INTRODUCTIONTO THE DNA ISOLATION METHOD Deoxyribonucleicacid (DNA) isolation is a type of DNA purification method that combines theusage of physical and chemical methods to obtain pure DNA molecules fromvarious sample cells. Thefirst DNA extraction was made by a Swiss doctor named, Friedrich Miescher in theyear 1868, where he found some precipitate, which now known as DNA, was formedwhen he performed experiments to understand the chemical compositions ofleucocytes (Dahm, 2007). DNA extractions are always done along with gelelectrophoresis to observe the DNA bands of the sample DNA.
The DNA bands showthe fragments of DNA, which the more intense the bands shown, the larger thefragment of DNA is observed.A pure sample of DNA can beused to detect genetic disease in newborn, analyze forensic evidence found atcrime scene, aid in the identification of body (war victim, rapist, etc.) andorganisms such as plant and animal species.
For different living tissuessuch as plants, animal, and microorganisms, there are different method of DNAextraction. It also depends on the age and size of the sample. Ultimately, theaim is to separate DNA in the nucleus of the cell from other componentspresent.
Mainly,DNA isolation consists of five steps namely lysis; breaking open the cells torelease nucleic acid, DNA isolation; separation from DNA from proteins andother cellular debris, precipitation of DNA with alcohol, DNA purification andlastly analysis of quality and quantity (Boom et al., 1990). There are 4 DNA extractionmethods that are commonly used (Hoff-Olsen et al., 1999):1. Organic (variations of phenol/chloroform) – havemany liquid chemical processes but produces a high yield and clean extractedDNA sample.2. Inorganic Chelex or silica method – simple and lowcost that uses one-tube extraction process where Mg2+ bindsto resin beads and yields a single-stranded DNA product.3.
Solid phase extraction methods – simpleextraction 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 – processwith many steps used to separate sperm from other cells using DTT; to analysebiological evidence from sexual assault cases (Drobnic, 2003)ROLE OF CHEMICAL USED AND FOR DIFFERENT ORGANISMS Allspecimen is disrupted by mechanical force, using pestle and mortar. Lysis iscarried out in a salt solution containing detergent.
Both cellular membranesand detergent have amphipathic characteristic; having both hydrophilic andhydrophobic region and due to this, detergents are able to break apart themembrane. The isolation ofnucleic acids from plant tissues differs from methods used for animal andmicrobial specimens due to the cellular structure of plant material.The Extraction BufferTheselection of buffer for the initial cellular structure rupture depends largelyon the tissue type. The general function of buffer solution is to dissolvecellular membrane, deactivation of DNase and RNase and to assist in the removalof the contaminants. Plants have cell walls comprised mostly of celluloseand complex polysaccaharide and high content of RNA and secondary metabolite(Porebski et al., 1997). Buffer solutions for plant DNA isolation areExtraction Buffer A (EBA), Extraction Buffer B (EBB) and sodium dodecylsulphate (SDS). Extraction Buffer A (EBA) contain hexadecyltrimethylammoniumbromide (CTAB) that helps to remove membrane lipids and promote cell lysis.
Tris removes the polysaccharides on cell membrane, and facilitates in increasemembrane permeability, while maintaining pH stability of the solution.Polyvinylpyrrolidone (PVP) in EBA helps in removing phenolic compounds in plantcells, ?-mercaptoethanol and ascorbic acid also help in removing polyphenols inthe plant extract. ?-mercaptoethanol is also a strong reducing agent thatdenature proteins of the cells by breaking the disulphide bonds. Sodium dodecylsulphate (SDS) removes excess lipid membranes and DNA associated proteins, alsothe cellular proteins to purify the isolated DNA. The function of EDTA is actsas chelating agents and chelates the magnesium ions. Magnesium ion are neededfor DNase activity.
