The document discusses different types of DNA extraction methods. It begins with a brief history of DNA extraction, from Miescher's initial isolation of nucleic acid in 1869 to the development of the phenol-chloroform method in the late 20th century. The document then covers key aspects of the DNA extraction process, including lysing the cell wall/membrane and nuclear membrane using chemicals or enzymes. It discusses several common DNA extraction methods in detail, such as the phenol-chloroform method, enzymatic methods using proteinase K, and solid-phase extraction using silica columns. The solid-phase method is highlighted as being widely used due to its speed, ease of use, and production of high-quality DNA.

1. DIFFERENT TYPES OF DNA EXTRACTION METHODS
Specialist Biologist
Mudhafar Qader Saber

2.  Different types of DNA extraction methods are
available for different cell types. For example, the
DNA extraction method for plant DNA is different
from that of the blood.
 In this article, we are going to discuss different
types of DNA extraction methods which are mostly
used in genomic labs. We will focus on the principle
and the mechanism of each type of DNA extraction
method.

3. HISTORY OF DNA EXTRACTION
 The first DNA extraction attempt had performed by Friedrich Miescher in
1869. He had isolated the cell material and named it as the “nuclei” later
on his student named it as a “nucleic acid”. Although he accidentally
developed a method for isolation of nucleic acid, he was not sure that
what he isolated was DNA or not.
 Later on, in 1958 Meselson and Stahl developed a full-function protocol
for DNA extraction. The density gradient centrifugation protocol was the
first protocol described by isolating DNA from E.coli bacteria.
 The protocol of the proteinase K enzyme method of DNA extraction was
developed by Lahiri and Nurenberger in 1991. They also modified the
protocol by using the Nonidet P40 and SDS. However, the use
of proteinase K in DNA extraction was reported earlier by Miller et al., in
1988.
 The phenol-chloroform isoamyl alcohol method which is most popular in
recent days was developed by Joseph Sambrook and David W. Russell.
 The PCI method becomes so popular that most of the researcher using
them in their daily extraction. The yield and consistency of PCI are much
decent.

4. WHAT IS DNA EXTRACTION
 Extracting DNA from the cell” this is the simplest
explanation for DNA extraction.
 Several definitions of DNA extraction are enlisted here,
 “Isolating DNA by disrupting cell wall/cell membrane and
a nuclear membrane is called a DNA extraction”.
 “Isolation of DNA by breaking the cell membrane and
nuclear membrane with the help of chemicals, enzyme
or physical disruptions is defined as a DNA extraction”.
 Or simply, “isolating nucleic acid from rest of the cell
organelle is called nucleic acid extraction or DNA
extraction”.

5. HOW TO OBTAIN DNA
 Well, I don’t have the exact label to define this section,
but still, I want to discuss this portion with you because it
is important to understand how DNA is obtained from
the cell.
 First, we will understand the anatomy of the cell,
roughly. The cell is made up of cytoplasm and cell
membrane/ cell wall. The cytoplasm contains several
organelles such as mitochondria, ribosomes, nucleus,
endoplasmic reticulum etc.
 The animal cell does not have the cell wall, plant cell
and (most) bacterial cells contain the cell wall. I am not
going to discuss each and every component of the cell
wall or cell membrane because that is not the point of
discussion.

7. LYSIS OF CELL WALL/ CELL MEMBRANE:
 Chemical disruption
 enzymatic disruption
 Mechanical disruption
 Lysis of nuclear membrane:
 Chemical lysis
 Enzymatic lysis
 Removing cell debris
 Centrifugation


9.  The nuclear membrane and cell membrane is made up of
protein and lipids almost. Hence the same types of chemicals
can work for both.
 Chemicals such as SDS, CTAB, Tris and other detergents can
lyse the cell wall/ cell membrane by solubilizing it. Each
chemical has a different function to play which we will discuss
separately in each method.
 Enzymes such as proteinase K, peptidase, protease
disrupt proteins by digesting it. The enzyme works better than
any other chemicals because it directly targets bonds between
the amino acids and digests the protein.
 Once the cell wall or cell membrane lysed, there are no
compartments inside the cell hence all the cell organelles are
mixed into the solution. By doing high-speed centrifugation
DNA remains in the solution and the other cell debris settled
into the bottom of the tube.

