INTRODUCTION TO THE DNA ISOLATION METHOD
Deoxyribonucleic acid (DNA) isolation is a type of DNA purification method that combines the usage of physical and chemical methods to obtain pure DNA molecules from various sample cells. The first DNA extraction was made by a Swiss doctor named, Friedrich Miescher in the year 1868, where he found some precipitate, which now known as DNA, was formed when he performed experiments to understand the chemical compositions of leucocytes (Dahm, 2007). DNA extractions are always done along with gel electrophoresis to observe the DNA bands of the sample DNA. The DNA bands show the fragments of DNA, which the more intense the bands shown, the larger the fragment of DNA is observed.
A pure sample of DNA can be used to detect genetic disease in newborn, analyze forensic evidence found at crime scene, aid in the identification of body (war victim, rapist, etc.) and organisms such as plant and animal species.
For different living tissues such as plants, animal, and microorganisms, there are different method of DNA extraction. It also depends on the age and size of the sample. Ultimately, the aim is to separate DNA in the nucleus of the cell from other components present.
Mainly, DNA isolation consists of five steps namely lysis; breaking open the cells to release nucleic acid, DNA isolation; separation from DNA from proteins and other cellular debris, precipitation of DNA with alcohol, DNA purification and lastly analysis of quality and quantity (Boom et al., 1990).
There are 4 DNA extraction methods that are commonly used (Hoff-Olsen et al., 1999):
1. Organic (variations of phenol/chloroform) – have many liquid chemical processes but produces a high yield and clean extracted DNA sample.
2. Inorganic Chelex or silica method – simple and low cost that uses one-tube extraction process where Mg2+ binds to resin beads and yields a single-stranded DNA product.
3. Solid phase extraction methods – simple extraction process in which the DNA binds to paramagnetic or silica beads. (e.g., Promega’s DNA IQ (Eminovic et al., 2005, DNA IQ manual)
4. Differential extraction – process with many steps used to separate sperm from other cells using DTT; to analyse biological evidence from sexual assault cases (Drobnic, 2003)
ROLE OF CHEMICAL USED AND FOR DIFFERENT ORGANISMS
All specimen is disrupted by mechanical force, using pestle and mortar. Lysis is carried out in a salt solution containing detergent. Both cellular membranes and detergent have amphipathic characteristic; having both hydrophilic and hydrophobic region and due to this, detergents are able to break apart the membrane. The isolation of nucleic acids from plant tissues differs from methods used for animal and microbial specimens due to the cellular structure of plant material.
The Extraction Buffer
The selection of buffer for the initial cellular structure rupture depends largely on the tissue type. The general function of buffer solution is to dissolve cellular membrane, deactivation of DNase and RNase and to assist in the removal of the contaminants.
Plants have cell walls comprised mostly of cellulose and complex polysaccaharide and high content of RNA and secondary metabolite (Porebski et al., 1997). Buffer solutions for plant DNA isolation are Extraction Buffer A (EBA), Extraction Buffer B (EBB) and sodium dodecyl sulphate (SDS). Extraction Buffer A (EBA) contain hexadecyltrimethylammonium bromide (CTAB) that helps to remove membrane lipids and promote cell lysis. Tris removes the polysaccharides on cell membrane, and facilitates in increase membrane permeability, while maintaining pH stability of the solution. Polyvinylpyrrolidone (PVP) in EBA helps in removing phenolic compounds in plant cells, ?-mercaptoethanol and ascorbic acid also help in removing polyphenols in the plant extract. ?-mercaptoethanol is also a strong reducing agent that denature proteins of the cells by breaking the disulphide bonds. Sodium dodecyl sulphate (SDS) removes excess lipid membranes and DNA associated proteins, also the cellular proteins to purify the isolated DNA. The function of EDTA is acts as chelating agents and chelates the magnesium ions. Magnesium ion are needed for DNase activity. Sodium chloride, NaCl neutralize the negative charges on DNA so that molecules can come together. NaCl is in both EBA and EBB buffer solution. Higher concentrations of Na+ ions produce a cloudy solution on addition of alcohol. Less concentrated solutions caused less DNA to precipitate.
