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21 views27 pages

Lab - 1

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herojm10
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DNA EXTRACTION

Molecular Biology
4th stage
MLT Department
Molecular biology: is the branch of biology that concerns the molecular basis of
biological activity in and between cells, including molecular synthesis,
modification, mechanisms and interactions. The central dogma of molecular
biology describes the process in which DNA is transcribed into RNA, then
translated into protein.
DNA Extraction

• DNA extraction is a procedure used to isolate DNA from


the nucleus of cells.
• Purpose of DNA Extraction
To obtain DNA in a relatively purified form which can be
used for further investigations, i.e. PCR, sequencing, etc
DNA Analysis

Downstream techniques can:


http://www.biocompare.com/itemdetails.asp?itemid=687320

* Reveal how organisms are related

* Identify cryptic species

* Locate mutations in DNA

http://www.cdc.gov/hemochromatosis/training/images/dna.jpg
➢DNA extraction is used to isolate…
Mitochondrial DNA
Genomic DNA

➢DNA can be extracted from almost


any intact cellular tissue
Skin,
blood,
saliva,
semen,
mucus,
muscle tissue, http://www.davidkfaux.org/shetlandislandsmtDNA.html
bone marrow, etc.

http://faculty.uca.edu/~benw/biol1400/pictures/
Basic steps forDNA extraction

1.Breaking the cells open, commonly


referred to as cell disruption or cell
lysis, to expose the DNA within. This is
commonly achieved by grinding or
treating the sample with lysis buffer .
Purposes of theExtraction Buffer

1 2

1. Dissolve cellular 2. Assist in the removal of


membranes contaminants
Basic steps for
DNA extraction
2. Removing
membrane lipids
by adding a
detergent.
Basic steps for DNAextraction
3.Removing proteins by adding a protease (optional but
almost always done).

4. Precipitating the DNA with an alcohol — usually ice-cold


ethanol or isopropanol. Since DNA is insoluble in these
alcohols, it will aggregate together, giving a pellet upon
centrifugation. This step also removes alcohol-soluble salt.
Most Commonly used DNA Extraction
Procedures

A. Organic (Phenol-Chloroform) Extraction


B. Inorganic (Proteinase K and Salting out)
C. NCM / Nylon membrane i.e. kit method(Collection,
Storage, and Isolation)

NCM: nitrocellulose membrane


A. ORGANIC EXTRACTION REAGENTS
• Cell Lysis Buffer - Non-ionic detergent ,Salt, Buffer, EDTA
designed to lyse outer cell membrane, but will not break
down nuclear membrane.
•EDTA (Ethylenediaminetetraacetic disodium salt) is a
chelating agent of divalent cations such as Mg2+. Mg2+is a
cofactor for DNase nucleases. If the Mg2+is bound up by
EDTA, nucleases are inactivated.
ORGANIC EXTRACTION REAGENTS
• Proteinase K - it is usual to remove most of the protein by
digesting with proteolytic enzymes such as proteinase K,
which are active against a broad spectrum of native proteins,
before extracting with organic solvents. Proteinase K is
approximately 10 fold more active on denatured protein.
Proteins can be denatured by sodium dodecyl sulfate (SDS)
or by heat.
• Phenol/Chloroform- remove proteinaceous material.
• TE Buffer - Tris-EDTA Buffer: TE buffer is often used to store DNA and
RNA. EDTA in TE chelates Mg2+ and other divalent metals ions
necessary for most causes of DNA and RNA degradation, suppressing
these processes. Tris is a buffering agent to keep the solution at a
defined pH.
• Ethanol or isopropanol: which help in precipitation of DNA.
ORGANIC EXTRACTION
PROCEDURE
1.Cell Lysis Buffer – This
buffer will lyse cell
membrane, nuclei are
intact, pellet nuclei.
2.The nuclei is
Resuspended in a buffer
containing Sodium
Dodecyl Sulfate (SDS) and
Proteinase K. This will
Lyse nuclear membrane
and digest protein.
3. DNA released into
solution is extracted
with phenol-
chloroform to remove
proteinaceous
material.
4.DNA is precipitated from the aqueous layer by the
addition of ice cold 95% ethanol and salt.
5.Precipitated DNA is washed with 70% ethanol, dried
under vacuum and resuspended in TE buffer.
INORGANIC DNA EXTRACTION

➢ Does not use organic reagents such as phenol or chloroform.


➢Digested proteins are removed by salting out with high
concentrations of Lithium chloride (LiCl).
➢However, if salts are not adequately removed, problems could occur
with the procedure due to alteration of DNA mobility (band shifting)
(later on the gel).
INORGANIC DNA EXTRACTION
Procedure:
1. Cell Lysis Buffer Containing Proteinase - lyse cell membrane, lyse
nuclear membrane and digest protein at high temperature e.g. 65℃
for 2 hours. Temperature helps denature proteins, and Proteinase K
auto digests itself.
2. To remove proteinaceous material, Lithium chloride (LiCl) is added
to a final concentration of 2.5 M, and incubated on ice. Proteins
precipitate out and are pelleted by centrifugation.
3. DNA remains in solution. Transfer supernatant to a new
tube, care must be taken not to take any of protein pellet.
4. DNA is precipitated by the addition of room temperature
isopropanol. LiCl will not precipitate with DNA.
5. Precipitated DNA is washed with 70% ethanol, dried under
vacuum and resuspended in TE buffer.
C. Membrane based
method(i.e. kit
method)
• A spin column using a
silica-based extraction
method is used. This
does not require the use
of hazardous chemicals.
Nucleic acids are
attracted to the silica
bead under high
chaotropic salt
concentrations.

Chaotropic agents are cosolutes that can disrupt the hydrogen bonding network between water
molecules and reduce the stability of the native state of proteins by weakening the hydrophobic
effect.
STEPS
• The stages of the method are:

• Lyse – The cells of a sample are broken open with a lysis procedure.
• Bind – A buffer solution is then added to the sample along with
ethanol or isopropanol. This forms the binding solution. The binding
solution is transferred to a spin column and the column is put in a
centrifuge. The centrifuge forces the binding solution through a silica
gel membrane that is inside the spin column. If the pH and salt
concentration of the binding solution are optimal, the nucleic acid will
bind to the silica gel membrane as the solution passes through.
STEPS
• Wash – The flow-through is removed and a wash buffer is added to
the column. The column is put in a centrifuge again, forcing the wash
buffer through the membrane. This removes any remaining impurities
from the membrane, leaving only the nucleic acid bound to the silica
gel.
• Elute – The wash buffer is removed and an elution buffer (or simply
water) is added to the column. The column is put in a centrifuge
again, forcing the elution buffer through the membrane. The elution
buffer removes the nucleic acid from the membrane and the nucleic
acid is collected from the bottom of the column.
OVERVIEW
• The lysate is combined with alcohol and
placed into the spin column, which is
inserted into a tube.
• The removal of proteins and divalent
cations is accomplished using multiple
buffer washes and centrifugation steps.
• Removal of cations, such as Mg2+,
prevents nucleases from further
degrading the DNA.
• Pure DNA is eluted from the membrane
into sterile water or TE buffer.

Cations: Positive ions

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