DNA isolation is also called: DNA extraction or DNA purification
DNA Isolation: what is it and how does it help my studies?
DNA isolation is the process by which DNA is separated from viruses, cells, or sample matrices. DNA (ribonucleic acid) is a polymeric molecule essential in various biological roles in coding, decoding, regulation and expression of genes. DNA is often extracted for the purpose of downstream PCR applications.
Nucleic acid isolation is a routine procedure for many research and clinical laboratories.
The initial procedures in nucleic acid isolation depend on the nature of the starting specimen and the future use of a particular sample. Some test systems include sample-collection vessels and reagents that begin the nucleic isolation in transport to the laboratory. Details of sample collection are important because of the diversity of sample types (viruses, bacteria, nucleated cells). Use of an improper collection tube or transport method will compromise the test results.
For some bacteria and fungi with cellular wall polymers, manufacturers have developed enzymes that can digest these molecules easily. This result can also be achieved by grinding or mixing with glass beads.
Nucleic acid can also be isolated from cells in suspension, such as blood or bone marrow aspirates. In this case, the procedure takes place thanks to differential density centrifugation or differential osmotic fragility of RBCs and WBCs. In both cases the goal is to isolate the mononuclear white blood cells and performing the nucleic acid isolation. Generally there are three methods for isolation, organic, inorganic and solid-phase.
After release of nucleic acid from the cell, further purification requires removal of contaminant molecules (proteins, lipids, carbohydrates, and cell debris). For organic isolation, this is accomplished using a combination of high salt, an organic mixture of phenol and chloroform in an acidic solution (low pH).
Organic isolation decreased in popularity due to the use of harsh chemicals like phenol. On the other hand, inorganic isolation makes use of low pH and high salt concentrations to purify the nucleic acid. This achieves the same result of specific precipitation of proteins and contaminants.
Lastly, solid-phase isolation takes advantage of the silica-based solid matrices to specifically bind and hold to nucleic acid during washing. This is accomplished by lysing the cells, acidifying the supernatant that contains nucleic acid, followed by adsorption of the nucleic acid into the solid matrix (facilitated by low pH and high salt conditions), finished with washing and eluting the DNA.
The initial procedures in nucleic acid isolation depend on the nature of the starting specimen and the future use of a particular sample. Some test systems include sample-collection vessels and reagents that begin the nucleic isolation in transport to the laboratory. Details of sample collection are important because of the diversity of sample types (viruses, bacteria, nucleated cells). Use of an improper collection tube or transport method will compromise the test results.
For some bacteria and fungi with cellular wall polymers, manufacturers have developed enzymes that can digest these molecules easily. This result can also be achieved by grinding or mixing with glass beads.
Nucleic acid can also be isolated from cells in suspension, such as blood or bone marrow aspirates. In this case, the procedure takes place thanks to differential density centrifugation or differential osmotic fragility of RBCs and WBCs. In both cases the goal is to isolate the mononuclear white blood cells and performing the nucleic acid isolation. Generally there are three methods for isolation, organic, inorganic and solid-phase.
After release of nucleic acid from the cell, further purification requires removal of contaminant molecules (proteins, lipids, carbohydrates, and cell debris). For organic isolation, this is accomplished using a combination of high salt, an organic mixture of phenol and chloroform in an acidic solution (low pH).
Organic isolation decreased in popularity due to the use of harsh chemicals like phenol. On the other hand, inorganic isolation makes use of low pH and high salt concentrations to purify the nucleic acid. This achieves the same result of specific precipitation of proteins and contaminants.
Lastly, solid-phase isolation takes advantage of the silica-based solid matrices to specifically bind and hold to nucleic acid during washing. This is accomplished by lysing the cells, acidifying the supernatant that contains nucleic acid, followed by adsorption of the nucleic acid into the solid matrix (facilitated by low pH and high salt conditions), finished with washing and eluting the DNA.
How is DNA isolated?
Solid-phase DNA isolation uses beads or columns to capture and release DNA at desired times. The sample or cellular / viral lysate is incubated with binding beads or columns, that selectively bind to DNA and thereby allows for the removal of proteins, salts, PCR inhibitors, and other contaminants.
- Magnetic beads selectively bind to DNA in the lysate and are then separated from the lysate using a magnet. These beads are washed to remove contaminants, before the DNA is eluted off of the beads, resulting in isolated DNA.
- Columns selectively bind to DNA in the lysate and allow non-DNA components of the lysate to flow through the column. The column is then washed to remove contaminants, before the DNA is eluted off of the column, resulting in isolated DNA.
Direct PCR / extraction-free PCR and the improved, Next Generation Direct PCR, allows for downstream PCR applications without the need for RNA or DNA isolation.
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