Taq enzyme

These observations confirmed that strong interactions happened among SWCNTs, DNA templates, and Taq enzyme in the course of the PCR. SWCNTs could improve the PCR efficiency, which indirectly demonstrate that CNTs could improve the bio-activity of Taq enzyme, and may own bio-catalytic function in a variety of biochemical reactions. 

CNTs can be functionalized with protein via non-covalent bond (Li et al., 2005 Kim et al., 2003 Mitchell et al., 2002). For example, (beta-lactamase I, that can be immobilized inside or outside CNTs, doesn t change enzyme s activity (Vinuesa and Goodnow, 2002). Taq enzyme can attach to the outside of CNT, and doesn t change its activity (Cui et al., 2004). Peptide with Histidine and Tryptophan can have selective affinity for CNT(Guo et al., 1998). Monoclonal antibody can attach to SWNTs. Protein-modified CNTs can be used to improve its biocompatibility and biomolecular recognition capabilities (Um et al., 2006). For example, CNTs functionalized with PEG and Triton X-100 can prevent nonspecific binding of protein and CNTs. Biotin moiety is attached to the PEG chains Streptavidin can bind specifically with biotin-CNT (Shim et al., 2002). 

For example, Asuri and his co-workers (Asuri et al., 2007) reported that the proteases attached to the surface of SWCNTs can create self-cleaning surfaces that resist protein adsorption. This kind of SWCNT-enzyme composite exhibited 30 times higher overall catalytic activity than control composites where the proteases were conjugated to a non-nanoscale graphite support. Importantly, the enzymes preserved more than 90% of their initial activity over 30% days in the liquid buffer, with only negligible amounts of enzymes leaching out. The result demonstrates that the nanocomposites of SWCNTs and polymers can act as hosts for enzymes and can prevent protein contamination on the surface of medical devices. We consider that SWCNTs may have the function as biocatalyst to improve the enzyme activity attached to the surface of CNTs. Our previous work also indirectly hints that CNTs could improve the bioactivity of enzymes such as Taq enzyme Cui et al. (2004). However, there are also adverse reports. CNTs can... 

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... 

Enzyme Taq polymerase (or some other enzyme) adds new deoxyribonucleotides during strand elongation. Taq is added to the assay at 1 unit per 50 qL of reaction mixture. 

Molecular methods used to uncover mutations are subject to several variables. The anticoagulants used for blood collection can affect digestion with restriction enzymes and amplification reactions. The type of detergent used in cell lysis can affect amplification of DNA by inhibiting the DNA-amplifying enzyme such as the taq polymerase used in the polymerase chain reaction (116). The control of contamination is crucial in ensuring the quality of results obtained by molecular analysis (117). 

Several of the enzymes involved in the processes of repheating, transcription and reverse transcription are available commercially and are used by molecular biologists in the manipulation of nucleic acids. One of the most important of these is Taq polymerase (Taq), which is a thermostable DNA polymerase named after the thermophihe bacterium Thermus aquaticus from which it was originally isolated. This enzyme is especially important, as it is central to the technique known as PCR, which allows sophisticated, targeted in vitro amplification and manipulation of sections of DNA or RNA. DNA... 

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. 

Preparation of Taq polymerase-Taq-start antibody complex 1 mix 1 1 Taq-start antibody Taq polymerase enzyme and incubate at 22°C for 10 min. 

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). 

A high profile example of biocatalysis patenting is that of Taq polymerase, the thermostable DNA polymerase enzyme isolated from the thermophilic microorganism... 

The PCR is a three-step cyclic process that repeatedly duplicates a specific DNA sequence, contained between two oligonucleotide sequences called primers (154,155). The two primers form the ends of the sequence of DNA to be amplified and are normally referred to as the forward and reverse primers. The forward primer is complementary to the sense strand of the DNA template and is extended 5 to 3 along the DNA by DNA polymerase enzyme (Fig. 27). The reverse primer is complementary to the antisense strand of the DNA template and is normally situated 200-500 base pairs downstream from the forward primer, although much longer sequences (up to 50 kbase) can now be amplified by PCR. The process employs a thermostable DNA polymerase enzyme (such as the Taq polymerase from Thermus aqualicus BM) extracted from bacteria found in hot water sources, such as thermal pools or deep-water vents. These enzymes are not destroyed by repeated incubation at 94 °C, the temperature at which all double stranded DNA denatures or melts to its two separate strands (155). 

It is practical to use PCR reagents in 10 x working solutions that will be diluted by a factor of ten for final concentration in the PCR samples. An exception is the thermostable enzyme Taq polymerase, which is stored at a much higher concentration. All reagents are available from numerous companies. 

A sample of double-stranded DNA is denatured. One of the resulting single strands is used as a template to direct the synthesis of a complementary strand of radioactive DNA using a suitable DNA polymerase. The "Klenow fragment" of E. coli, DNA polymerase I, reverse transcriptase from a retrovirus, bacteriophage T7 DNA polymerase, Taq polymerase, and specially engineered enzymes produced from cloned genes have all been used. 

Other Class A polymerases. The Thermus aquati-cus (Taq) polymerase is best known for its widespread use in the polymerase chain reaction (PCR Fig. 5-47). Like E. coli I the enzyme is a large multidomain protein. The structure of the catalytic domains of the two enzymes are nearly identical, but the Taq polymerase has poor 3 -5 editing activity.276 The enzyme has been carefully engineered to improve its characteristics for use in the PCR reaction.277... 

Restriction enzymes, 1 unit/pY. Keep stored in freezer until ready to use. Recommended enzymes are BamYtt. and coRI for A. phage DNA,. fooRI and Taq for plasmids (pBR322 or ColEl), and Hpal for Ad 2 DNA. 

The association of Taq polymerase with the ends of the PCR product stencally hindering the restriction enzyme... 

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