10.  Moreover, the DNA extraction method varies depending
upon the type of cells. Take look at some example here,
 The cell having a soft cell wall: some of the bacteria
have a very smooth and soft cell wall. For example,
M.tuberculosis has a smooth cell wall. By only heating
the bacterial solution we can lyse cell wall.
 The supernatant can directly be used for the PCR.
Even, by putting the bacterial culture directly into the
PCR tube for 15 minutes at initial denaturation we can
directly get the good quality of result in PCR.
 However, the addition of a simple lysis buffer during
heating will increase the yield and quality of DNA.
 Our series of articles on PCR,

11.  The cell having a harder cell wall: Plant cells have pectin and other
polysaccharides present in their cell wall. This pectin protects the cell
from mechanical damage. Therefore pectin provides additional strength
to the cell wall of the plant.
 Some of the fungus, algae and bacteria also have hard cell wall for
surviving in harsh conditions. For extracting DNA from this type of cells,
we have to modify protocol with a combination of mechanical- chemical-
enzymatic method.
 Mechanical lysis is an important step in the isolation of DNA from pectin-
rich cells. For doing this mortar pestle, grinding and liquid nitrogen is
used.
 Read more on plant DNA extraction method.
 The cell having a cell membrane: The cell wall is not present in animal
cells. A combination of the enzymatic and chemical method is most
suitable for the DNA extraction from animal cells, however, each method
individually performs the best.
 The phenol-chloroform method is one of the best choices for animal
cells.

12. HOW TO DO MECHANICAL LYSIS
 ake plant tissue and grind it with a mortar and pestle.
Grind it well, and add liquid nitrogen carefully and again
grind it even harder until the tissue becomes powder.
Finally, add lysis buffer or DNA extraction buffer. Grind it
till it becomes homogeneous. The tissue is ready for
DNA extraction.
 Additionally, we can add an enzyme to this
homogenized solution and incubate it for an hour. The
combination of mechanical- chemical and enzymatic
lysis helps to extract good quality of DNA from the plant
cell. This combination most probably gives the best
result.


13. DIFFERENT TYPES OF DNA EXTRACTION METHODS

14. CHEMICAL OR SOLUTION-BASED DNA
EXTRACTION METHOD:
 Different types of organic and inorganic solutions are used in
the chemical or solution-based DNA extraction method.
 The steps of the DNA extraction remain the same in all the types
of DNA extraction methods.
 SDS, CTAB, phenol, chloroform, isoamyl alcohol, Triton X100,
guanidium thiocyanate, Tris and EDTA are several common
chemicals used in the solution based DNA extraction method.
 The solution-based ( also called chemical) DNA extraction
method is subdivided into organic solvent-based DNA
extraction and inorganic solvent-based DNA extraction.
 The organic solvent-based DNA extraction method is based on
the use of organic substances such as phenol and chloroform.
Due to the harmful nature of the phenol and chloroform, the
method is restricted.
 Nonetheless, phenol-chloroform DNA extraction method is one of
the best methods among all.
 Among the inorganic DNA extraction, two are most popular: use
of proteinase K and use of salt.

15.  The proteinase K DNA extraction method facilitates high DNA
yield but the method is time-consuming. Also, if not
maintained well in a cold chain, the proteinase K cannot be
utilized for a longer period of time.
 The lower stability of the enzyme is another major issue in this
method.
 Salting out DNA extraction method is safer than the PCI
method. The use of salts such as sodium chloride, potassium
acetate and ammonium acetate helps in the DNA extraction.
 However, the method is more aggressive in combination with
proteinase K.
 Use of the different salt in DNA extraction can increase the
yield but the purity is not good enough. We had prepared two
different salt solution in this article: phenol-chloroform DNA
extraction method.

16. PHENOL-CHLOROFORM METHOD OF DNA EXTRACTION:
 This method is one of the best methods of DNA
extraction. The yield and quality of DNA obtained by
the PCI method is very good if we perform it well.
The method is also called as a phenol-chloroform
and isoamyl alcohol, PCI method of DNA extraction.
 The major chemicals of PCI DNA extraction
methods are lysis buffer, Phenol and chloroform.

17.  The lysis buffer contains Tris, EDTA, MgCl2, NaCl, SDS, and
other salts. Here the components of lysis buffer help in lysis of
cell membrane as well as the nuclear envelope. The protein
portion of the cell denatured with the help of chloroform and
phenol which are organic in nature.
 Role of chemicals:
 Tris: DNA is pH sensitive, Tris buffer maintains the pH of the
solution. Also, it interacts with the lipopolysaccharides of the
cell membrane and makes them permeable, this will help in
lysis of the cell membrane.
 EDTA: EDTA is a chelating agent and can be used to block
DNase activity. DNase is an enzyme which lyses the DNA.
However, every enzyme required cofactor to work properly.
 The chelator EDTA blocks the activity of DNase by blocking
the cofactor binding site. It will work best in combination with
Tris.

18.  SDS: Sodium dodecyl sulphate is an anionic
detergent which helps cell membrane and nuclear
envelope to break open.
 NaCl: the Na+ ion of NaCl creates the ionic bond
with the negative charge of DNA and neutralize it. It
will help DNA comes together and protect from
denaturation.
 MgCl2: overall, it protects the DNA. MgCl2 block
the negative charge of the lipoproteins of the cell
membrane. After the lysis of cell, there is no
compartment in the cell hence it protects DNA by
mixing with other cell organelles.

19.  Phenol: it precipitates the protein impurities.
 The SDS removes the negative charges from the amino acid and disrupts the confirmation of a
protein. Therefore, the protein loses its structure and stabilized by using the SDS.
 The combination of phenol, chloroform and isoamyl alcohol helps in the removal of protein.
After centrifugation, the phenol settles in the bottom of the tube and DNA in the aqueous phase
while the denatured protein remains between both layers as a whitish cloud.
 So care must be taken to collect nucleic acid from this method. The collected nucleic acid is
precipitated with the help of chilled alcohol (isoamyl alcohol). We can add salt as well to
increase the yield of the DNA.
 Finally, the DNA is dissolved in TE buffer. Because of the use of the organic solvents, this
method is also named as an organic solvent-based DNA extraction method.
 The PCI method of DNA extraction is widely accepted. Even the forensic departments trust PCI
method rather than a Kit method. The quantity of DNA obtains by PCI method is very high. We
can obtain 800 to 900 ng of DNA with great quality.
 If we prepare all chemicals very well and perform it sincerely we can always get a good result
by this method.
 However, the amount of sample required for PCI DNA extraction is high. It is difficult to isolate
DNA from the samples such as hair and nail. Also, the purity of DNA becomes a major issue if
not performed well. Read more on Phenol chloroform DNA extraction: Basics, preparation of
chemicals and protocol

21. AN ENZYMATIC METHOD OF DNA EXTRACTION:
 Actually, this method is a combination of a salt method as well as
enzymatic method. Here the extraction buffer is used before going
further on enzymatic digestion.
 The extraction buffer composition may vary from lab to lab, however, the
major components are Tris, EDTA, NaCl, sodium lauryl and SDS. Here
phenol, chloroform or isoamyl alcohol is not used. Instead, the enzyme
proteinase K is utilized for digesting the sample.
 The sample is incubated with proteinase K for 2 hours this will digest all
the protein present inside the sample.
 Immediately after the proteinase K digestion, the sample is precipitated
by chilled alcohol. By centrifuging sample, all other cell debris are
removed. Finally, the DNA pellet is dissolved in TE buffer.
 This method of DNA extraction is rapid and easy. We can use ready to
used DNA extraction buffer. Even the yield is very high. However, the
quality of DNA is a major concern for this method.
 We had covered a whole article on this method. Read it here: Proteinase
K DNA extraction method

