Question

Can you please write about DNA extraction from blood and preparation of blood samples for DNA extraction. This is the protocol:

1.Take 750 μl of blood from each blood sample, and transfer it to new 1.5ml tube;
2. Mix 750 μl of blood with 1ml RBC lysis buffer, respectively (avoid stock contamination by not touching the walls of tube by pipette tip);
3. Centrifuge at 2300 rpm for 5 minutes;
4. Take out the liquid, keep the pellet;
5. Mix 1 ml of RBC lysis buffer with pellet in 15ml centrifuge tube;
6. Add PBS up to 14 ml by pipette controller (increase volume and contrast);
7. Centrifuge at 2200 rpm, at 4°C, for 5 minutes
8. Discard the supernatant, keep the pellet;

9. Add 1 ml of RBC lysis buffer;
10. Add PBS up to 10 ml;
11. Centrifuge at 2200 rpm, at 4°C, for 5 minutes;
12. Discard the supernatant and resuspend the pellet in 1 ml of Trizol;
13. Store the samples at -20°C

Can you explain the protocol, why are we doing these steps. Also if you can say the function of each chemical:

• RBC lysis buffer
• PBS
• Trizol

Thank you in advance. I really really need your help.

Answer 1

Using precipitation chemistry the whole blood DNA isolation acts by precipitating DNA out of lysate via high salt concentration and by addition of an alcohol like ethanol and ispropyl alcohol. It seperates DNA from other biomolecules and compounds like lipids and carbohydrates. They also seperate DNA from any exogenously added chemical compounds which is present in our blood. They can be one step and 2 step lysis methods:

a) Two step lysis method- RBC'S lysed with an anionic detergent in presence of a DNA stabilizer, WBC'S lysed, gDNA and cellular DNA will be released into the solution by RNA digesting treatment, c) Proteins are removed by salt precipitation, d) DNA precipitated is washed and resuspended in hydration buffer.

This method is used in puregene kits and in the first step the RBC's are lysed using sodium dodecyl sulphate or SDS and on the second step it ensures WBC's to release cell nucleus, genomic DNA, and RNA. Ribonuclease treatment removes contaminating rna's leaving dna and protein molecules behind supernatant. In next step, proteins aare removed using concentrated salts which precipitate proteins from lysed cells. As the cation concentration increases due to salt addition and free water becomes less available and protein solubility decreases, and proteins are salted out. DNA remains in sipernatant and is collected.

b) one step lysis method- RBC's and WBCs are lysed nuclei and mitochondria isolated from wbc by centrifugation( cell lysis), pellet resuspended in denaturation buffer with a chaotropic salt and protease( contaminant removal), DNA precipitated washed and resuspended in hydration buffer9 DNA recovery)

Its used in flexi gene kits the red and white blood cells are lysed in 1 step and works via detergent mix capable of lysing both cell types. This step releases nuclei and mitochondria from wbc's collected via centrifugation leaving rna in supernatant. Next step is to remove contaminated proteins in which white cell nuclei and mitochondria are treated with denaturation buffer which contains chaotropic salt and protease which simultaneously denatures and digests proteins in cell nucleus and mitochondrion leaving digested proteins and genomic dna in solution.

In both of these methods dna is recovered by alcohol precipitation and rehydrated using a tris or tris EDTA buffer solution.

1) rbc lysis buffer- they allows for preferential lysis of red blood cells from whole blood and majorityn of cells in whole blood permits the concentration of nucleated white blood cells. Its ideal for isolation of dna and rna from blood.

2) PBS- pbs saline is commonly used in biological research and this buffer helps to maintain a constant pH. A pH of 7.4 is maintained. Osmolarity and ion concentration of the solution usually match those of the human body.

3) trizol- its used for rna isolation from cells and tissues and works by maintaining rna integrity during tissue homogenization and disrupting and breaking down the cell and cell components.