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LATEST BIOCHEMICAL TECHNIQUES
Capillary electrophoresis is an analytical technique that separates ions based on their electrophoretic mobility with the use of an applied voltage. The electrophoretic mobility is dependent upon the charge of the molecule, the viscosity, and the atom’s radius. In conventional electrophoresis, electrically charged analytes move in a conductive liquid medium under the influence of an electric field. The rate at which the particles moves is directly proportional to the applied electric field; the greater the field strength, the faster the mobility. If two ions are the same size, the greater charge will move the fastest. For ions of the same charge, the smaller particle has less friction and overall faster migration rate. The technique of capillary electrophoresis was designed to separate species based on their size to charge ratio in the interior of a small capillary filled with an electrolyte. Capillary electrophoresis is used most predominately because it gives faster results and provides high resolution separation. It is a useful technique because there is a large range of detection methods available.
Electrophoresis is the process whereby the movement of ions is produced under the influence of an applied voltage across a field that the ions exist. In electrophoresis, ions that are negatively charged will move or migrate towards the positively charged electrode while ions that are positively charged will migrate towards the negatively charged electrodes. This is the basis for the capillary electrophoresis operates.
TERMINOLOGIES * Rate of migration: What makes Capillary Electrophoresis a powerful tool in separation science is the phenomena whereby every ion will migrate at different rates. This difference is based on its quantity of charge compared to its relative hydrodynamic size. Hydrodynamic size very closely relates to the mass of the molecule. It is commonly called the “Charge to Mass Ratio”. A simpler term that is used to describe the molecules affinity for its opposite electrode is commonly called “Electrophoretic Mobility”. These motilities’ can be exploited for incredible separations of very closely structured molecules. * Ionic Mobility: The actual mobility of an ion takes into account the environment that the ion exists in during the separation process. For example, electrophoretic mobility will differ from actual mobility when viscosity changes and of course the amount of voltage that is applied. In simpler terms, this makes the attraction much greater or weaker to the opposite charged electrode. The more voltage, the more attraction and greater the speed. * Electro-Osmotic Flow (EOF): Capillary Electrophorsis has incredible efficiency or ability to separate similarly structured compounds. This is due to EOF. When a voltage is applied across a tube filled with an electrolyte solution (a solution that conducts electricity), the solution begins to move toward the cathode. This is not similar to the chromatographic pump, but it does provide the flow of materials past a detector like the pump in HPLC. This should not be confused with electrophoretic mobility described above. It is a separate phenomenon and is exploited in Capillary Electrophosis for maximum flexibility in separation power. Both EOF and Electrophoretic mobility can occur at the same time working in opposite directions to provide greater resolution.
A typical capillary electrophoresis system consists of a high-voltage power supply, sample vial, source vial, destination vial, a capillary, a detector, electrodes, an output device and handling device. Some instruments include a temperature control device to ensure reproducible results. This is because the separation of the sample depends on the electrophoretic mobility and the viscosity of the solutions decreases as the column temperature rises.
The source vial, destination vial and capillary are filled with an electrolyte such as an aqueous buffer solution. To introduce a sample, the capillary inlet is placed into a vial containing the sample and then returned to the source vial (sample is introduced into the capillary via capillary action, pressure or siphoning).
Each side of the high voltage power supply is connected to an electrode. These electrodes help to induce an electric field to initiate the migration of the sample from the anode to the cathode through the capillary tube. It is important to note that all ions, positive or negative, and pulled through the capillary in the same direction by electroosmotic flow. The analytes separate as they migrate due to their electrophoretic mobility and are detected near the outlet end of the capillary. The output of the detector is sent to a data output and handling device such as an integrator or computer. The data is then displayed as an electropherogram, which reports detector response as a function of time. Separated chemical compounds appear as peaks with different migration times in an electrophorogram.
Capillaries are typically of 50 µm inner diameter and 0.5 to 1 m in length. The capillary tubes can be filled with many different matrices depending on the sample type. Samples are applied to the capillary tubes when the cathode buffer is moved aside and sample chamber placed at the opening of the capillary tube. The applied potential is 20 to 30 kV, this result in reduced sample run times. Either pressure is applied to the sample and 10 - 100 nL is injected or an electrical current is applied through the sample and only the charged molecules enter the capillary. Detection of the migrating molecules is accomplished by shining a light source through a portion of the tubing and detecting the light emitted from the other side
Once the electrophoretic separation is completed, the contents of the capillary are flushed out and fresh matrix fills the tube. Replacing the matrix within the capillary minimizes the possibility of contaminating samples between runs. Separation is accomplished as in conventional gel electrophoresis but the capillary allows higher resolution, greater sensitivity, and on-line detection.
