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.

 

 

 

Phenol-Chloroform
Extraction

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.

Resuspending
DNA

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.

 

CONCLUSION

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.