INTRODUCTION

The
genetic coding called DNA manage to show information that required in
diagnostics purpose such as medicine which is the latter application and in
scientific used , scientist used it for introduction of DNA into cells and
animal or plants . Other than that, in forensic, DNA isolation helps in
identification of individuals and also crucial for heredity. The type of DNA
can be divided to genomic and plasmid. “a successful nucleic acid purification
required four important steps such effective disruption of cells or tissue,denaturation
of nucleoprotein complexes, inactivation of nucleases and DNA extraction away
from contamination” (Doyle, 1996). Isolation of source
of DNA need to be specify as the method and chemical used for each organisms is
different from another depend on its source as it have different cell
composition and structure, size and age DNA isolation target is to separate DNA
located in the nucleus from the cellular components. The source of DNA can be extracted
from living or dead organisms such as blood, bones, bacteria, plant tissue and others.

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The
first process would be lysis which cell composition gone through mechanical
disruption. As the result, the cell and it is broken and open up release
nucleic acids in the form of DNA. The first way is using a tissue homogenizer
which particularly using mortar and pestle. This way is more likely to use in
DNA extraction of plant and insect as the organisms comes in a big size and
have a tough cell wall .Then, lysis also can undergo by using detergent and
enzyme’s to unrestricted DNA and dissolve contaminating protein .It is
necessary to separate DNA from the contaminating protein and cellular debris as
enzyme which is proteinase k will degrade it. This process also required for
protein denaturation and removal. “When lipids, proteins, polysaccharides and other
impurities in the DNA preparation were present, it will interfere the DNA
analysis methods by reducing the quality of DNA” (vlab.amrita.edu, 2017)

DNA
is still mix up with cell parts so the precipitating the DNA with alcohol is
needed by adding ice-cold ethanol or isopropanol .At First, “Na+ ions  neutralize negative charges on the DNA
molecules to makes them more stable and less water soluble” (The Basics of
DNA Extraction, 2017).DNA will precipitate in aqueous as it
is not soluble in alcohol and visible in form of stringy white precipitate .Purification
follow up is DNA pallet rinsed with alcohol to remove unwanted material and
cellular debris. At this point the purified DNA is re-dissolved in water..
Lastly, the whole process can be conclude by confirmation of the presence and
quality of the DNA. The analysis process can be test by Agarose gel
electrophoresis and nanodrop test to identify the present and the quality of
DNA sample from any source.

CONTENT

Each
organisms have its own structure, composition, size and age. This would affect
the procedure and method of DNA extraction as the organisms required different
chemical to undergo reaction. Some chemical cannot digest or degrade certain
composition of cell due to its characteristic but certain chemical can be used
in all type of organisms as the chemical can react and show the good result. This
chemical divide into three purpose which is to dissolve the cellular membranes,
inactivation of DNase and RNase and assist in the removal of the contaminants. Through
the purpose, CTAB or Cetyl trimethylammonium bromide use in source like animal,
plant and soil and water as in soil and water contain microbe like bacteria and
others .CTAB is a surfactant and has two different kind of moiety which included
hydrophobic and a hydrophilic. This help in formation of micelles.

 The plasma membrane has similar structure to
the CTAB molecule which assist in breaking the plasma membrane because CTAB
molecule can pull out the phospholipids and incorporate themselves but in
specific CTAB most likely to use for plant as it effectively remove
polysaccharides and polyphenols this is because plant have cell wall composed
with cellulose and complex polysaccharides while SDS (Sodium dodecyl sulphate)
purpose is the same as CTAB but majorly use in animal and microbe .Plant also
have high contain of RNA and metabolic. Other than that, CTAB/NaCl buffer which
composed of the mix CTAB and sodium chloride (salts) use in plant and animal to
disturb the cell membrane by binding to the lipids. Then trap them into micelles
and NaCl, will bind to the backbone of DNA to neutralize the charges. The
changes of polarity makes the DNA insoluble in water.