1. DNA FINGERPRINTING: DNA fingerprinting is a useful tool for identifying the genotype of living organisms by determining their DNA sequence. For this technique, genomic DNA must be amplified by PCR. Capillary electrophoresis separates this amplified DNA with a one base pair resolution and creates specific peaks for each nucleotide to map the DNA sequence. 2. PHARMACEUTICAL ANALYSIS: Capillary electrophoresis can be used for pharmaceutical analysis of basic drugs and related substances. The principle of separation of Capillary Electrophoresis allows for analysis of a wide variety of samples. The selectivity of the method provides extraordinary resolution to Capillary Electrophoresis. For example, capillary electrophoresis greatly simplifies the analysis of products containing basic amine functional groups. Use of bare-fused silica capillaries at an acidic pH produces a non-reactive capillary surface while highly ionizing an analytes’ amines; the assay for basic drug analysis is thus simplified. 3. PROTEIN CHARACTERIZATION: Through a pH gradient established by the electric field, capillary electrophoresis is able separate the amphoteric molecules such as proteins. Samples migrate to their individual isoelectric points (pI) and stop; each sample is thereby focused into a tight zone which is mobilized past the detector by pressure or through chemical means. This technique is in protein identification. As capillary electrophoresis is easily to automate, it saves time and labor over gel electrophoresis.
Southern blot is a method characteristically used for detection of a specific DNA sequence in DNA samples. Southern blotting is the transfer of DNA molecules, usually restriction fragments, from an electrophoresis gel to a nitrocellulose or nylon sheet (referred to as membrane) in such a way that the DNA branding pattern present in the gel is reproduced on the membrane. During the transfer, the DNA becomes immobilized on the membrane. In essence, southern blotting is a method for detection of a specific restriction fragment against a background of many other restriction fragments.
In this procedure, the DNA is isolated from each source and then digested with a specific restriction enzyme. The DNA restriction fragments are then loaded onto an agarose gel and the fragments separated by electrophoresis according to size, with the smaller fragments migrating faster than larger fragments. The DNA is then transferred from the fragile gel to a nylon filter. The nylon filter (nitrocellulose powder or sheets) have the ability to bind DNA. The radioactively labeled nucleic acid probe is added. The probe binds to complementary DNA segments.
In summary, the principle is based on the specific base-pairing between the DNA immobilized on the blotting membrane (i.e. the target) and the labeled probe, and the latter having a sequence that is complementary to the target.
DNA is applied to an agarose gel, and electrophoresis separates the fragments of DNA according to size. The gel is then placed atop a thin sponge wick resting in a dish of salt solution and a special filter (nitrocellulose) is placed on top of the gel. A stack of absorbent material (usually paper towels) is placed on top of this stack. The absorbent material draws the salt solution from the dish into the wick and through the gel by capillary action, which transfers the DNA fragments into the filter. This procedure is called a “Southern transfer”. The filter now contains the DNA fragments in the same pattern as the gel, but is more easily manipulated. The filter is placed in a standard “seal-a-meal” bag, containing a solution of radioactively-labeled DNA probe for a particular gene sequence. The probe binds to the filter only where a complementary DNA sequence is located. After washing to remove unbound probe, a piece of X-ray film is placed over the hybridized filter and left for several hours to several days. The radioactive label produces a black band on the film where it has stuck to the complementary DNA, producing an autoradiogram. If a labeled size marker has been used, the exact size of the fragments can be determined.
Various modifications have been introduced to the Southern blot over the years to improve the efficiency of DNA transfer from gel to membrane. The major improvement has been the introduction of nylon membranes, whose advantages over their nitrocellulose counterparts includes; * Nylon membranes are less fragile than nitrocellulose sheets. The nitrocellulose sheet has the tendency to crack if handled roughly during Southern blotting and usually disintegrate if attempts are made to carry out more than two or three hybridization analyses with the same blot. Nylon membranes cannot be damaged by handling and a single blot can be hybridized up to ten times. This limit is not due to eventual breakage of the membrane, but to the gradual loss of the blotted DNA during repeated hybridizations. * Under certain conditions (a positively charged membrane and an alkaline transfer buffer) the transferred DNA becomes covalently bound to the nylon membrane during the transfer process. This is not the case with the nitrocellulose membrane, which initially binds DNA in a semi-permanent manner, with immobilization occurring only when the membrane is baked at 808oC. Transfer onto a positively charged nylon membrane can therefore reduce the possible loss of DNA that might occur by leaching through the membrane during the blotting process. It is also quicker as the transfer time is reduced from 18 hours to 2 hours. * Nylon membranes efficiently bind DNA fragments down to 50bp in length, whereas nitrocellulose membranes are effective only with molecules longer than 500bp.
Nitrocellulose has however not been completely old-fashioned, as it has one significant advantage compared with nylon membranes: a reduced amount of background hybridization, especially with probes that have been labeled with nonradioactive markers. Other changes to the original Southern blotting procedure have been introduced to speed up and improve the efficiency of the transfer.
The commonest application of Southern blotting is identification and cloning of a specified gene. Southern blotting of genomic DNA is used to identify one or more restriction fragments that contain the gene being sought and after cloning and tentative identification of the desired recombinant by colony or plaque hybridization probing, Southern blotting of restricted clone DNA is used to confirm the clone identification and possibly locate a shorter restriction fragment, containing the sequence of interest from within the cloned DNA.
The second major application of Southern hybridization is in the technique called restriction fragment length polymorphism (RFLP) mapping, which is important in construction of genome maps. Treatment of genomic DNA from different individuals with a single restriction enzyme does not always give the same set of fragments because some restriction sites are polymorphic, being present in some individuals but absent in others, usually because a point change in the nucleotide sequence changes the restriction site into a sequence not recognized by the restriction enzyme. These polymorphic sites can be used as markers in genetic mapping.
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