Introduction to the Extraction of Human Genomic DNA Using Salting Out Method
There are many methods available for the extraction of human DNA, one of the popular methods is the salting out method. here are some available DNA extraction methods:
- Phenol-Chloroform Extraction
- Salting-Out Method
- Chelex Resin Extraction
- Silica-Based DNA Extraction:
- Spin Column-Based DNA Extraction Kits
- Detergent-Based DNA Extraction:
- Magnetic Bead DNA Extraction
- Solid-Phase DNA Extraction
- CTAB (Cetyltrimethylammonium Bromide) Method
- Proteinase K Digestion:
- GuSCN (Guanidine Isothiocyanate) Method:
- Heat Shock Method
- Inorganic Method (Silica and Guanidine)
- PAXgene Blood DNA Kit
- Manual Stir Bar Sorptive Extraction (mSBSE)
These extractions of DNA methods provide row materials for many downstream applications such as PCR, sequencing, and genetic analysis.
I. Overview of the Salting Out Method:
salting methods is relies on salt manipulation and selectively precipitation of protein and get the purified Genomic DNA in solution. this technique has many advantages such as:
- simplicity
- cost-effectiveness
- minimal requirement for specialized equipment
- making it accessible for laboratories with varying resources.
II. Principle of Operation:
This process begins with the lysis of cells or tissues, releasing genomic DNA into a solution. This initial step is facilitated by the addition of a cell lysis buffer, often Tris-HCl and EDTA, to maintain an appropriate pH and inhibit nucleases that can degrade DNA. Then, introducing a high salt solution induces protein precipitation, effectively removing cellular proteins from the mixture.
III. The following steps in the salting out method are:
Cell Lysis
Cellular material is initially lysed to release genomic DNA. This step is often enhanced by the addition of proteinase K, an enzyme that digests proteins and further facilitates DNA release.
Protein precipitation
A high salt solution, usually containing sodium chloride (NaCl), is added to the lysate. Increased salt concentration induces precipitation of cellular proteins, which are then removed by centrifugation.
DNA Precipitation: The supernatant enriched with genomic DNA undergoes a further precipitation step by adding an appropriate DNA precipitation buffer. This causes genomic DNA to form visible particles during centrifugation.
Washing and resuspension: DNA pellets are washed using ethanol or isopropanol to remove residual contaminants. After removing the wash solution, the purified genomic DNA pellets are air-dried or vacuum-dried and then resuspended in an appropriate buffer.
IV. Applications and Considerations:
DNA obtained by the salting-out method is suitable for a variety of molecular biology applications, including PCR, restriction enzyme digestion, and sequencing. Researchers should consider factors such as sample size, the need for high molecular weight DNA, and downstream applications when choosing DNA extraction methods.
V. Conclusion:
In conclusion, the salt-extraction method is a robust and accessible approach for the extraction of human genomic DNA. Its simplicity, cost-effectiveness and compatibility with various downstream applications make it desirable.
Extraction of Human genomic DNA using the salting out method
You will need
- 30 μl of whole blood collected into EDTA/ACD, buffer vial
- Red Blood Cell lysis solution
- Nuclei lysis solution
- Protein precipitation solution
- 100% Isopropanol
- Img/ml RNAse
- 70% ethanol
EDTA/ACD, buffer
Blood is collected in a vial containing an anticoagulant called EDTA (Ethylenediaminetetraacetic acid) or ACD (Acid-Citrate-Dextrose). These anticoagulants prevent blood clotting and preserve the integrity of cellular components.
1. Prevention of blood clotting
EDTA (Ethylenediaminetetraacetic Acid):
EDTA is a chelating agent, meaning it binds to metal ions. In the context of blood collection, EDTA chelates calcium ions (Ca^2+).
Calcium ions play an important role in the coagulation cascade, a series of events leading to blood clotting. By binding to calcium, EDTA inhibits clotting factors and prevents blood clots from forming.
ACT (Acid-Citrate-Dextrose):
ACD works by keeping the blood in a non-clotting state through a combination of citrate and dextrose.
Citrate chelates calcium ions, which inhibits the clotting process.
Dextrose provides energy to preserved cells, maintaining their viability during storage.
2. Preservation of cellular components:
Both EDTA and ACD help maintain the integrity and function of the cellular components in the blood sample.
EDTA:
EDTA helps protect the structure of cells by inhibiting the clotting process, which can lead to cell damage during clot formation.
It inhibits enzymes that can degrade cellular components such as nucleases that break down DNA and RNA.
ACD:
Citrate in ACD not only prevents blood clotting but also has a cell-stabilizing effect.
Dextrose acts as an energy source for cells, helping to maintain their metabolic function and structural integrity.
3. Cumulative Impact:
The addition of EDTA or ACD to blood collection vials ensures that the blood remains in a liquid state, which prevents clot formation during sample collection and subsequent processing.