 PVP or polyvinyl pyrrolidone included in
extraction of animal and plant use to minimize the effect of metabolites and also
help in remove phenolic substances by forming hydrogen bonds. Next, EDTA and
Tris EDTA buffer usually use in all types of organisms but in animal only use
Tris EDTA. TE buffer helps in solubilize DNA or RNA. It is also protecting it
from degradation. EDTA main function is to lyse the outer
membrane of the cell.In contrast it will not the nuclear membrane and if Mg+
binds to it will inactivate the nucleuses . “DNA extraction methods generally
use EDTA to chelate divalent cations, through inhibiting nuclease activity” (Panyutin I.G. and Hsieh,P., 1994), and will be lost
during standard DNA extraction procedures.

 

Other
than that, Tris-HCl buffer contain mix of Tris buffer and HCL helps in breaking
upon the cell of most types of source like TE buffer as most lysis buffers
contain salts that regulate the acidity and osmolarity of the lysate. Mercaptoethanol
chemical use only in animal as the function is to helps in denaturing proteins
by breaking the disulphide bonds between the cysteine residues and for removing
the tannins and polyphenols present in the crude extract while ?-
Mercaptoethanol use in plant that have strong reducing agents characteristic which
will remove tannins and other polyphenol often see in the crude plant extract.
Next, the function of ascorbic acids in plant DNA extraction is to “decreased
pH of extraction buffer while inhibit the activity of polyphenol oxidase that
will converts colourless polyphenols to coloured black-brown compounds tannins
and melanin” (Tushar BORSE, Prashant JOSHI and
Sushama CHAPHALKAR, 2011) while proteinase k in form of enzyme use
to breakdown the contaminating protein in animal and microbe and inactivates
nucleases rapidly.

This
is to avoid degrade DNA or RNA to occurs in purification. As in cold ethanol
and cold isopropanol undergo the same purpose which increases the yield of DNA.
Low temperatures protect the DNA by slowing down the activity of enzymes that
could break it apart and precipitate it quickly. DNA is insoluble in these
alcohols, therefore it will aggregate DNA by giving a white pellet after being
centrifuge. In addition ,the function of phenol , chloroform/isoamyl and
isoamyl is based on theory phenol is a weak acid helps to equilibrated with
buffer to bring the pH into optimum PH either acidic for RNA purification or
slightly alkaline for DNA purification and also assist in the extraction to
dissolved phenol while aids denature proteins in the aqueous solution.

The
buffer makes saturated phenol gain a density that slightly higher than water. As
for chloroform/isoamyl will ” increases the overall density of that phase,
helping to prevent phase inversion and  it’s
also reduce the interphase.The adding chloroform to the mix helps reduce the
fuzzy border between the two phases that composed of the compound” (plank, 2010) that cannot
determine the phase which is in protein and DNA .Then chlorofoam itself will “extract
the nucleic acid solution by aside washing with a volume of phenol” (Gaikwad) . Chloroform “forces
a separation of the organic and aqueous phases and assist the removal of aqueous
phase with minimum cross contamination from the organic phase”. Finally the
function of sodium acetate and potassium acetate is to increase of ionic
strength while as a salt for precipitation of DNA, potassium acetate
precipitates sodium dodecyl sulphate (SDS) and SDS-bound proteins to allow
their removal from DNA.

CONCLUSION

As
a conclusion, DNA extraction for every source is different in term of chemical
usage in the procedure. Each chemical have its own function in order to extract
the DNA as the source such as microbe have lipid bilayer outer membrane and a
cytoplasm containing a circular chromosome. It is also have proteins, inorganic
salts , metal ions, sugar molecules, and other cell element .while animal have
lipid bilayer outer membrane and cytoplasm containing proteins, sugars, lipids,
and inorganic ions of various types and function. Lastly ,All DNA extraction
procedure include the basic steps of disruption of the cell wall, cell membrane
and nuclear membrane to release the DNA into solution. Then it is “followed by
precipitation of DNA while protect removal of the contaminating biomolecules
such as the proteins, polysaccharides, lipids, phenols and other secondary
metabolites” (Gaikwad)