1. DNA Extraction from human blood
Practical Of Genetics
DNA Extraction from human blood

2. Objectives Isolation of genomic DNA from human blood
Analysis of isolated DNA using
Agarose gel electrophoresis

3. DNA (deoxyribonucleic acid)
Is a nucleic acid, made of carbon, hydrogen, oxygen, nitrogen, and phosphorous.
A fundamental molecule found in all living things.
Carries the genetic information in the cell.
DNA is in the nucleus of almost every cell in your body.

4. The more closely related organisms are, the more similar their DNA.
The length of DNA per cell is about 100,000 times as long as the cell itself.
However, DNA only takes up about 10% of the cell’s volume. This is because DNA is specially packaged through a series of events to fit easily in the cell’s nucleus.

5. DNA Extraction
DNA extraction is a routine procedure to isolate & collect DNA.
DNA extraction is the first step for subsequent molecular or forensic analysis.
DNA from a single cell is not visible to the naked eye.
However, when DNA is extracted from multiple cells, the amassed quantity can easily be seen and looks like strands of mucous-like, translucent cotton.

6. DNA can be extracted from almost any intact cellular tissue
Skin
bone marrow
Blood
Saliva

7. Why you isolate DNA Rapid detection of genetic disorders in a patient.
Analyze forensic evidence.
Study a gene involved in cancer.
DNA fingerprinting to identify individuals (Paternity test).

9. For extraction DNA some substances were used:
EDTA (Ethylenediamine tetracetic acid):
known as a chelating agent, In other words, it binds divalent cations such as Mg and ca.
This ion is used as a cofactor in nuclease enzymes.
Mg-ion present in DNase as a cofactor and responsible for DNase action that degrade the DNA, here EDTA bind with Mg-ion and nullify the action of DNase.

10. Tris :
In the solution acts as a buffer and raises the pH of the solution in preparation for the acids added in the subsequent steps of the DNA extraction procedure.

11. SDS (Sodium Dodecyl Sulfate) :
Is a biological detergent.
which disrupts the lipid layers, helps to dissolve membranes & binds positive charges of chromosomal proteins (histones) to release the DNA into the solution.

12. NaCl (Sodium chloride) :
NaCl provides Na+ ions that will block negative charge from phosphates on DNA.
Enables DNA to come closer together and coalesce.

13. Alcohol:
DNA will be precipitated by adding cold alcohol to the cell extract, DNA will come out of the suspension and may be seen and collected on a glass rod.

14. Materials Tric-CL 20 mM, Ph 7.6 Ethanol 70% Isopropanol NH4HCO3 NH4CL
Cell lysis buffer:
10 mM Tris-Cl (Ph 8)
1 mM EDTA (Ph 8)
0.1% (w/v) SDS
Red Cell Lysis Buffer (RCLB)
NH4HCO3
NH4CL

15. Protocols
Draw 5 ml of blood using Vacutainer tube (with anticoagulant )
Keep cool until preparation is performed, but do not freeze. Highest yield will be achieved by extracting within 24 hours.
Empty blood into a 15 ml Falcon tube; add 10 ml of Red Cell Lysis Buffer (RCLB) and mix completely by inversion.

16. Spin 10 minutes at 1,200g in a clinical centrifuge.
Discard supernatant and resuspend cell pellet in 10ml RCLB; repeat step 4.
Discard supernatant; resuspend cell pellet (it should be white now) in 1.8 ml of White Cell Lysis Buffer (WCLB). It should be extremely gelatinous.

17. To perform final extraction, add 150 μl of saturated NaCl (~ 6M NaCl) to 400 μl of white cell lysate to precipitate.
Vortex and invert to mix; place on ice for 10 minutes.
Centrifuge 5 minutes at 12,000 RPM.
Add the supernatant to a 1.5 ml tube (550 μl); add 1000 μl absolute (100%) ethanol. Mix by inversion - the DNA precipitate should be visible at this point.

18. Spin 5 minutes at 12,000 RPM; discard supernatant.
Wash with 1 ml 70% ethanol; air dry for 15 minutes. Leave overnight at 4°C to resuspend completely.