DNA Extraction: Every living thing contains DNA, because it is th e blueprint of life.

The extraction of DNA from cells, its purification and furt her analysis have a vital role in the field of forensics and biotechnology. For the purposes to detect genetic disorders, DNA fingerprinting and the production of genetically engineered organisms that can produce beneficial products such as insulin, antibiotics, and hormones, isolation of DNA has primary importance. Methods of DNA Extraction: There are many different met hods and techniques of genomic DNA isolation. In general, each method consists o f disruption and lysis of the initially taken material, removal of proteins and other contaminants and in last recovery of the DNA. Proteins are removed by dige stion with proteinase K, salting out, organic extraction, or binding of the DNA to a solid-phase support (either using silica technology or anion-exchange). Usu ally DNA recovery is carried out by the process of precipitation, this process i s achieved by using ethanol or isopropanol. The choice of a method is dependent upon many factors, such as the required quantity and molecular weight of the DNA , required purity for applications, and time and expenses. The process of DNA is olation from other cellular components can be divided into following four stages : a) Disruption b) Lysis c) Proteins and contaminants removal d) DNA recovery. Mostly a&b stages are combined in some methods. Following methods are commonly used for DNA extraction: 1. Salting-out Method: In this method high concentration of salt such as potassium acetate or ammonium acetate is used for the precipita tion of proteins and other contaminants from the cell lysate. Centrifugation is applied for precipitates removal and then DNA is recovered by alcohol precipitat ion. This method is inefficient in removal of proteins and other contaminants. D NA quantity and quality may vary using this method. Precautions: i. Before the usage of DNA in downstream application repeated alcohol preci pitation is often necessary. ii. With the increase in concentration of the certain ions, the solubility o f proteins may also increases instead of decrease. In this case either a differe nt ion or an alternative purification method should be used. 2. Preparation of Crude Lysate: An easy method for g enomic DNA isolation is to incubate cell lysate at high temperature (e.g. 90C for 20 minutes), or by carrying out a proteinase K digestion, and then use this lysa te directly in downstream applications. This simple technique is only suitable f or a limited number of applications. Usually the subsequent lysate contains enzy me-inhibiting contaminants, such as salts, and sometimes pH of DNA is not optimu m. Precautions: i. Proteinase K should be inactivated completely, because incomplete inacti vation results into false negative results and high failure rates. ii. Recovered DNA by using this method should not be stored; proteinase K of ten causes DNA degradation. 3. Organic Extraction Method: This is a conventional technique of DNA isolation. In this method contaminants from cell lysate are rem oved by using organic solvents. Detergent is used to lyse the cell, and then mix

ed with chloroform, phenol and isoamyl alcohol. Alcohol precipitation is applied to recover DNA from aqueous phase. This is a time-consuming technique and also the toxic compounds are used in this method. Furthermore, this may not give prec ise results and required yields. In addition, this technique is not useful for s ensitive applications such as PCR and for high-throughput applications, as the r ecovered DNA may contain residual phenol or/and chloroform, which can restrain e nzyme reactions in downstream applications, and also this technique is not autom ated.

Precautions: i. The accurate salt concentration and precise pH must be followed during e xtraction to ensure that DNA resides in the aqueous phase and contaminations are separated into the organic phase. ii. Toxic waste must be disposed of with care and in accordance with hazardo us waste guidelines. 4. Cesium chloride (CsCl) Density Gradient Method:

In this technique a cesium chloride (CsCl) density gradient uses suspended DNA mixed with ethidium bromide and purified CsCl. Centrifugation is applied on this material for several hours and then DNA is recovered with isopro panol. The density of DNA is a function of base composition and sequence. Theref ore a highly repeated DNA sequence will form a sharp band in CsCl density gradie nts at a characteristic density. The resolution of this process may be increased or altered by binding ligands to the DNA. For example, netropsin binds specific ally to A + T-rich regions of DNA and reduces their density. Another useful liga nd is Ag+, which must then be centrifuged in Cs2SO4 gradients to avoid precipita tion of AgCl. 5. Anion-exchange Chromatography: C olumn chromatography has recently evolved to give a fast and efficient substitut e method of more laborious methods for isolating high quality DNA, such as CsClgradient centrifugation. This technique follows the use of a column made of a un ique anion-exchange resin that selectively binds nucleic acids, allowing fast se paration of DNA from RNA, carbohydrates, proteins, and metabolites. A moderate-s alt buffer is applied to wash out the contaminants from the column, and DNA is e luted. Precautions: i. Do not over dry, as it will make the pellet difficult to dissolve. ii. While dissolving, do not pipet high-molecular-weight DNA up and down, as this may cause shearing. iii. To prevent shearing lysate must be handled gently. 6. Silica-based Method:

DNA isolation by using the techniq ue of silica adsorption is an important method that is used in modern technologi es that use micro-channels. This type of separation is based upon the principle that DNA molecules bind to silica surface in the presence of specific salts and under certain pH conditions. A sample is placed into the chip and lysed. The sub sequent mixture which consists of proteins, DNA, phospholipids, etc. , is then p assed through the channel where the DNA is absorbed by silica surface in the pre sence of solutions with high ionic strength. The highest DNA adsorption efficien cies occur in the presence of buffer solution with a pH at or below the pka of t he surface silanol groups. Precautions: i. Make sure that wash solution is made up of 95% or 100% ethanol.

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To avoid evaporation, wash buffer should be stored at -20C.