnost povećati DNA sekvenci od 10-40 kb će donijeti brzinu i jednostavnost PCR za genocid i sekvenciranje i mapiranje olakšati studij molekularne genetike (26). Općenito govoreći, uvjeti za dalekometno PCR uključivati kombinaciju izmjene standardnih uvjeta s dva-polymerase sustav. This provides optimal levels of DNA polymerase and 3’to 5’ exonuclease activity which serves as a proofreading mechanism (16). Ovo osigurava optimalno razinama DNA polymerase i 3'to 5 'exonuclease aktivnosti koje služi kao mehanizam lektura (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 koji automatski regulirati temperature za PCR biciklizam su uvedeni 1986 (Slika 6). Osim toga na napredak u PCR Reagensi, novi instrumenti za automatizirano termalne biciklizam i za analizu PCR proizvodi su razvijeni. Nova termo-cyclers imaju povećane stope grijanje, hlađenje, i prijenos topline na izmijenjeni reakcija plovila. reakcija plovila primao po prvi naraštaj termalne cyclers (ili čak i kupelji vode i grijanje blokova) su standard microfuge plastične cijevi. PCR pojačanje u tanke kapilarne cijevi dozvoljeno brzo termalne biciklizam, DNA i sintezu na 20s. Brzina promjene temperature postignute u tim sustavima je dopušteno je precizna definicija Optima temperatura za svaki pojedini korak u PCR ciklusa. Nova generacija termalne cyclers također smjestiti više uzoraka, preciznije termalne profili, te su programabilan (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). Jedan od najmanje poštovati doprinosa na široko rasprostranjena primjena PCR je razvoj pouzdanog automatizirani kemije za oligonucleotide synthesis. Do nedavno, izgradnju jednog oligonucleotide bio je bitan zadatak koji bi mogao biti samo u izvedbi jednog vještog organski kemičar. Sada je je moguće kupiti ili jedan oligonucleotide sintetizator koji može biti operiran je tehničar ili oligonucleotides se od komercijalnih ili akademskih izvor. Multiplex oligonucleotide synthesis strojevi su konstruirani s ciljem smanjenja ukupni trošak sinteza (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). Kao oligonucleotides definirati eventualne PCR products, ima malo sumnje da je u odsutnosti svoje spremni supply, PCR ne bi uživali u širokom prihvaćanju da je dobio danas (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. Istraživači dogovoreno da se početkom na dizajn PCR početnice je teško i nepouzdana. Računalne programe Izmislili su poduzeti sve od dizajna kriterije u obzir. 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). Jedan od prvih programa napisano za upaljač dizajn je Olga koja je napravio korištenje implementacije Digitalnog istraživanja GEM (Graphics Environment Manager) na Atari ST (29). Olga was specifically suited to the polymerase chain reaction (PCR) allowing simultaneous analysis of two primer sequences. Olga je posebno prilagođen za PCR (PCR) koji omogućuje simultano analiza sekvenci dva fitilj. 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). Prednost Olga je bila pod uvjetom da se u jedan program analize za direktni ponavlja, sekundarne strukture i upaljač dimerizacija, kao i nekoliko korisnih 'završavanja' alate za radnike koji rade na poslovima optimizacije i oligonucleotide PCR Sinteze. Primer3 program na Whitehead Institut je sada misli da se najpouzdanijih i svestrani alat trenutno je dostupan (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 sada mogu biti izvedena dopusta pojačanje DNA fragmenata do nekoliko kilobases u dužini od više od milijun puta njihove početne izobilju. Procedura je vrlo automatable i zahtijeva samo nekoliko sati od početka thermocyling za analizu proizvoda. To je bio ne slučaj ranije, i praktične zahtjeve za izvođenjem PCR je uvelike pojednostavljena jer je prvi rukopisi od metoda (13). Danas, većina od početnih hitches ili inefficiencies od PCR je razrađen (8). Nadalje, PCR je proširuju kako bi uključile više od 270000 članci (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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