Sodium chloride, NaCl neutralize the negative charges onDNA so that molecules can come together. NaCl is in both EBA and EBB buffersolution. Higher concentrations of Na+ ions produce a cloudy solution onaddition of alcohol. Less concentrated solutions caused less DNA to precipitate.
Phenol-ChloroformExtractionDNAsolution usually contains contaminants that are mainly made up of protein. Topurify it, Phenol-chloroform extraction is used. Firstly, the nuclei acidsolution is isolated by washing it continuously with a certain volume of phenolfollowed by phenol: chloroform: isoamyl alcohol with the ratio of 25:24:21 andlastly chloroform: isoamyl alcohol with the ratio 24:1Aftercentrifudge process, the content will be separated by three layers, namelyaqueous phase, interphase and organic phase. Denatured contaminants will beaccumulated at the organic phase and interphase phase while DNA molecules arepreserved in the aqueous phase. Chloroform and phenol acts as protein denaturantand are able to denatures proteins and dissolves denatured proteinsLysisbuffer that contain detergent and proteinase K is used for DNA extraction ofleech, water and soil samples. This helps them to release their DNA whereasmixture of carbohydrae enzymes is used to digest the cell wall of plant sample.Proteinase K helps to digest contaminating proteins because during theisolation of DNA or nucleic acids in general, there are a lot of contaminatingprotein presents and they must be removed. PrecipitationNuclei AcidThemost common way used is by alcohol precipitation.
Monovalent salt is neededbecause the nuclei acid will be diluted in it, followed by addition of alcoholand gently mixed. Precipitation of nuclei acid is spontaneous and throughcentrifugation, pellet will form. Supernatant will be removed after. 70%ethanol is used to wash the remaining of salt and alcohol. Saltwill interrupt the hydrogen bond between water and DNA molecules. sodium acetatepH 5.2 (plant specimen), sodium chloride (soil and water sample), ammoniumacetate, lithium chloride and potassium chloride are some type of common saltused.
Sodium acetate and potassium acetate helps in precipitate the proteinsfully away from DNA to prevent the proteins bound to the DNA again.Thenext step is by adding cold isopropanol or ethanol. This is because when thereis presence of cations, ethanol will induce a structure change in DNA moleculesthat can cause them to aggregate.
Absolute isopropanol and 70% ethanol are usedto concentrate and de-salting DNA in aqueous solution because DNA is notsoluble in both substances, thus is easier for DNA to precipitate in alcoholicsolution. 70% ethanol is used to dissolve excess salt while preserving DNA. Coldtemperature is used to inhibit DNA enzymes activity. ResuspendingDNAAllsample uses TE buffer to resuspended the nucleic acid pellet. TE solution isalso used to solubilize DNA while protecting it from degradation.
Purificationof DNA The DNA is purified by incubating the nucleicacid solution with RNase A (10mg/ml) at 37° C and reprecipitation followingphenol: chloroform extraction to remove the RNase. CONCLUSIONIn conclusion, we knowthat DNA extraction is the isolation of nucleic acid from a cell taken fromdifferent organisms such as human, plant, animal or microorganisms. Sample thatdiffers in size, source and age have different method of DNA isolation. Beingcareful in handling the biological materials is important to ensure that thesample will not be crossover or contaminated during DNA extraction process, DNA isolation processrequires careful handling of biological materials so that the sample will notbe contaminant or crossover. Due to phosphate groups in nuclei acid, DNA ishighly negatively charged, so it is stabilized by magnesium in cell whenunwound.
There is 4 commontechnique 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 manysamples. These techniques have to be properly selected so that the quality ofthe DNA extracted in optimized.
In conventional method,different sample had different buffer solution of the lysis process becausethey have different structure of cell. It is important to know the function ofeach buffer solutions, it can help a better understanding of how the DNAextraction process happen. It is because if there is error in adding buffersolution, DNA might not be extracted properly causes no result during AGEprocess. DNA isolation is veryhelpful in many areas such as body and species identification, forensicevidence analysis and also the study of cancer gene.