Genomic DNA Extraction from Eukaryotic Cells
Transcript of Genomic DNA Extraction from Eukaryotic Cells
Practical Molecular Biology Lab. 02 October 25, 2020
Genomic DNA Extraction from Eukaryotic Cells
Objectives (Purposes):
What is the Genomic DNA Extraction?
What is the Purpose of Genomic DNA Extraction?
How is DNA Extraction done and which type of Eukaryotic Cells you are extracted?
What are the Practical Applications of DNA Extraction?
What are the Problems within DNA Extraction?
Genomic DNA extraction:
Genomic DNA extraction is the isolation and purification of DNA and it is
the key stage in the processing of the samples within most Molecular Genetics
or it is the first and foremost step involved in Molecular Biology.
Macromolecules (Biomolecules):
Carbohydrates.
Lipids.
Proteins.
Nucleic acids (DNA & RNA).
Purpose of Genomic DNA Extraction:
To obtain DNA in a relatively (amounts) purified form which can be used
for further investigations, i.e. Gene Cloning, PCR (Polymerase Chain
Reaction) amplification, Southern Blotting Technique, Microsatellite
Analysis and DNA Sequencing, etc.
DNA extraction is used to isolate:-
Mitochondrial DNA.
Genomic DNA (Nucleus DNA).
Plastid DNA (like Plant cells).
DNA can be extracted from…
Cells or tissues
Environmental samples
Principles of DNA Isolation & Purification
i. DNA can be isolated from any nucleated cell.
ii. DNA is a giant anion in solution.
Sources of DNA include:
Buccal cells, Blood cells, Culture cells, Biopsies (Tissues), Urine Sample and
Forensic samples i.e. body fluids, hair follicles, saliva, bone, teeth root and
semen…. etc.
Basic Protocol of DNA Extraction
Most DNA extraction protocols consist of three parts:
A step for breaking or opening (is lysing) the cells to release and solubilize the
DNA using:
1. Mechanically (Physically) by utilizing mortar and pestle or blender.
2. Chemically or Enzymatically by utilizing liquid nitrogen, NaCl, SDS
(Sodium Dodecyl Sulphate), EDTA ([disodium] Ethylene Diamine Tetra
Acetate), CTAB (Hexadecyl Trimethyl-Ammonium Bromide) and
Enzymes like (Lysozyme, RNase and Proteinase K).
A step for removing all of the proteins and RNA, contaminants or inhibitors
and precipitation by phenol chloroform or Salt.
A step to re-suspend and precipitate the purified DNA by ethanol or
isopropanol.
Main processes during DNA Extraction
Five processes are used to remove and purify the DNA from the rest of the cell:
1. Lysis.
2. Precipitation (by Centrifugation).
3. Resuspension.
4. Purify DNA (Concentrated).
5. Filtration.
Overview of DNA Extraction
Practical Molecular Biology Lab. 02 October 25, 2020
Two Practical Methods of Genomic DNA Extraction:
Many different techniques have been developed for extracting DNA from
animal, plant, bacterial cells and environmental samples. Some methods are very
complicated while others are quite simple. Our choice of technique depends on
the specific sample that we are working with and your requirements for the quality
of DNA extracted.
However, in our lab we will extract DNA from human blood sample by
performing the following two methods:
1. Phenol chloroform method.
2. Salting out method.
Materials and Functions of Solutions:
Ѽ DNA Buffer (DNA Lysis Buffer): (0.14 M NaCl + 0.01 M Sodium Citrate)
(its prepared by dissolving 8.19gm of NaCl with 2.2gm of sodium citrate in a
liter of D.W.), DNA buffer is used to detach protein (Histone and non-histone
proteins) from DNA strands and inhibit DNase enzyme, which digests DNA.
Ѽ Fresh or Frozen Human Blood Samples.
Ѽ Liquid Nitrogen (Cell wall breakdown and inactivate enzymes for tissue
samples like plant tissues, Biopsies, Bacterial Cells).
Ѽ Salt/EDTA buffer solution (2.6 M NaCl + 0.5 M EDTA pH= 7.5), it
contributes positively charged atoms that neutralize the normal negative
charges of DNA and to chelate divalent cations such as Mg2+ or Ca2+, which
are cofactors for many DNA-degrading enzymes and then forms EDTA-
nucleic acid complex.
