specific PCR polymerase - (Oct/17/2007 )

I learned a kind of specific PCR polymerase in a post in this forum. It can be work for some very difficult amplification PCR experiment. But I can not find it right now.
Does anyone know what it is. You can list any kind of polymerases. I can figure out which one is. Thank you very much.

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pfu-ultra? or phusion??
both work great!

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Hi,

Firstly, I have recently found this page http://www.pcrstation.com/pcr-polymerases/ that quickly explains different types of polymerases.

If you need High fidelity PCR, I advice you to choose PFU DNA Polymerase, an enzyme found in the hyperthermophilic archaeon Pyrococcus furiosus. I use Pyrobest from Taqara, when I need to decrease replication errors.
The main difference between Pfu and alternative enzymes is Pfu's superior thermostability and 'proofreading' properties compared to other thermostable polymerases. 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. This means that Pfu DNA polymerase-generated PCR fragments will have fewer errors than Taq-generated PCR inserts. As a result, Pfu is more commonly used for molecular cloning of PCR fragments than the historically popular 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. 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. Pfu DNA polymerase is hence superior for techniques that require high-fidelity DNA synthesis


Secondly, for the different kinds of PCR, you have, perhaps, learned about Touchdown PCR, nested PCR or Hot Start PCR.

- Touchdown PCR is a method of PCR by which primers will avoid amplifying nonspecific sequence. The temperature at which primers anneal during a cycle of polymerase chain reaction determines the specificity of annealing. The melting point of the primer sets the upper limit on annealing temperature. At temperatures just below this point, only very specific base pairing between the primer and the template will occur. At lower temperatures, the primers bind less specifically. Nonspecific primer binding obscures polymerase chain reaction results, as the nonspecific sequences to which primers anneal in early steps of amplification will "swamp out" any specific sequences because of the exponential nature of polymerase amplification.
The earliest steps of a touchdown polymerase chain reaction cycle have high annealing temperatures. The annealing temperature is decreased in increments for every subsequent set of cycles (The number of individual cycles and increments of temperature decrease is chosen by the experimenter). The primer will anneal at the highest temperature which is least-permissive of nonspecific binding that it is able to tolerate. Thus, the first sequence amplified is the one between the regions of greatest primer specificity; it is most likely that this is the sequence of interest. These fragments will be further amplified during subsequent rounds at lower temperatures, and will out compete the nonspecific sequences to which the primers may bind at those lower temperatures. If the primer initially binds to the sequence of interest at a low temperature, subsequent rounds of polymerase chain reaction can be performed upon the product to further amplify those fragments.

- Nested PCR intends to reduce the contaminations in products due to the amplification of unexpected primer binding sites. This method involves two successive runs of PCR with two sets of primers: the target DNA undergoes the first run of PCR with the first set of primers. The product from the first reaction undergoes a second run with the second set of primers. It is very unlikely that any of the unwanted PCR products contain binding sites for both the new primers, ensuring the product from the second PCR has little contamination from unwanted products of primer mis-binding and alternative primer target sequences.

- Hot Start PCR reduces non-specific amplification and increases PCR product target yield. In this procedure amplification cannot occur until the reaction temperature is above that where non-specific annealing of primers to targets occurs. This block in amplification is usually accomplished by using a DNA polymerase that is inactive until higher temperatures are reached. It is commonly performed by using included chemical modifications, wax-barrier methods, and inhibition by a taq-directed antibody.

I wish I could help you.

SB

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Yes. It is phusion. Is there any special requirement for setting up the PCR using phusion? I am trying an amplification of GC rich region.

Thank you guys for your input. 81tox, your input expand my knowledge of PCR. Thanks.

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