Taq, DNA polymerase

FIGURE 13.21 Polymerase chain reaction (PCR). Oligonucleotides complementary to a given DNA sequence prime the synthesis of only that sequence. Heat-stable Taq DNA polymerase survives many cycles of heating. Theoretically, the amount of the specific primed sequence is doubled in each cycle. 

Taq DNA polymerase from Thermus aquaticus) has made it unnecessary to add fresh enzyme for each round of synthesis. Because the amount of target DNA theoretically doubles each round, 25 rounds would increase its concentration about 33 million times. In practice, the increase is actually more like a million times, which is more than ample for gene isolation. Thus, starting with a tiny... 

Some investigators described artifactual DNA sequence alterations after formalin fixation, when testing DNA samples extracted from FFPE tissues. Williams et al.46 reported that up to one mutation artifact per 500 bases was found in FFPE tissue. They also found that the chance of artificial mutations in FFPE tissue sample was inversely correlated with the number of cells used for DNA extraction that is, the fewer cells, the more the artifacts. However, they mentioned that these artifacts can be distinguished from true mutations by confirmational sequencing of independent amplification products, in essence comparing the product of different batches. Quach et al.47 documented that damaged bases can be found in DNA extracted from FFPE tissues, but are still readable after in vitro translesion synthesis by Taq DNA polymerase. They pointed out that appropriate caution should be exercised when analyzing small numbers of templates or cloned PCR products derived from FFPE tissue samples. 

It is important to note that even at room temperature, Taq DNA polymerase can incorporate 0.25 nucleotides per second. Hence, the aliquot of master mix... 

Yang X, Chen Z, Meng X, Li B, Tan X (2007d) Inhibition of DNA restrictive endonucleases and Taq DNA polymerase by trimalonic acid Cm. Chin. Sci. Bull. 52 1802-1806. 

Make a PCR master mix as described containing excess amonnt of dNTP and Taq polymerase enzyme. For instance, we fonnd that if mnltiplexing for amplification of promoters of five genes has to be performed at the same time, 0.2 pL 10 pM dNTP and 1.25 U Taq DNA polymerase (Invitrogen) for each gene in the reaction are reqnired. For each gene, consider lOpL of this PCR master mix. 

Polymerase chain reaction (PCR) Master Mix containing Taq DNA polymerase, deoxynncleotide 5 -triphosphate (dNTP), MgCl, and reaction buffers (Promega). 

Urs, U. K., MuraU, R. and Krishna Murthy, H. M. (1999). Structure of taq DNA polymerase shows a new orientation for the structure-specific nuclease domain. Acta Crystallogr. D 55,1971—1977. 

Kits and enzymes Superscript reverse transcriptase (Invitrogen), Maxiscript and Megascript in vitro transcription kits (Ambion, Austin, TX). Taq-DNA polymerase, T4-polynucleotide kinase, EcoRI and Hindlll restriction enzymes (Invitrogen). 

During the amplification cycles of the PCR, the Taq DNA polymerase copies the DNA between the primers. Because numerous areas throughout the bacterial chromosome are amplified, many potential genetic differences can be detected. Figure D.4 shows a chip analysis of two samples of bacterial DNA amplified by rep-PCR. The gel electrophoresis results clearly show that the bacteria samples are different strains, as there are band differences appearing at approximately... 

Molecular biology Taq DNA polymerase for PCR/Sigma 2000 proteins/used in PCR marine Thermococcus littoralis, Archaea... 

Setup a PCR reaction starting with 0.05-0.2 pmol of template DNA, 50 pmol of each primer, 10 pL 10X PCR buffer, 10 pL 10X cINTP mix, 0.5 mM MnCl2, 5 U Taq DNA polymerase, and water to a final volume of 100 pL. The manganese solution should be added just prior to the polymerase (see section 2.4, note 1). 

Corresponding lOx polymerase buffer with MgCl2 (for Taq DNA polymerase) or MgSC>4 (for Vent DNA polymerase)... 

There are various PCR protocols with associated degrees of fidelity. A low-fidelity, high-yield protocol using Taq DNA polymerase (NEB) for amplification of a template such as that in Figure 8.2 is as follows ... 

Pfu DNA polymerase can be substituted for Taq DNA polymerase, but the manufacturer (Stratagene) recommends using the supplied buffer. 

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