Thermostable polymerases

PGR amplification of a DNA sequence is faciHtated by the use of a heat-stable DNA polymerase, Taq polymerase (TM), derived from the thermostable bacterium Thermus aquaticus. The thermostable polymerase allows the repeated steps of strand separation, primer annealing, and DNA synthesis to be carried out ia a single reactioa mixture where the temperature is cycled automatically. Each cycle coasists of a high temperature step to deaature the template strands, a lower temperature annealing of the primer and template, and a higher temperature synthesis step. AH components of the reaction are present ia the same tube. 

Fig. 5. Illustration of the 5 -nucleotidase (TaqMan) assay for allele discrimination. (A) The allele discrimination assay employs two unlabeled PCR primers and two doubly fluorescent labeled PCR probes for visuaUzation of a mutant allele. The target sequence is initially denatured and amplified in the presence of each of the primers and probes. Increasing polymerization in the presence of a thermostable polymerase which contains a 5 proofreading function allows cleavage of one fluorescent indicator from an appropriate probe during the cycling reaction. (B) Probes are designed with a fluorescent reporter and a quencher moiety. AmpUfication reactions are spiked with additional fluorescent quenchers in order to render the reaction initially dark to the photomultipUer mbe or diode. The probes are designed...

For PCR, it is important to use a licensed thermostable enzyme with a proofreading facility and to reduce the cycle number to the minimum number that provides acceptable yield after the post-PCR manipulations. The precautions are necessary, because the error rate for some thermostable polymerases is quite high and the number of errors that accumulate increases with cycle number. 

DNA polymerase is required for synthesis. There are several different polymerases available, each with its 6Wn characteristics that will affect its efficacy in the PCR. Since many labs have studied the properties of the different polymerase in current use, much is known about their efficiencies in PCR and rate of base misincorporation (Table 7.1). The thermostable polymerases (Tag, Vent) are preferred in PCR over the heat-labile enzymes... 

Polymerases catalyze the synthesis of complementary nucleic acid polymers using a parent strand as a template. In vitro, these enzymes can extend an oligonucleotide primer that is annealed to a template strand. Extension requires that the 3 OH of the extending end is free, and that nucleotide triphosphates (NTPs) are present. Extension stops if you rrm out of template or NTPs, or if no 3 OH groups are. available at the extending end. Thermostable polymerases, such as Thermus aquaticus (Taq) DNA polymerase, are essential reagents for the automation of many nucleic acid amplification procedures. 

By modifying reaction conditions, PCR can be adjusted for the WalkThrough synthesis using thermostable polymerase for the short, nascent DNA fragments. To adapt PCR to the WalkThrough synthesis provides a more convenient way for more... 

DNA polymerases are different enzymes able to synthesize a new DNA strand on a template strand. DNA polymerases are important enzymes involved in the DNA repair and replication. The following thermostable polymerases are the most common used enzymes for PGR. 

This is last phase in a PCR cycle. During this phase the thermostable polymerase catalyze the synthesis of the targeted DNA using the dNTPs. The optimal extension temperature ranges between 65 °C and 72 °C. Extension time depends on the size of the amplified sequence. Eor each kilobase of amplified sequence, 45-60 s are needed. 

The inclusion of one ss DNA block copolymer as a primer, in combination with a second conventional ODN primer, the plasmid pBR322 (as template), the thermostable polymerase from the bacterium Thermus aquaticus, and the four dNTPs, led to the production of several ds DNA diblock copolymers with extended nucleic acid segments ds DNA-h-PEG ds DNA-h-PS ds DNA-h-PPO and ds DNA-b-PNlPAM (entries l-29in Table 35.1). The lengths of the DNA block were adjusted by the annealing sites of the primer pairs on the template, and varied between 87 and 1578 bp. For the PCR, it was necessary to develop an optimized cycling protocol whereby, in order to achieve an effective amphfication, the aimeahng time was extended to 4 min, compared to periods of only 30 s for denaturation and extension. 

S rRNA metazoan phylogeny PCR with thermostable polymerase PCR with universal primers Whole mount in situ hybridization Engrailed antibody 4D9 Amphioxus Hox gene expression Amphioxus Hox gene cluster map 18S rDNA metazoan phylogeny Amphioxus ParaHox gene cluster... 

An important technical difference between NGS techniques is whether the input DNA or RNA must be amplified in quantity before sequencing. This matters because the methods of amplifying DNA/ RNA can introduce errors in the sequence and also introduce biases in the sequences that are amplified. Typical thermostable polymerases, used for amplifying DNA/RNA in polymerase chain reactions (PCR), introduce mutations at a rate of 1 in 9000 nucleotides. This rate sounds rare, but the multiple amplification cycles necessarily (up to 40) can yield a number of sequence errors. Furthermore, particular types of sequences called microsatellites, such as long stretches of adenine nucleotides, may cause an even higher error rate. Likewise, polymerases often amplify GC-rich sequences at lower efficiency... 

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