1.1 DNA Structure DNA is a polymer made of subunits called as nucleotides. Each nucleotide consists of a deoxiribose sugar, a phosphste, and a nitrogenous base (Genetics from Genes to Genomes). Watson and Crick proposed the structure for DNA (shown schematically in Figure 1 a). This is the presence of two polynucleotide strands coiling around a common axis and those strands linked together by a specific hydrogen bond scheme between the purine and pyrimidine bases (Figure 1 b), viz. adenine (A) with thymine (T) and guanine (G) with cytosine (C). The carbon atoms of the deoxyribose sugar are distinguished from atoms of the deoxyribose within the nucleotide base by the use of primed numbers from 1-5. The phosphodieser bonds always form a covalent link between the 3' carbon of one nucleoside and the 5' carbon of the following nucleoside. The consistent orientation of the nucleotide building blocks gives a chain overall direction, such that the two ends of a single chain are chemically distinct. At the 5' end, the sugar of the terminal nucleotide has a free 5' carbon atom and at the other 3' end of the chain, it is the 3' carbon of the final nucleotide that is free (Genetics from Genes to Genomes). In the model, two DNA chains spiral around an axis with the sugar-phosphate backbones on the outside and pairs of bases (one from each chain) meeting in the middle. Although both chains wind around the helix axis in a right-handed sense, chemically one of them runs 5' to 3' upward, while the other runs in the opposite direction of 5' to 3' downward. In short, the two chains are antiparallel. The base pairs are essentially flat and perpendicular to the helix axis, and the planes of the sugars are roughly perpendicular to the base pairs. As the two chains spiral about the helix axis, they wrap around each other spiral about the helix axis, they wrap around each other once every 10 base pairs, or once every 34Ã… (Genes to genomes). In a space-filling representation of the model, the overall shape is that of a cylinder with a diameter of 20Ã… whose axis is the axis of the double helix. The backbones spiral around the axis like threads on a screw, but because there are two backbones, there are two threads, and these two threads are vertically displaced from each other. This displacement of the backbones generates two grooves, one much wider than the other, that also spiral around the helix axis. Biochemists refer to the wider groove as the major groove and the narrower one as the minor groove. The two chains of double helix are held together by hydrogen bonds between complementary base pairs, A-T and G-C. Since the overall shapes of the two base pairs are quite similar, either pair can fit into the structure at each position along with DNA. Moreover, each base pair can be accommodated in the structure in two ways that are the reverse of each other: an A purine may be on strand 1 with its corresponding T pyrimidine on strand 2 or the T pyrimidine may be on strand 1 and the A purine on strand 2. In addition A-C and G-T pairs do not fit well together; that is, they do not easily form hydrogen bonds. The DNA molecule is essentially a polynucleotide or a polymer chain formed by phosphate diester groups joining b-D-deoxyribose sugars through their 3 and 5 hydroxyl groups (Figure 3). The backbone of the DNA molecule thus consists of six single bonds about which rotations can take place (also indicated in Figure 3) giving rise to various possible conformations/structures for the polymeric chain. As mentioned above, the canonical Watson-Crick DNA model is a two-stranded helical structure, in which the two chains are held together by hydrogen bonds between the purine (A,G) and pyrimidine (T,C) bases. There are 10 nucleotides per turn, separated by + 36 rotation and 3.4 Ã… translation along the helix axis,in each of the two chains and the two chains are aligned in mutually anti-parallel orientations (Figures 1 a and 4) (Manju Bensal) DNA can inter-convert between two well-defined forms, viz. A and B (Figure 2). The molecular structures corresponding to these two forms were later shown to be essentially similar in their handedness, chain orientation and hydrogen bonding scheme. Subsequently it has become clear that the DNA molecule has an enormous ability to undergo structural changes depending on its environment by twisting, turning and stretching, leading to a pantheon of DNA structures6. Several of these structural polymorphs of DNA have now been experimentally characterized using X-ray diffraction, NMR or other spectroscopic studies and are found to vary considerably from the Watson-Crick type structure (Manju Bensal).
1.2 Principle of Agarose Gel Electrophoresis Electrophoresis is defined as movement of small ions and charged molecules in solution under the influence of an electric field (Gel Electrophoresis of Nucleic Acid, A Practical Approach). Agarose gel electrophoresis is a widely used method that separates molecules based upon charge, size and shape. It is particularly useful in separating charged biomolecules such as DNA, RNA and proteins (Lab Electrophoresis). Agarose gel electrophoresis possesses great resolving power, yet is relatively simple and straightforward to perform. The gel is made by dissolving agarose powder in boiling buffer solution. The solution is then cooled to approximately 55oC and poured into a mol containing a comb that makes well when solution is polymerised (Lab Electrophoresis). Electrophoresis is carried out in the gels cast either in tubes or as slabs. A number of gel materials have been used successfully, including agar, agarose, and polyacrylamide. Agar and agarose gels are made by heating the granular material in the appropriate electrolyte buffer, casting the gels and allowing them to set on cooling. The resolving power of these gels depends on the concentration of dissolved agar or agarose; dilute gels are used for very large DNA molecules and more concentrated gels for low molecular weight DNA (Gel Electrophoresis of Nuclecic Acid, A Practical Approach). Samples are prepared for electrophoresis by mixing them with components that will give the mixture density, such as glycerol or sucrose. This makes the samples denser than the electrophoresis buffer. These samples can then be loaded with a micropipette or transfer pipet into wells that were created in the gel by a template during casting. The dense samples sink through the buffer and remain in the wells (Lab Electrophoresis). A direct current power supply is connected to the electrophoresis apparatus and current is applied. Charge molecules in the sample enter the gel through the walls of the wells. Molecules having a net negative charge migrate towards the positive electrode (anode) while net positively charged molecules migrate towards the negative electrode (cathode). Within a range, the higher the applied voltage, the faster the samples migrate. The buffer serves as a conductor of electricity and to control pH. The pH is important to the charge and stability of biological molecules (Lab Electrophoresis). The rate of migration depends on the size and shape of the molecule, the charge carried. In an electric field at moderate pH, negatively-charged DNA molecules migrate towards the anode. A fractionation is achieved because large molecules move more slowly through the gel than small molecules and selection of DNA within a given size range is obtained by selecting a gel of appropriate pore size. Electrolytes used in electrophoresis generally consist of an aqueous buffer, containing a chelating agent such as ethylenediaminetetractate (EDTA) and a nuclease inhibitor such as sodium dodecyl sulphate (SDS). A number of factors affect the fractionation of RNA. Increasing the current leads to higher rates of migration, but the flow of current also results in the production of heat, which, if excessive, adversely affects the separation by causing trailing and broadening of the zones.
1.3.1 Silver Staining Ethidium bromide staining is the conventional laboratory technique for the detection of DNA. Switzer et al (1979) originally introduced silver staining technique for the detection and analysis of proteins. Currently, silver staining is sometimes used to detect DNA fragments, including short interspersed nuclear elements, VNTR detection, and SNPs in various experiments (Yan-Chung Han et al, 2008). The new method is much efficient and sensitive for polymorphism DNA analysis and the detection of small amount of nucleic acid (Sommerville and Wang, 1981; Boulikas and Hancock, 1981; Goldman and Merril, 1982; and Guillemette and Lewis, 1983) but more versatile silver staining is needed for analysis of complex DNA profiles generated in DNA amplification fingerprinting and DNA sequencing (Anolles and Gresshoff, 1994). The ethidium bromide staining of DNA is time-consuming as they require a lot of preparation and handling of several solutions prior to use and needs expensive and bulky fluorescence imaging equipment. Furthermore, the sensitivity, color uniformity, and storage time of the staining gels are not ideal (Yan-Chuang Han et al, 2008). Moreover, Ethidium bromide used for staining of DNA as a conventional method is a carcinogenic substance and a cost of waster disposal. Many modifications to silver staining method have been reported since the introduction of silver staining method for the analysis of proteins and nucleic acid analysis in agarose and polyacrylamide gels. It has been reported that some procedures used to stain nucleic acids in polyacrylamide gels are not suitable for agarose gels due to differences in the chemical compositions of both matrices. The agarose matrix has the disadvantage of nonspecific depositions of silver ions resulting in high background (Willoughby and Lambert 1983, Peats 1983, and Lasne et al 1983). Those protocols developed in order to reduce the nonspecific stain in agarose gels involve time-consuming pretreatment steps with K2Cr2O7 or Na2S2O3 (Zalazar et al, 2001). During image development almost all staining procedures reduce silver ion to colloidal silver, which is then deposited in the immediate vicinity of the staining substratum. For optimal image contrast, the level of silver reduction in the gel matrix must be kept to a minimum level. This is performed by appropriate modulation of the speed of the reduction process, which depends mainly on the pH, the absolute and relative concentrations of silver and reducing agents, and the rate constant of the reaction (Anolles and Gresshoff, 1994). Silver staining is also useful for the microarray technology in order to impede the interference of fluorescent label with the hybridisation process. This advantage is achieved by application of silver staining after the hybridization process. Silver staining also eliminates the need of fluorescence imaging equipment in microarray technology by means of using a film scanner. The aims of the present study are to optimize a silver staining protocol performed for a commercially obtained DNA molecular weight marker, in which the procedure is modified. The detection limit of silver staining is investigated and is compared with ethidium bromide staining. Moreover, some of the siver staining methods are varied and are compared with ethidium bromide staining. The silver staining protocol is modified inorder to increase the sensitivity and reduce background staining. After a suitable protocol is optimized DNA are deposited on filter paper at various dilutions and stained with the optimized. This has implications for the development of portable biosensors with label free detection.