This liquid phase is important for downstream applications such as DNA extraction, as it allows efficient separation of various blood components without coagulation interference.
Briefly, both EDTA and ACD act as anticoagulants by chelating calcium ions, which disrupt the clotting cascade. In addition, these anticoagulants contribute to the preservation of cellular components by inhibiting enzymes and providing a supportive environment for the cells in the collected blood sample. This preservation is necessary to obtain high-quality samples for various laboratory analyses.
RBC lysis solution
Red blood cell (RBC) lysis solution is another reagent used in the DNA extraction process that is designed to interact specifically with red blood cells while preserving other blood cells. This solution creates a hypotonic environment characterized by low osmotic pressure, which leads to the lysis or rupture of red blood cells. In this environment, red blood cells absorb water, causing them to swell and eventually undergo rupture.
The need to remove red blood cells is due to the fact that they do not have a nucleus (nucleus) and lack certain cellular components, including nuclei. Their primary function is to transport oxygen through the body via hemoglobin. Although smaller and more easily degraded compared to white blood cells, the removal of red blood cells is important for DNA extraction because it removes unwanted components from the sample.
The hypotonic nature of the solution selectively affects red blood cells, allowing for their efficient lysis while minimizing the effect on other blood cells, particularly nuclei such as white blood cells. This selective degradation ensures that the extraction process focuses on the desired nuclear cells containing genomic DNA for downstream applications.
The specific composition of red blood cell (RBC) lysis solutions
The specific composition of red blood cell (RBC) lysis solutions varies depending on the manufacturer or the specific protocol used. However, a typical formulation may include the following elements:
Ammonium Chloride (NH4Cl):
Ammonium chloride helps create a hypotonic environment, which leads to lysis of red blood cells. It contributes to the disruption of red blood cells, it does not significantly affect the nuclear cells.
Potassium Bicarbonate (KHCO3):
Potassium bicarbonate is often added to maintain a constant pH during the digestion process.
EDTA (Ethylenediaminetetraacetic Acid):
EDTA is a chelating agent that binds to divalent cations such as calcium ions. Its addition helps prevent clotting by inhibiting calcium-dependent clotting factors.
Distilled water:
Distilled water is often used as a solvent to create the hypotonic solution required for lysis of red blood cells.
Buffer Agents:
Buffers may be added to maintain the pH of the solution, ensuring optimal conditions for the degradation process.
It is important to note that some variants of RBC lysis solutions may contain additional components or slight variations in concentrations of specified substances. Additionally, commercial kits or protocols from different manufacturers may contain proprietary formulations with specific additives to improve performance or stability.
Researchers generally follow established protocols or kit instructions when using RBC lysis solutions, and can confirm the specific formulation by referring to the documentation provided with the solution or kit.
The Nuclei Lysis solution
The Nuclei Lysis solution is another reagent in the DNA extraction process designed to break down the cell membrane and nuclear membrane, particularly in white blood cells. This process releases DNA into the solution. The Nuclei Lysis solution contains detergents that interact with the cell membrane and nuclear membrane, disrupting the phospholipid layers or bilayers.
The specific composition of the nucleolysis solution varies depending on the DNA extraction protocol or instrument used. However, a typical nuclease solution may contain the following components:
Detergent:
Detergents are added to disrupt the lipid bilayers of cell and nuclear membranes. They break the hydrophobic interactions that hold these membranes together, leading to the release of cellular contents, including genomic DNA.
Salt:
Salt is often added to the nucleus lysis solution to create an isotonic environment and to maintain the osmotic balance and stability of cellular components.
Buffer Agents:
Buffers are added to maintain the pH of the solution within an optimal range. This helps stabilize the reaction conditions during the decomposition process.
EDTA (Ethylenediaminetetraacetic Acid):
EDTA can also chelate divalent cations such as calcium and magnesium ions. Chelating these ions prevents nucleation and helps maintain the stability of the released DNA.
Protease inhibitors:
Protease inhibitors can be added to prevent the degradation of proteins released during the degradation process. It helps preserve the integrity of cellular proteins, which may be important for some downstream applications.
Reducing agents:
Some protocols include reducing agents that help break disulfide bonds in proteins and release cellular contents.
The Protein Precipitation Solution
The protein precipitation solution typically contains protease, salts, and other components that facilitate the removal of proteins from the solution. This step is crucial for purifying DNA because, in the solution after cell lysis, both DNA and proteins are present. To isolate DNA, the proteins must be removed. The protein precipitation solution commonly contains salts, such as ammonium acetate or ammonium sulfate, along with other reagents that alter the solubility of proteins. These changes in solubility induce the precipitation of proteins, allowing their separation from the DNA in the subsequent centrifugation step.
