• PCR PCR
• Learn about PCR Preberite več o PCR
• Types of PCR Vrste PCR
• PCR Links PCR Povezave
• PCR News PCR Novice
• PCR Troubleshooting PCR Odpravljanje težav
• PCR Problems PCR Težave
• PCR Books PCR Knjige
• PCR Forum PCR Forum
• Biology Resouces Biologija Resouces
• Tell A Friend Povej prijatelju
• Bookmark Us! Bookmark Us!
• Advertise on PCR Station! Oglašujte na PCR postaja!

PCR ENZYMES: PCR Polymerases PCR encimi: polimeraze PCR

PCR Polymerases - The Ultimate Guide Polimeraze PCR - The Ultimate Guide

copyright 2006 PCR STATION copyright 2006 PCR POSTAJA

PCR Polymerases: An Introduction Polimeraze PCR: uvod

The choice of the DNA polymerase employed by PCR is determined by the goals of the experiment. Izbira je DNA-polimeraza, ki jih zaposlujejo PCR je določena s ciljem poskusa. There are now a plethora of commercially available enzymes to choose from that differ in their thermal stability, processivity, and fidelity. Obstajajo številni sedaj na voljo komercialno encimov, da izberejo, da se razlikujejo glede na njihove termične stabilnosti, processivity, in zvestobe. The most commonly used and most extensively studied enzyme is Taq DNA polymerase. Najpogosteje uporabljeno in najbolj obširno proučevali encim Taq DNA-polimeraza.

Initial PCR Polymerases Začetno polimeraze PCR

The DNA polymerases used originally in the first PCR reactions were extracted from the bacterium Escherichia coli . DNA polimeraze uporabljene prvotno v prvi PCR reakcije so bile povzete iz bakterije Escherichia coli. Although this enzyme was an invaluable tool for many molecular biology research uses in the past and even today, it had many disadvantages in the original PCR. Čeprav se ta encim, je neprecenljivo orodje za mnoge molekularne biologije raziskovalne namene v preteklosti in še danes, je bilo veliko pomanjkljivosti v originalni PCR. In PCR, the pcr reaction must be denatured by heating the double-stranded DNA product after each cycle. Unfortunately, heating also irreversibly inactivated the E. coli DNA polymerase. V PCR, PCR reakcijo, mora biti denaturirana, s segrevanjem v dvojno DNA po vsakem ciklu proizvoda. Na žalost, ogrevanje tudi ireverzibilno inaktivirana v E. coli DNA-polimeraza. Fresh aliquots of enzyme had to be added manually at the start of each cycle. Sveže alikvote encima, je treba dodati ročno na začetku vsakega cikla. With 30-40 cycles of PCR, this was a very laborious and boring task. S 30-40 ciklov PCR, je to zelo naporen in dolgočasen nalogo.

What was required was a DNA polymerase that remained stable during the DNA denaturation step performed at 90°C or hotter. Kaj je bilo zahtevano je DNA-polimeraza, ki so ostale stabilne v času denaturacije DNA korak izvede pri temperaturi 90 ° C ali bolj vroč. A discovery made the solution much easier. A odkritje je rešitev precej lažja. The bacterium Thermophilus aquaticus was isolated from water hot springs. Bakterije thermophilus aquaticus je bil izoliran iz vode, izviri tople vode. This bacterium was able survive and proliferate in the extremely high water temperatures of the hot springs. Ta bakterija je sposobna preživeti in se razmnožujejo v izjemno visoke temperature vode v toplice. Isolation of the DNA polymerase from this bacterium yielded a PCR polymerase that was not rapidly inactivated at high temperatures. Osamitev DNA polimeraze iz bakterije to ustvarilo polimeraze PCR, da ni bilo hitro inaktivira pri visokih temperaturah. Gelfand et al. Gelfand et al. at Cetus corporation successfully purified and cloned this PCR polymerase now called Taq ( T hermophilus aq uaticus in short) Polymerase. na Kit družba uspešno očistili in klonirane te PCR zdaj imenuje Taq polimeraza (T hermophilus aq uaticus v kratkem) polimerazo.

