TO THE DNA ISOLATION METHOD
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 such as 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
DNA isolation consists of five steps namely lysis; breaking open the cells to
release nucleic acid, DNA isolation; separation of DNA from proteins and other
cellular debris, DNA precipitation 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). The first method is the
organic method, where different of phenol or chloroform is used. This method
includes many liquid chemical process but a high and clean DNA sample is
extracted. Next is inorganic Chelex or silica method. This method is low cost and
simple as it uses one-tube extraction process where single-stranded DNA is
yield when resin beads are bind with Mg2+.
The third method is called the solid phase extraction. It is a simple
extraction method where DNA binds to paramagnetic or silica beads. Lastly is
the differential extraction method. It is a method that used DTT to separate
the other cells from sperm cell. It is widely used to analyse biological evidence
from sexual assault cases (Drobnic, 2003)
ROLE OF CHEMICAL USED AND FOR DIFFERENT ORGANISMS
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 present of cell wall.
The Extraction Buffer
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. Function of Sodium chloride, NaCl is to neutralize the
negative charges on DNA to make easier for DNA molecules to cluster together. NaCl is in both EBA and EBB buffer solution. Cloudy
solution is produced if there are a higher concentration Na+ ions with the adding
of alcohol. DNA will be less precipitation if there are less concentration solutions.
solution usually contains contaminants that are mainly made up of protein. To
purify it, Phenol-chloroform extraction is used. Firstly, the solution of
nuclei acid is isolated by continuously washing it with a volume of phenol
followed with the ratio of 25:24:21of phenol: chloroform: isoamyl alcohol and
lastly with the ratio 24:1 of chloroform: isoamyl alcohol
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
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.
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.
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.
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.
sample uses TE buffer to resuspended the nucleic acid pellet. TE solution is
also used to solubilize DNA while protecting it from degradation.
37° C, RNase A (10mg/ml) is used to incubate with nuclei acid solution. This step
is followed with using phenol: chloroform extraction to remove the RNase for
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, when unwound, magnesium will stabilize it.
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
DNA isolation is very
helpful in many areas such as body and species identification, forensic
evidence analysis and also the study of cancer gene.