Introduction the cells to release nucleic acid, separation

Introduction to the DNA isolation method

DNA molecules in a cell can be extracted or isolated for various
aims such as DNA amplification and analysis via electrophoresis. DNA isolation
was done in order to separate DNA from other materials such as proteins, fats,
and carbohydrates. DNA isolation involve breaking open the cells to release
nucleic acid, separation of DNA from proteins and other cellular debris,
precipitation of DNA with alcohol, purification of DNA and analysis of quality
and quantity. There are several things to note in the process of DNA isolation,
DNA must produce without any contaminants such as proteins and RNA; the method
should be effective and applicable to all species of the method performed
should not alter the structure and function of DNA molecules; and the method
should be simple and fast.

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The first step of DNA isolation is disruption of cellular structure
to form a lysate. Lysis is carried out in a salt solution containing detergents
to denature proteins. The selection of buffer for the initial cellular
structure rupture depends largely on the tissue type. The isolation of nucleic
acids from plant and algae tissues differs from methods used for animal and
microbial specimens due to the cellular structure of plant material. Plants and
macroalgae have cell walls comprised mostly with cellulose, complex
polysaccharide and high content of RNA. The disruption of cells or cell lysis
to expose the DNA within is achieved by grinding, sonicating or treating the
samples with lysis/extraction buffer.

After the lysis step, the lysate is subjected to separation of
soluble DNA from cell debris and other insoluble material and purification of
the DNA from soluble proteins and other nucleic acids. The proteins are
denatured by the organic mixture during the separation step, hence DNA is
retained in the aqueous layer and organic solvent is at the bottom of the tube.
Ethanol or isopropanol is added to the separated upper layer supernatant
obtained in separation step. A precipitate will visible when the alcohol layer
is stirred gently. The quality and quantity of DNA could be assessed by Agarose
gel electrophoresis (AGE).




Role of chemicals used and for different organism type

            First, the chemical used for DNA isolation from leech (annelida)
and insect is lysis buffer (50 ml). The contents in the buffer is 1% Cetyl
trimethylammonium bromide (CTAB), 5% (w/v) PVP (Polyvinyl Pyrrolidone), 1.4 M
NaCl and 20 Mm 2-Mecaptoethanol (use 14.3 M stock). This buffer is used for the
aim of breaking open cells. Second, the chemical used is Tris EDTA buffer (100
ml). This buffer contains 10 Mm Tris HCL (pH 8.0) (use 1 M stock) and 1 Mm EDTA
(use 0.5 M stock). The function is to solubilize DNA or RNA and protecting it
from degradation.

The other reagents are 70% ethanol, proteinase K, isopropanol and
phenol: chloroform: isoamyl alcohol. First, the 70% ethanol is used to rinse
the DNA pellet. The function is to remove some of the salts from the pellet. Second,
function proteinase K is to remove or digest contaminating protein present in
the solution to its component amino acids. Third, function of isopropanol is
giving a pellet upon centrifugation since DNA is insoluble in these alcohol, it
will aggregate together. Fourth, the function of phenol that adding along with
chloroform is to ensure a clear separation between aqueous and organic phases.
This is important to obtain a pure nucleic acid when aqueous phase is removed
from the solution.

Next is chemicals used for DNA isolation from plants are extraction
buffer A (EBA), extraction buffer B (EBB), TE buffer, 20% (w/v) sodium dodecyl
sulphate (SDS), potassium acetate, sodium acetate, 70% ethanol and absolute

The function of extraction buffer A (EBA) and buffer B (EBB) is
same. The function is to make sure the protein dissolve in aqueous layer so
that it is not make any precipitation in the alcohol with DNA. Third, the
function of TE buffer is same for DNA isolation from insects which is to
solubilize DNA and RNA and protecting it from degradation. Fourth, the function
of 20% sodium dodecyl sulphate (SDS) is used for cell lysis and release of cell
contents. Fifth, the function of potassium acetate is used as a salt for ethanol precipitation of DNA, potassium acetate precipitates
sodium dodecyl sulfate (SDS) and SDS-bound proteins to allow their removal from DNA. Next, the function of sodium
acetate is to
help separate plasmid DNA from chromosomal DNA. Then, 70% ethanol is used to
rinse the DNA pellet as I mention before in chemical role used for insects.
Lastly is absolute isopropanol. Absolute isopropanol is useful for
precipitations. Salts are generally less soluble in isopropanol than in ethanol
so they have more of tendancy to co-precipitate with the DNA.

Lastly is the chemical used for DNA
extraction for microbial from sand and water. The chemicals used is CTAB/NaCl, Tris EDTA (TE) buffer,
EDTA buffer 0.5M, Tris-HCl buffer 1 M, SDS buffer 10%, proteinase K,
chloroform/isoamyl alcohol and cold isopropanol.

The function of CTAB/NaCl is facilitates the separation of polysaccharides
during purification. Then, the function of Tris EDTA (TE) buffer is to
solubilize DNA or RNA and protecting it from degradation. Then, EDTA buffer is responsible for chelation of divalent ions. It stops
the action of DNases found in cytoplasm of cells. Cells and nucleus need to be
disrupted. So, DNA comes in contact with DNases present in the cytoplasm. Next
is Tris-HCl buffer. Most lysis buffer contains salts like Tris-HCl, it helps to
regulate the acidity and osmolarity of the lysate. SDS buffer, proteinase K,
chloroform/isoamyl alcohol and cold isopropanol has the same roles and
functions as before that I have mention above.



            As a
conclusion, there are many purpose of DNA isolation and also have many
different reagents. The reagents used are based on what type of sample that we
used. I learn that the basic process of isolation of DNA from various
sources such as microbe from sand and water, insects, leeches and plants. Different
types of DNA require different methods of isolation and different types of
chemicals used. Different DNA samples produced different amounts of DNA. Molecules
can interfere with DNA extraction, they bind to the DNA and prevent the DNA
from precipitating where a lot of DNA is extracted and there are fewer
interfering substances. I can conclude that DNA extraction have many
importance. It is used to diagnose many medical
conditions. It can also be used for genetic engineering of both plants and
animals. Many
agricultural companies use genetic extraction to create DNA that they then
modify to make a particular genetic strain of a crop that is resistant to
various chemicals or pests. DNA extraction can also be used to gather
evidence in a crime investigation.