This allowed a 30-40 cycles of PCR amplification to be performed without the need to open the PCR reaction tube and add fresh polymerase. Ta dovoliti, da v 30-40 ciklih PCR ojačanja, ki jih je treba opraviti brez potrebe, da se odpre reakcije PCR epruveto, in dodamo sveže polimeraza. This also reduced potential contamination that could be introduced when adding polymerase manually over 20-30 times in the original PCR reactions. To tudi zmanjša potencial kontaminacije, ki bi se lahko uvedli, ko se sešteva s polimerazo ročno 20-30 krat več kot v prvotni reakcije PCR. Also, due to the nature of the thermophilic bacterium and the polymerase, Taq functioned optimally at temperatures around 72°C, allowing DNA synthesis to be performed at much higher Tudi zaradi svoje narave nastavljena tako termofilnih bakterije in polimerazo, Taq deluje optimalno pri temperaturi približno 72 ° C, kar omogoča sinteze DNA, ki se izvajajo na precej višji
temperatures than was possible with the E. coli enzyme. temperature, kot je mogoče z E. coli encima. This had the advantage of allowing the template DNA strand to be copied at a much higher fidelity due to higher strigency of PCR primer annealing. To je prednost, ki omogoča matrice DNK usmeritev, ki jih želite kopirati na veliko višji zvestobe zaradi višjih strigency PCR primer prileganje. This further reduced non-specific products which had affected many earlier PCR reactions. To nadalje zmanjša število nespecifičnih proizvodov, ki so prizadele veliko prej reakcije PCR.

TAQ DNA Polymerase Taq DNA-polimeraza

Taq Polymerase also simply termed "Taq", is a thermostable DNA polymerase used in polymerase chain reaction (PCR) to catalyze the DNA replication reaction in the PCR cycle. Taq polimerazo tudi preprosto označijo kot "Taq", je Termostabilan DNA-polimeraza, uporabljenih v verižna reakcija s polimerazo (PCR) za spodbujanje DNA replikacije reakciji PCR v ciklu. In this manner of amplification, PCR is able to examine for the presence or absence of a gene of interest in a biological sample. Na ta način ojačanja, PCR se lahko preuči za ugotavljanje prisotnosti ali odsotnosti gena za obresti v biološkega vzorca.

Molecular model of Taq DNA Polymerase. Molekulska model DNA Taq polimerazo. Crystal structure of Taq DNA-Polymerase Shows A New Orientation For The Structure-Specific Nuclease Domain. Kristalno strukturo Taq DNA-polimeraza kaže novo usmeritev za strukturo in posebne nukleaze domene.

Taq DNA polymerase replaced E.Coli DNA Polymerase in PCR, due to its thermostable properties. Taq DNA-polimeraza nadomesti E. Coli DNK polimerazo v PCR, zaradi svoje Termostabilan lastnosti. Taq was first isolated from Thermus aquaticus , a bacterium that lives in hot springs and hydrothermal vents. Taq je bil prvič izoliran iz Thermus aquaticus, bakterija, ki živi v vroči izviri in hydrothermal zračnike.

Taq was the first polymerase that was able to withstand the denaturing conditions (over 90 °C) required during PCR cycling. Taq polimeraza je bil prvi, ki je sposobna prenesti denaturacijo pogoji (nad 90 ° C), ki so potrebne v obdobju PCR kolesarjenje.

Taq has an e nzymatic halflife at 95°C of about 40 min. Taq je e nzymatic Halflife pri 95 ° C približno 40 min. For more properties of Taq, see the table below. Za več lastnosti Taq, glej spodnjo tabelo.

One of Taq polymerases' major disadvantages is its low replication fidelity. Eden od Taq polimeraze "velike pomanjkljivosti je njegova majhna replikacije zvestobe. As Taq does not have 3' to 5' exonuclease proofreading mechanism to replace an accidental mismatch in the newly synthesized DNA strand, Taq produces more errors than proofreading polymerases such as Pfu. Kot Taq nima 3 'na, 5' exonuclease lektoriranje mehanizem za nadomestitev naključnega neusklajenosti v novo sintetizirane verige DNA, Taq proizvede več kot napake, kot so lektoriranje polimeraze PFU.

Taq DNA polymerase also is unique in that it produces PCR products with A (Adenine) overhangs. Taq DNA-polimeraza je edinstvena tudi po tem, da izdeluje produktov PCR z A (Adenin) overhangs. This was found to be quite useful, and was exploited to produce TA Cloning and TOPO cloning (Invitrogen). To je bilo ugotovljeno, da je zelo koristno, in je bil izkoriščen za proizvodnjo VK Kloniranje in TOPO kloniranje (Invitrogen). These methods employ a cloning vector (or plasmid) which possesses T (Thymine) overhangs. Te metode zaposlujejo kloniranje vektor (plazmid ali), ki ima T (timin) overhangs. This allows ligation using topoisomerase or DNA ligase to quickly be accomplished with the A overhangs of the PCR product To omogoča topoizomeraze uporabljate ligation ali DNK ligase na hitro se doseže z A overhangs produkta PCR

PFU DNA Polymerase PFU DNA-polimeraza

Pfu DNA polymerase is an enzyme found in the hyperthermophilic archaeon Pyrococcus furiosus, where it functions in vivo to replicate the organism's DNA. PFU DNA-polimeraza je encim, ki ga najdemo v hyperthermophilic archaeon Pyrococcus furiosus, kjer se funkcije in vivo, da posnema organizma v DNK. In vitro, Pfu is used to quickly amplify DNA in the Polymerase Chain Reaction, where the enzyme serves the central function of copying a new strand of DNA during each extension step. In vitro PFU se uporablja za hitro širiti DNK v verižno polimerizacijo, kjer encim služi centralnih funkcij: kopiranje nov del DNA med vsakim podaljšanjem korak.