Ѽ CTAB: is a cationic detergent that it binds strongly to DNA forming nucleic
acid complex, solubilize membranes and thus displacing protein and
preventing DNA degradation.
Ѽ Cold ethanol 95% (DNA does not dissolve in alcohol, that is why when
alcohol is added the DNA either floats or precipitate, when it is floating you
can see the white foggy fibers of DNA in the Alcohol solution).
Ѽ ACT (Ammonium Chloride Tris Buffer Solution): Red blood cells lyse
and removing hemoglobin.
Ѽ PBS (Phosphate Buffer Saline) Solution: White blood cells collect and lyse.
Ѽ 10% SDS (Sodium Dodecyl Sulphate): Lipid degradation, this detergent aids
the process of lysis by removing lipid molecules and thereby causes disruption
of the cell membranes and nuclear envelops.
Ѽ Phenol chloroform: to deproteinize a cell extract. These organic solvents
precipitate proteins but leave the nucleic acids (DNA and RNA) in aqueous
solution.
Ѽ Proteinase K Enzyme: It breaks polypeptides down into smaller units, which
are more easily removed by phenol.
Ѽ RNase Enzyme: The only effective way to remove the RNA is with the
enzyme ribonuclease, which rapidly degrades these molecules into
ribonucleotide subunits (may be useful for PCR running as dNTPs).
Ѽ TRIS EDTA (TE):
I. Procedures of Manually Method:
DNA Extraction from Blood cell samples by (Phenol Chloroform Method):
1. Take 5ml the venous blood sample from the human in anticoagulant
(K3EDTA) test tube.
2. Add 4-5 ml of sterile ACT solution to the whole blood sample (2ml) to lyse
the red blood cells (R.B.C.s) and then the solution is gently shaken to change
the color of the solution from blood red to a dark clear red.
3. Five minutes after the additional of ACT solution a clear pellet of white blood
cells (nucleated cells) are collected by spinning at 4000 rpm for 5 minutes
(repeat the previous step until clear pellet appear of the bottom test tube).
4. After centrifugation the supernatant solution is discarded and pelleted WBCs
are re-suspended by adding of 1ml of phosphate buffer solution (PBS) and then
spin 2 or 3 times at 4000 rpm for additional 4 minutes.
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5. One volume (for example 1 ml) of WBCs pelleted is mixed with two volumes
of DNA lysis buffer, in sterile stoppered test tube, by inverting several times
and the mixture is left at 4Cº for 10-30 minutes.
6. Centrifuge at 4000 rpm for 10 minutes to obtain nuclear pellet.
7. Re-suspend in 1ml of salt/EDTA buffer by overtaxing briefly.
8. Add 200 µl of 10% SDS to re-suspend extracted pellet and then add 10 µl of
proteinase K and then incubate overnight at 37 Cº.
9. Add 1 ml of phenol or chloroform or phenol chloroform and mixed on a rotary
mixer for 10 min, followed centrifugation at 4000 rpm for 5 min.
10. The supernatant is removed to another test tube and add 2-3 ml of cold ethanol
(absolute) to precipitate nucleic acid DNA and RNA.
11. Removed RNA from the solution by adding RNase enzyme.
12. Hooked out by glass hook and then dissolve in deionized distilled water or Tris
EDTA buffer (100-200 µL).
13. Dissolve the pellet DNA in D.W. or TE solution and incubate in water bath
at 37°C for 15 min. DNA must be stored in a slightly basis TE buffer to prevent
depurination, and the EDTA chelates any Mg2+ helping to inactivate DNases.
14. This solution was transferred to Eppendorf tube (micro-centrifuge tube) and
kept at 4°C as a stock DNA sample, however for long term storage, -20Cº is
preferable.
15. If the DNA containing solution is too concentrated, make a dilution.
16. Store the DNA for further tests but avoid repetitive freeze thawing of DNA,
since this can cause degradation.
DNA Storage:
Extracted DNA samples are usually stored at -80Cº; it can be stored for more
than 2 years at this degree temperature. It can be stored at -20Cº also, but storing
it under this degree for extended period of time, several months, may reduce
sample’s yield of DNA. So the samples quality & quantity start to decline and
eventually DNA is not recovered.
II. Procedures of Kit Method: