Lab. 6DNA extraction

from human blood

 • Be introduced to the laboratory

techniques involved in DNA extraction.

• Test DNA integrity using gel electrophoresis.

• DNA is used every day by scientists and lawyers to help in criminal investigation, paternity test, researches, …. etc.

• Your DNA is your “genetic fingerprint”, this means that your DNA is like no one else’s in the world.

• DNA is a nucleic acid, made of carbon, hydrogen, oxygen, nitrogen, and phosphorous.

• DNA can be considered the hereditary “code of life” because it possesses the information that determines an organism’s characteristic and is transmitted from one generation to the next.• You receive half of your genes from your mother and half from your father.

The more closely related organisms are, the more similar their DNA. • Day to day, DNA’s job is to direct the functioning within the cells of your body.

• DNA is in the nucleus of almost every cell in your body. • 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.

• The structure of DNA, the double helix, is wrapped around proteins, folded back onto itself, and coiled into a compact chromosome.

• Individual chromosomes can be studied using microscopes, but the double helix of a chromosome is so thin that it only be detected through innovative, high-tech procedures.

• Chromosomal DNA from a single cell is not visible to the naked eye.

• However, when chromosomal DNA is extracted from multiple cells, the amassed quantity can easily be seen and looks like strands of mucous-like, translucent cotton.

• (Ethylenediamine tetracetic acid) is 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 and must be made unavailable to the cells if we want to end up with nucleic acids as an end product.

• 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.

• (Sodium Dodecyl Sulfate) is a biological detergent which causes the cell membrane to break down further and emulsifies the lipids and proteins of the cell by disrupting the polar interactions that hold the cell membrane together, and forms complexes with these lipids and proteins causing them to precipitate out of the solution.

• (Sodium chloride) enables nucleic acids to precipitate out of an alcohol solution because it shields the negative phosphate end of DNA causing the strands to come closer together and coalesce.

• 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.

Basic Steps in Isolating DNA from Clinical

Specimens• Separate WBCs from RBCs.• Lyse WBCs.• Denature/digest proteins.• Separate contaminants (e.g., proteins,

heme) from DNA.• Precipitate DNA.• Resuspend DNA in final buffer.

Procedure

1. transfer 300μl of blood to 900μl of Cell Lysis Solution in 1.5ml microcentrifuge tube.Important: Blood must be collected in EDTA, heparin or citrate anticoagulant tubes to prevent clotting.

Eppendorf tubes

2 .Invert the tube 5–6 times to mix.

3. Incubate the mixture for 10 minutes at room temperature (invert 2–3 times once during the incubation) to lyse the red blood cells.

4. Centrifuge at 13,000g for 20 seconds.

Centrifuge

5. Remove as much supernatant as possible without disturbing the visible white pellet. Approximately 10–20μl of residual liquid will remain in the 1.5ml tube.

6.Vortex the tube vigorously until the white blood cells are resuspended (10–

15 seconds).7. Add 300μl of Nuclei Lysis Solution. Pipet the solution 5–6 times to lyse the white blood cells. 8. Add 100μl of Protein Precipitation Solution to the nuclear lysate, and vortex vigorously for 10–20 seconds.

9.Centrifuge at 13,000g for 3 minutes.

10. transfer the supernatant to a clean 1.5ml microcentrifuge tube containing 300μl of isopropanol.

11. Gently mix the solution by inversion until the white thread-like strands of DNA form a visible mass.

12 .Centrifuge at 13,000g for 1 minute.

13. Decant the supernatant, and add 300μl of 70% ethanol to the DNA.

14. Gently invert the tube several times to wash the DNA pellet.

15 .Centrifuge at 13,000g for 1 minute.16. Decant the ethanol and invert the

tube on clean absorbent paper and air-dry the pellet for 10–15 minutes.

16. Add 100μl of DNA Rehydration Solution to the tube and rehydrate the DNA.

17 .Incubating the solution at 65°C for 1 hour or overnight at 4°C.

Freeze at -20 or -70°C for long-term storage .

Gel electrophoresis:• Prepare 1% a garose gel and mix 10 l

DNA samples with loading dye buffer as described in Lab 3, Transfer the DNA samples into the wells of the gel using a pipette. Loading DNA samples must be done slowly and smoothly to prevent sample from squirting up into the running buffer and diffusing away.