22. SOLID-PHASE DNA EXTRACTION METHOD:
 Nowadays all the DNA extraction kits available are
based on the unique chemistry of the solid/ liquid
phase DNA extraction.
The silica is the solid substance which binds with
DNA during purification along with it, different
solutions are used to purify the DNA.
The solid phase silica is one of them.
The main advantage of silica gel-based DNA
extraction is that it is rapid and gives “PCR ready
DNA” for the downstream applications. No
extraction or precipitation steps are required
therefore this method is superior among all.

23.  The image represents the general process of Spin column
method for DNA extraction.
 The silica-based solid-phase DNA extraction method is now
commercially available and it is most routinely used in
diagnostic laboratories. Because of its good quality DNA yield
and minimal simple operating system, it is widely accepted.
 The lysis buffer breaks the cell membrane and the nuclear
envelope. The proteinase K digests all the protein.
 In the very first step, the sample is incubated with a cell lysis
buffer or called a DNA extraction buffer.
 Along with it, a small amount of proteinase k is added to the
sample. All the other impurities are removed by centrifugation.
Here the DNA remains bounded with silica and other
impurities passes through the silica column.
 Now the DNA can be washed twice for improving the purity in
it. The aqueous phase contains the impurities are discarded
by discarding the collection tube.

24. SILICA COLUMN-BASED DNA EXTRACTION METHOD
 The silica column-based DNA extraction method is very
unique and different from other DNA extraction methods.
 In the PCI or proteinase K method we have to centrifuge
sample many times and have to collect aqueous phase
or pellets depending upon the step of extraction.
Sometimes we have to collect aqueous phase or
sometimes we have to collect pellets.
 The silica-based DNA extraction method works on the
unique chemistry of interaction between silica and DNA.
A positively charged silica particles bind with the
negatively charged DNA and hold it during
centrifugation.
 The method was first described by McCormick in 1989.
However, the idea was developed in 1979, when silica
was used in DNA purification by Vogelstein.

26.  The image represents the general process of Spin column method for DNA
extraction.
 The silica-based solid-phase DNA extraction method is now commercially available
and it is most routinely used in diagnostic laboratories. Because of its good quality
DNA yield and minimal simple operating system, it is widely accepted.
 The lysis buffer breaks the cell membrane and the nuclear envelope. The
proteinase K digests all the protein.
 In the very first step, the sample is incubated with a cell lysis buffer or called a DNA
extraction buffer.
 Along with it, a small amount of proteinase k is added to the sample. All the other
impurities are removed by centrifugation. Here the DNA remains bounded with
silica and other impurities passes through the silica column.
 Now the DNA can be washed twice for improving the purity in it. The aqueous
phase contains the impurities are discarded by discarding the collection tube.
 Finally, the DNA is dissolved into the TE buffer. The method is fast reliable,
accurate and consumes less time as compared to other methods.
 Other DNA extraction methods are DNA extraction using the anionic resins,
magnetic bead DNA extraction method and CsCl density gradient DNA extraction
method.

28. DNA EXTRACTION STEP
STEP MATERIAL REQUARIMENT
Lysis of cell wall/ cell membrane and
Lysis of nuclear membrane
Tris, MgCl2, EDTA, NaCl, SDS, CTAB,
Triton X100
Digestion of protein CTAB, SDS, phenol, chloroform,
Nonidet P40, different chaotropic, urea,
guanidium isothiocyanate, guanidium
thiocyanate, N-Lauroyl sarcosine
Precipitation of DNA Isopropanol, ethanol, methanol, NaCl,
sodium acetate
Washing of DNA Any alcohol
Dissolving DNA TE buffer, distil water