Thermostability, DNA

Polymerase chain reaction (PCR) The process by which a specific sequence of DNA can be amplified (copied many times) in vitro. It requires a pair of primers and template DNA, thermostable DNA polymerase (e.g. Taq polymerase), deoxynucleotide triphosphates and a thermocycler. The process can amplify large... 

The thermostabilization of biomolecules is a result of the mutual contribution from fundamental interactions [e.g., hydrophobic forces (4, 5) or ionic interactions (3, 6, 7)] that stabilize individual molecules and prevent their aggregation (6), structure modifications [such as DNA superhelicity (8, 9) and posttrans-lational modification of proteins], interactions with an environment (10), intermolecular interactions (11), and oligomerization (12). The possible dependence of fundamental interactions, for example, hydrophobic forces, on temperature may also affect stability. However, it remains a subject of controversy as to how and to what extent the dependence of the interaction strength on temperature should be taken into account (13-16). This article reviews the very basic level of protein and DNA thermostability. 

Figure 2 Base stacking provided by the correlations in nucleotide sequences is the major mechanism of DNA thermostability. Upper row. Real amino acid sequence and original codon bias. Middle row. The effect of codon interface is removed through the reshuffling of protein sequences while retaining the actual codons used for each amino acid. Bottom row. Codon bias in natural protein sequences is removed by using synonymous codons with equal probabilities. ApG and CpT pairs in the sense strand and ApG pairs in the antisense strand of DNA are underlined if they are located inside one codon. For example (upper row), the first ApG pair in the sense strand is in the Lys codon, whereas the second ApG pair is on the border between the codons of Leu and Val.

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. 

In our previous works the fact of AR-DNA interactions, resulting in modifications of physicochemical properties of this biopolymer with formation of supramolecular complexes has been described [Davydova et al., 2005]. The AR-DNA interactions also leads to B —> A transition of DNA, increase the thermostability of these complexes and improving the resistance of DNA to some external influences [Davydova et al., 2006, 2007]. 

While many diseases have long been known to result from alterations in an individual s DNA, tools for the detection of genetic mutations have only recently become widely available. These techniques rely upon the catalytic efficiency and specificity of enzyme catalysts. For example, the polymerase chain reaction (PCR) relies upon the ability of enzymes to serve as catalytic amplifiers to analyze the DNA present in biologic and forensic samples. In the PCR technique, a thermostable DNA polymerase, directed by appropriate oligonucleotide primers, produces thousands of copies of a sample of DNA that was present initially at levels too low for direct detection. 

A gene (erstEl) encoding a thermostable esterase was isolated from Escherichia coli cells that had been transformed by DNA libraries with metagenomes from environmental samples isolated from thermal habitats. The enzyme belonged to the hormone-sensitive lipase family, could be overexpressed in E. coli, was active between 30 and 95°C, and used 4-nitrophenyl esters with chain lengths of C4-C16 (Rhee et al. 2005). 

Stability of several enzymes like proteases from thermophilic micro-organisms can be increased in aqueous-organic biphasic systems. Owusu and Cowan [67] observed a strong positive correlation between bacterial growth temperature, the thermostability of free protein extracts, and enzyme stability in aqueous-organic biphasic systems (Table 1). Enzymes, like other cell components (membranes, DNA, (RNA ribosomes), are adapted to withstand the environmental conditions under which the organism demonstrates optimal growth. 

Saiki, R. K., etal. (1988). Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239,487-491. 

In the metagenomic approach, DNA was directly extracted from uncultured samples followed by cloning and expression [3]. For example, by combination of directed evolution with the metagenome approach, an a-amylase mutant with optimal activity at pH 4.5 and optimal thermostability at 105 °C was discovered for starch liquefaction and EtOH production [4]. 

Since rolling circle amplification takes place at a constant temperature, there is no need for the target amplification process to take place in a thermal cycler, which is required to regulate the temperature for different parts of the reaction. The type of DNA polymerase to be used in RCA is not limited to thermostable enzymes, like the PCR-based diagnostics. On the other hand, the RCA method requires the environment to be free of contaminations as the RCA arrays are highly sensitive. Wiltshire [22]... 

Taq polymerase is a thermostable DNA polymerase which was originally isolated from the bacterium Thermus aquaticus, which lives in hot springs. 

Automated programmable instruments that can carry out the repeated thermal cycles necessary for PCR and that can accommodate multiple samples simultaneously are now widely available. The procedure is usually performed with thermostable DNA polymerases. PCR is widely used to facilitate detection of minute amounts of viral DNA. The technique can also be used to detect specific point mutations, provided the approximate site of mutation is known. One limiting feature of this approach arises from the fact that the bacterial polymerases frequently make errors when synthesizing new strands and so can introduce mutations that are not present in the original sample. 

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... 

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