DNA solution usually contains contaminants that are mainly made up of protein. To purify it, Phenol-chloroform extraction is used. Firstly, the nuclei acid solution is isolated by washing it continuously with a certain volume of phenol followed by phenol: chloroform: isoamyl alcohol with the ratio of 25:24:21 and lastly chloroform: isoamyl alcohol with the ratio 24:1
After centrifudge process, the content will be separated by three layers, namely aqueous phase, interphase and organic phase. Denatured contaminants will be accumulated at the organic phase and interphase phase while DNA molecules are preserved in the aqueous phase. Chloroform and phenol acts as protein denaturant and are able to denatures proteins and dissolves denatured proteins
Lysis buffer that contain detergent and proteinase K is used for DNA extraction of leech, water and soil samples. This helps them to release their DNA whereas mixture of carbohydrae enzymes is used to digest the cell wall of plant sample. Proteinase K helps to digest contaminating proteins because during the isolation of DNA or nucleic acids in general, there are a lot of contaminating protein presents and they must be removed.
Precipitation Nuclei Acid
The most common way used is by alcohol precipitation. Monovalent salt is needed because the nuclei acid will be diluted in it, followed by addition of alcohol and gently mixed. Precipitation of nuclei acid is spontaneous and through centrifugation, pellet will form. Supernatant will be removed after. 70% ethanol is used to wash the remaining of salt and alcohol.
Salt will interrupt the hydrogen bond between water and DNA molecules. sodium acetate pH 5.2 (plant specimen), sodium chloride (soil and water sample), ammonium acetate, lithium chloride and potassium chloride are some type of common salt used. Sodium acetate and potassium acetate helps in precipitate the proteins fully away from DNA to prevent the proteins bound to the DNA again.
The next step is by adding cold isopropanol or ethanol. This is because when there is presence of cations, ethanol will induce a structure change in DNA molecules that can cause them to aggregate. Absolute isopropanol and 70% ethanol are used to concentrate and de-salting DNA in aqueous solution because DNA is not soluble in both substances, thus is easier for DNA to precipitate in alcoholic solution. 70% ethanol is used to dissolve excess salt while preserving DNA. Cold temperature is used to inhibit DNA enzymes activity.
All sample uses TE buffer to resuspended the nucleic acid pellet. TE solution is also used to solubilize DNA while protecting it from degradation.
Purification of DNA
The DNA is purified by incubating the nucleic acid solution with RNase A (10mg/ml) at 37° C and reprecipitation following phenol: chloroform extraction to remove the RNase.
In conclusion, we know that DNA extraction is the isolation of nucleic acid from a cell taken from different organisms such as human, plant, animal or microorganisms. Sample that differs in size, source and age have different method of DNA isolation. Being careful in handling the biological materials is important to ensure that the sample will not be crossover or contaminated during DNA extraction process,
DNA isolation process requires careful handling of biological materials so that the sample will not be contaminant or crossover. Due to phosphate groups in nuclei acid, DNA is highly negatively charged, so it is stabilized by magnesium in cell when unwound.
There is 4 common technique used in DNA extraction procedures, namely the organic method, inorganic Chelex or silica, solid phase extraction method and differential extraction. All these methods had been successfully used in many laboratories with many samples. These techniques have to be properly selected so that the quality of the DNA extracted in optimized.
In conventional method, different sample had different buffer solution of the lysis process because they have different structure of cell. It is important to know the function of each buffer solutions, it can help a better understanding of how the DNA extraction process happen. It is because if there is error in adding buffer solution, DNA might not be extracted properly causes no result during AGE process.
DNA isolation is very helpful in many areas such as body and species identification, forensic evidence analysis and also the study of cancer gene.