Superiority of Pfu polymerase The main difference between Pfu and alternative enzymes is Pfu's superior thermostability and 'proofreading' properties compared to other thermostable polymerases. Večvrednosti PFU polimeraza Glavna razlika med PFU in alternativnih encimov je PFU's superior thermostability in "lektoriranje" lastnosti v primerjavi z drugimi Termostabilan polimeraze. Unlike Taq DNA polymerase, Pfu DNA polymerase possesses 3' to 5' exonuclease proofreading activity, meaning that it works its way along the DNA from the 3' end to the 5' end and corrects nucleotide-misincorporation errors. Za razliko od Taq DNA-polimeraza, PFU DNA-polimeraza ima 3 'na, 5' exonuclease lektoriranje dejavnosti, kar pomeni, da je del poti vzdolž DNK od 3 'konca na 5' koncu in popravlja nukleotid-misincorporation napake. This means that Pfu DNA polymerase-generated PCR fragments will have fewer errors than Taq-generated PCR inserts. To pomeni, da PFU DNA-polimeraza generirani fragmente PCR bo imela manj napak, ki nastanejo kot Taq-PCR vstavlja. As a result, Pfu is more commonly used for molecular cloning of PCR fragments than the historically popular Taq. Kot rezultat, PFU je bolj pogosto uporabljajo za molekularno kloniranje PCR fragmenti od nekdaj priljubljena Taq. Commercially available Pfu typically results in an error rate of 1 in 1.3 million base pairs and can yield 2.6% mutated products when amplifying 1kb fragments using PCR. Komercialno dostopne PFU običajno za posledico napačno stopnjo 1 v 1,3 milijona baznih parov in se lahko donos 2,6% mutirali proizvodov, kadar okrepitvijo 1KB fragmente PCR z uporabo. However, Pfu is slower and typically requires 1–2 minutes to amplify 1kb of DNA at 72° C. Using Pfu DNA polymerase in PCR reactions also results in blunt-ended PCR products. Vendar pa PFU je počasnejši in ponavadi zahteva 1-2 minut, da se ojači 1KB DNK pri 72 ° C. Uporaba DNA-polimeraza PFU v PCR reakcije se odraža tudi v odkrito-končalo produktov PCR. Pfu DNA polymerase is hence superior for techniques that require high-fidelity DNA synthesis, but can also be used in conjunction with Taq polymerase to obtain the fidelity of Pfu with the speed of Taq polymerase activity. PFU DNA-polimeraza je tako za vrhunske tehnologije, ki zahtevajo visoko zvestobo sinteze DNA, vendar se lahko uporabi tudi v povezavi s Taq polimeraza za pridobitev natančnosti PFU s hitrostjo Taq polimeraza dejavnost.

History Zgodovina

Scientists associated with the biotech company Stratagene, based in La Jolla, California discovered the superiority of Pfu over Taq in 1991. Znanstveniki so povezana z Stratagene biotehnološko podjetje, s sedežem v La Jolla, Kalifornija, odkrite večvrednosti PFU Taq več kot v letu 1991. They published their work in the journal Gene in December of that year (Gene. 1991 Dec 12;108(1):1-6). So njihova dela objavljena v reviji Gene v decembru tistega leta (Gene. 1991 dec 12, 108 (1) :1-6). US Patent 5,489,523 was granted over exonuclease-deficient Pfu in February 1996 while US Patent 5,545,552 over Pfu itself was granted in August 1996. US Patent 5489523 je bila dodeljena več kot exonuclease-pomanjkljiva PFU v februarju 1996, medtem ko US Patent 5545552 več PFU sam je bil odobren avgusta 1996.

PCR Polymerases Polimeraze PCR

Summary of Available PCR Polymerases and their properties. Povzetek Na voljo PCR-polimeraze in njihovih lastnosti.

PCR and Polymerases PCR in polimeraze

PCR has been performed on DNA larger than 10 kilobases, however the average PCR is only several hundred to a few thousand bases of DNA. PCR je bila izvedena na DNK, večji od 10 kilobases, vendar pa je povprečna PCR je le nekaj sto do nekaj tisoč baz DNA. The problem with long PCR is that there is a balance between accuracy and processivity of the enzyme. Problem z dolgimi PCR je, da obstaja ravnovesje med natančnostjo in processivity na encim. Usually, the longer the fragment, the greater the probability of errors. Ponavadi je več fragmentov, večja je verjetnost napak.

PCR Polymerases References Polimeraze PCR Literatura

1.

2.

3.

copyright 2006 PCR STATION copyright 2006 PCR POSTAJA