Aptamers are synthetic nucleic acids which are characterized for their specific binding to targets and as a consequence they are used in many affinity chromatographic applications in order to recognize molecules (Zhao, Li, Shao, and Le, 2008). The process which is used for the identification of aptamers to various chemical targets is SELEX (systematic evolution of ligands by exponential enrichment). More specifically, large amounts of nucleic acids with a functional role are being isolated in a repetitive way through in vitro selection and then amplification follows through polymerase chain reaction (Tombelli, Minunni and Mascini, 2005). The category of targets contains both nucleic acid and nonnucleic acid binding proteins, like for example growth factors and also enzymes, antibodies and peptides. Another characteristic of aptamers is that they have been related to the recognition of small molecules like for example amino acids, nucleotides, drugs and dyes. This collection between target and aptamer was the motive for in vivo and in vitro developments (Romig, Bell and Drolet, 1999). According to various reports aptamers were discovered in the beginning of 1990s. A number of studies have provided information about their generation, which has occurred all these years towards a broad range of targets such as peptides, amino acids, small molecules and proteins, including cell membrane proteins. The affinity exhibition of aptamers for their targets is high as it can be understood from the low nanomolar to picomolar range of their Kd values (Proske, Blank, Buhmann and Resch, 2005). Based on the study of Proske et al. (2005), the discrimination between isoforms, with a really close relationship or distinct structure states of the sam... ... middle of paper ... ...e. Solution to this problem has been given by using aptamers with both weak and strong binding affinity in order to avoid the restriction of affinity CE. One important advantage of using aptamers in affinity chromatographic applications, mainly for the analytes which are present in complex sample matrix, is the capture of the target analytes of interest in a selective and efficient way (Zhao et al., 2008). One particular requirement in order to improve the sensitivity of the applications by taking advantage of the preconcentration of targets is that targets with a strong binding to the aptamer affinity column have to be eluted in a suitable way. What it is also wanted is the capture and the elution of targets from the column stationary phase without any damage of the latter. As a result the affinity column can be used again for similar purposes (Zhao et al., 2008).
Amplification reaction was done in a 25.0 µL reaction mixture containing 0.4 µL DNA (from DNA extraction), 5.0 µL of 10X PCR reaction buffer, 14.2 µL of sterelized dH2O, 2.0 µL of magnesium chloride (MgCl2, 25 mM), 1.0 µL nucleotide/dNTP mix (10 Mm), and 0.4 µL of 5 u/µL Taq DNA polymerase for each primer namely respectively. The components and the volume used for the amplification reactions are listed in Table 3.2. For the reaction, PCR reaction was performed in a programmable gradient-enabled thermocycler (Bio-Rad MyCycler™ Thermal Cycler).
Zhang X., Tang H., Ye C. and Liu M. (2006). Structure-based drug design: NMR-based approach for ligand-protein interactions. Drug Discovery Today: Technologies. 3 (3), pp. 241-245.
Oligonucleotides and analogues: A practical approach. Edited by F Eckstein. Series editors: D. Rickwood and B.D. Hames. IRL PRESS. Oxford University Press. 1991.
3. Prospects for Antisense Nucleic Acid Therapy of Cancer and AIDS. Eric Wickstrom, Ed. Wiley-Liss, Inc., NY, 1991. pp 25-33, 35-51, 125-141.
To form a polynucleotide DNA, many nucleotides are linked together with 3`-5` phosphodiester linkages. In a compl...
Thus, it contains two active sites and has metal ions that play a key role in regulating catalytic activities and stabilizing enzyme-substrate complex. The active sites are 30Å apart from each other (7). As proposed by Gettins and Coleman using NMR studies (6), each active site of AP comprises of three metal binding sites, which acknowledged as M1, M2, and M3. Two zinc ions bind to M1 and M2 sites while a magnesium ion occupies M3. In each active site, two zinc ions are 4Å apart while magnesium ion is 5Å and 7Å away from two zinc ions (7). The structures of AP are shown in Figure 1, which specifically indicating metal binding sites and interaction with essential amino acids in active
... similarities between proteins exhibiting homology, and inspecting the AFP nucleic acid sequence in comparison with proteins showing similarities.
Due to the nature of amino acids, a titration curve can be employed to identify
One of these classes is DNA binding agents. They form a covalent bond with the DNA or stick to it noncovalently very tightly. A covalent bond is a chemical bond. It involves sharing of electron pairs in the middle of atoms. These electron pairs are known as shared pairs or bonding
An enzyme is a specialized protein that acts as a catalyst and facilitates complex metabolic processes. An enzyme, like any protein, is a polymer made up of a long chain of amino acids. The sequence of amino acids is determined by the DNA template in which it was made, and the amino acids are attached together by peptide bonds. Cross linking takes place between the R groups of the amino acids and forms a unique three dimensional molecules. The structure and spatial configuration of an enzyme, especially its binding site, is key to its optimal function and activity. This 3-dimensional structure can easily be altered by environment factors, such as salinity and pH. Each enzyme has a binding site in which chemical bonds are achieved with their
Ion exchange chromatography is a unique technique for effective separation of ions, amino acids, peptides, nucleotide and nucleic acids etc. This technique is widely used in the pre-fractionation or purification of a target protein from crude biological samples.
EDTA Titrations [homepage on the internet]. No date. [cited 2014 Mar 24]. Available from: http://bionmr.unl.edu/courses/chem221/lectures/chapter-12.ppt.
The first study, concerning the direct and covalent immobilization of Cibacron Blue on Sephadex G-200 and also the purification of yeast pyruvate kinase with this affinity sorbent, was conducted by Roschlau and Hess (1972). Later, there were many articles and reviews dealing with this concept, which has been used in a wide variety of applications in order to purify a wide range of proteins with a number of matrices having this blue ligand.
References Alberts, B., Johnson, A., Lewis, J., Raff, M., Roberts, K. and Walter, P., 2008. Molecular