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.

Practical Molecular Biology Lab. 02 October 25, 2020

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: