Nucleic acid Extraction methods.

Nucleic Acid Extraction methods :-

It can be characterized into two different types - 

• Solution - based method 
• Solid-phase based method 

Solid-phase based method

1. Guanidinium Thiocyanate-Phenol-Chloroform extraction 

Salt is common impurity in nucleic acid samples. It has always been required to be removed from nucleic acid samples before any analysis or process. Therefore, single or multiple purification steps needed to desalt the sample comprising the nucleic acid.

Phenol-chloroform extraction is one of the examples, which widely used in isolating nucleic acid. Although Phenol is flammable, corrosive and toxic carbolic acid can denatures proteins rapidly, it does not solved by using a Phenol:Chloroform :isoamyl alcohol (25:24:1), this will remove proteins, lipids, carbohydrates and cell debris. It gives aqueous phase with the mixture of phenol and chloroform. This biphasic emulsion separated by centrifugation. The upper phase contain DNA and collected by Precipitation with ethanol or isopropanol (2:1 or 1:1) after centrifuge DNA Precipitate. Excess salt or impurity removed by rinsed with 70% ethanol and centrifuge it, DNA aggregate as pellet, then dissolved with TE buffer or sterile distilled water.

The use of guanidinium isothiocyanate is RNA extraction was first mentioned by Ulrich et. al. (1977). The method was laborious, therefore it has been displaced by a single-step technique, which is known as Guanidinium-Thiocyanate-Phenol-Chloroform extraction develop by chomezynski and sacchi (1987),where by the homogenate is extracted with Phenol-chloroform at reduced pH. Guanidinium thiocyanate is a chaotropic agent used in protein degradation.

The principle of this single-step technique is that RNA is separated from DNA after extraction with acidic solution consisting guanidinium thiocyanate, sodium acetate, phenol and chloroform. In acidic condition, total RNA will remain in the upper aqueous phase of the whole mix, while DNA and proteins remain in the interphase or lower organic phase. Recovery of total RNA is then done by Precipitation with isopropanol.

2. Alkaline extraction 

This technique has been used to isolate plasmid DNA. It involves harvesting bacterial culture and consequently exposing the bacteria to a highly alkaline solution. The alkaline extract is generally mixed with the detergent Sodium Dodecyl Sulfate (SDS).

The principle of the method is based on selective alkaline denaturation of high molecular weight chromosomal DNA while covalently closed circular DNA remains double stranded. Bacterial proteins, broken cell walls and desaturated chromosomal DNA enmeshed into large complexes that are coated with dodecyl sulfate. Plasmid DNA can be recovered from the supernatant after the denatured material has been removed by centrifugation

3.CTAB extraction

The method mainly used for the plant nucleic acid extraction, the initial step is to grind or chop the sample to break down cell wall material and allow nucleic acid to come out, while cellular enzymes and chemicals remain inactivated. After grinding the sample resuspended in suitable buffer such as Cetyltrimethylammonium bromide (CTAB).

CTAB is non-ionic detergent that can Precipitate nucleic acids and acidic polysaccharides from low ionic strength solution. Meanwhile nucleic acid and some other polysaccharides are insoluble in solution of 2% CTAB at high pH. In solution of high ionic strength, CTAB will not Precipitate nucleic acid and form complex with protein. CTAB is therefore useful for purification of nucleic acid from organisms which produce large quantities of polysaccharides such as plants and certain gram negative bacteria.

This method also uses in later steps of organic solvents and alcohol precipitation. Insoluble particles are removed through centrifugation to purify nucleic acid. Soluble proteins and other materials are separated through mixing with chloroform and centrifugation. Nucleic acid must be precipitated after this form the supernatant and washed thoroughly to remove contaminating salts. The purified nucleic acid is then resuspended and store in TE buffer and sterile distilled water.

4. Ethidium Bromide (EtBr) - Cesium Chloride (CsCl) gradient centrifugation

Cesium Chloride (CsCl) is extremely dense salt. CsCl gradient centrifugation is complicated, expensive and time-consuming method. Compared to other purification protocols. The method is not suitable for mini preparation of plasmid DNA extraction, it required large scale culture. Nucleic acid can be concentrating by centrifugation in an EtBr-CsCl gradient after alcohol precipitation and resuspension. In centrifugation the dense solution (with a higher CsCl concentration) will settle to the bottom of the tube, maintaining the concentration gradient for the some time. Other molecules dissolved in the solution will separate themselves according to how dense they are, with the denser molecules going towards the bottom of the tube and less dense molecules moving towards the top. When DNA is in presence of EtBr it will bind to it and becomes fluorescent when under UV light. The EtBr also adjust the density of DNA so that it moves towards the centre of the tube upon centrifugation. Contamination will move to different position and therefore will be separated from the DNA, which can be easily seen and recovered since it will be fluorescent. Subsequent steps will then separate the DNA from the CsCl and EtBr. This method effective for separating plasmids, small DNA strands, mitochondrial DNA.

5. Chelex extraction 

This technique used in the field of forensics for DNA extraction from different sources like buccal swabs, blood stains cards and hair. This method uses a resin Chelex (Trade name), that binds to common inhibitors of the polymerase chain reaction (PCR) process. This yields a fairly crude sample, but one that preserves DNA and it useful for P R-based forensic analysis.

6. Purification of Poly(A)+RNA by oligo (dT)-cellulose chromatography

Poly(A)+ RNA is template for protein translation and most of the eukaryotic mRNAs carry tracts of it at their 3' termini. It makes up 1 to 2% total RNA and it can be separated by affinity chromatography on oligo(dT)-cellulose. Poky(A) tails from stable RNA-DNA hybrids with short chains of oligo(dT) that attach to various support matrices. High salt must be added to the chromatography buffer to stabilize the nucleic acid duplexes as only a few dT-A base pairs are formed. A lower-salt buffer is used after non-polyadenylated RNAs have been washed from the matrix. This buffer helps to destabilize the double-stranded structures and elute the poly(A)+RNAs from the resin.

There are two methods are used. 

1. Column chromatography - normally used for the purification of large quantities (>25μg) of non radioactive poly(A)+ RNA isolated from mammalian cells.
2. Batch chromatography - the preferred method when working with a small amount (<50μg) of total mammalian RNA. It will processed samples are radioactive or not this method carried out by fine grade of oligo(dT) cellulose at optimal temperature for binding and elution.

Solid-phase Nucleic Acid Extraction

It allows quick and efficient purification compared to conventional methods. Many of the problems that are associated with liquid-liquid extraction such as incomplete phase separation can be prevented. Solid phase system will absorb nucleic acid in the extraction process depending on the pH and salt content of the buffer. The absorption process is based on the following principles: hydrogen-binding interaction with a hydrophilic matrix under chaotropic conditions, ionic exchange under aqueous conditions by means of an anion exchanger, and affinity and size exclusion mechanisms. Silica matrices, glass particles, diatomaceous earth, and anion-exchange carriers are examples that have been utilized in solid-phase extraction methods as solid support. Four key steps involved in solid-phase extraction are cell lysis, nucleic acids adsorption, washing, and elution. 

1.Silica Matrices. 

The basis for most of the products related to nucleic acid purification is the unique properties of silica matrices for selective DNA binding. Types of silica materials including glass particles, such as glass powder, silica particles, and glass microfibers prepared by grinding glass fiber filter papers, and including diatomaceous earth.Hydrated silica matrix, which was prepared by refluxing silicon dioxide in sodium hydroxide or potassium hydroxide at a molar ratio of about 2:1 to 10:1 for at least about 48 hours, had been introduced in DNA purification. DNA binds to the inorganic matrix. 

The principle of silica matrices purification is based on the high affinity of the negatively charged DNA backbone towards the positively charged silica particles. Sodium plays a role as a cation bridge that attracts the negatively charged oxygen in the phosphate backbone of nucleic acid. 

Besides silica matrices, nitrocellulose and polyamide membranes such as nylon matrices are also known to bind with nucleic acids, but with less specificity. These materials are often used as solid-phase nucleic acid transfer and hybridization matrices. Polyamide matrices are more durable than nitrocellulose and are known to bind nucleic acids irreversibly. Nucleic acids can be immobilized on polyamide matrices in low ionic strength buffer. 

2. Glass Particle

Glass particles, powder and beads are useful for nucleic acid purification. For example, DNA isolation from agarose gels involved the use of chaotropic salts to facilitate binding of DNA to common silicate glass, flint glass, and borosilicate glass (glass fiber filter). The adsorption of nucleic acid onto the glass substrate occurs most likely based on the mechanism and principle that similar to adsorption chromatography. Nucleic acid purification can also be done on silica gel and glass mixture. This invention has discovered that a mixture of silica gel and glass particles can be used to separate nucleic acid from other substances in the presence of chaotropic salts solution.

3. Diatomaceous Earth

which is also known as kieselguhr or diatomite, has silica content as high as 94%. It has been used for filtration and in chromatography and it is useful for the purification of plasmid and other DNA by immobilizing DNA onto its particles in the presence of a chaotropic agent. The resulting diatomaceous earth-bound DNA is then washed with an alcohol-containing buffer. The alcohol–containing buffer is then discarded and DNA is eluted out in a low salt buffer or in distilled water. 

4. Magnetic Bead Based Nucleic Acid Purification

Magnetic separation is a simple and efficientway which is used in purification of nucleic acid nowadays. Many magnetic carriers are now commercially available. Particles having a magnetic charge may be removed by using a permanent magnet in the application of a magnetic field. Often, magnetic carriers with immobilized affinity ligands or prepared from biopolymer showing affinity to the target nucleic acid are used for the isolation process. magnetic particles that are produced from different synthetic polymers, biopolymers, porous glass or magnetic particles based on inorganic magnetic materials such as surface-modified iron oxide.The nucleic acid binding process may be assisted by the nucleic acid “wrapping around” the support. A magnet can be applied to the side of the vessel, which contains the sample mixture for aggregating the particles near the wall of the vessel and pouring away the remainder of the sample. 

Magnetic oligo (dT) bead is an alternative to other oligo (dT) matrices for the purification of poly(A)+ RNA from total RNA sample. The poly(A)+ RNA can be extracted by introducing magnetic beads coated with oligo (dT). RNA with a poly-A tail attached to the oligo (dT). The beads will then be drawn to the bottom of a tube removing mRNA directly from total RNA. The magnetic beads which are specially treated minimize the nonspecific binding of other nucleic acids and ensure the purity of mRNA. 

5. Anion-Exchange Material

Anion exchange resin is one of the popular examples that utilized the anion-exchange principle. It is based on the interaction between pos­itively charged diethylaminoethyl cellulose (DEAE) groups on the resin’s surface and negatively charged phosphates of the DNA backbone.The anion-exchange resin consists of defined silica beads with a large pore size, a hydrophilic surface coating and has a high charge density. The large surface area of resin allows dense coupling of the DEAE groups. The resin works over a wide range of pH conditions (pH 6–9) and/or salt concentration (0.1–1.6 M) which can optimize the separation of DNA from RNA and other impurities. Therefore, salt concentration and pH conditions of the buffers are one of the main factors that determine whether nucleic acid is bound or eluted out from the column.DNA can bind to the DEAE group over a wide range of salt concentration. Impurities such as protein and RNA are washed from the resin by using medium-salt buffers, while DNA remains bound until eluted with a high-salt buffer.