Views: 198 Author: Site Editor Publish Time: 2022-08-04 Origin: Site
Nucleic acid extraction and purification is the primary step for the downstream applications like sequencing, RT-PCR amplification, hybridization, cloning and biological detection. It is critical to ensure reliable results in molecular diagnostic tests and molecular biology research.
Typically, we have 3 purposes in nucleic acid separation from a variety of samples with complex components.
1. Remove inhibitors that will interfere downstream processes.
2. Increase target nucleic acid concentration to optimize assay performance
3. Improve the sample uniformity to ensure reliable assay results
Methods for Isolation and Separation of Nucleic Acid
Liquid-Phase and Solid-Phase separations:
The traditional liquid-phase extraction involves steps such as precipitation and centrifugation. Liquid-phase extractions are complicated, time-consuming, and labor intensive. Solid phase extraction uses solid-phase absorbent carriers to bind the nucleic acids, so that impurities such as proteins and lipids remain in the liquid-phase and can be removed as supernatant.
Solid phase separation methods are more preferred as they provide efficient phase separation, has low contamination risk, and requires lesser sample volume. The most used solid-phase extraction techniques are spin columns and magnetic bead-based extraction.
Spin column-based method
The most common spin column uses a silica membrane that selectively binds nucleic acids. The following steps are employed in silica membrane spin column;
Step 1 Lysis: To disrupt the cells, mix the sample and lysis buffer using a vortex mixer. Nucleic acid is separated from proteins and other impurities after efficient reaction.
Step 2 Incubation: put the mixture in an incubator where the proteins will be digested and dissolved, while nucleic acid is further released.
Step 3 Binding: The lysed sample along with the binding solution is then transferred to a spin column and centrifuged. This forces the solution through the silica membrane inside the column. The nucleic acids are retained by the silica membrane while the remaining solution passes through it.
Step 4 Washing: Add wash buffer and centrifuge again. This helps to wash away the impurities like cell debris and unwanted proteins.
Step 5 Elution: After air-drying, add elution buffer to detach the DNA from the silica column using a centrifuge again.
As seen from the above steps, frequent liquid handling is involved and the entire process can be laborious and time-consuming and needs to repeatedly use lab equipment like vortex mixer and centrifuge. So, spin column method is better suited for low throughput applications in the lab.
Magnetic bead method
Magnetic bead-based method is widely used in automated nucleic acid extractors together with a reagent kit containing lysis, wash and elution buffers. Following are the steps commonly used for automated magnetic bead extraction:
Step 1 Sample Loading: First, load the sample, magnetic beads and various buffers into the specific wells of a 96-well deep well plate.
Step 2 Lysis and Binding: Cells are lysed under heating and repeated stirring using the magnetic rod sleeve combs. The released nucleic acid binds to the magnetic beads. Magnetic rods lower down to collect and transfer the magnetic beads with the bound particles to the next wells for washing.
Step 3 Washing: Alcoholic wash buffers in the wash wells help to remove contaminants. Once the washed beads are collected using magnetic rods, it is held above the wells for some time to remove the alcohol before the elution stage.
Step 4 Elution: After alcohol is removed, transfer the magnetic beads to the well with the elution buffer. The bound nucleic acid is then released, and the magnetic beads are collected and removed. The pure nucleic acid extract can then be collected from the elution well.
Magnetic bead-based automated nucleic acid extraction integrates mixing, binding, washing and elution and delivers high-efficiency, high-quality extraction, suitable for assays that requires high-throughput and high yields in a short time. The most commonly available high throughput nucleic acid extractors available in the market offers a maximum throughput of 32 samples (like MultiEx032) or 96 samples (like MultiEx096).
If an automated high-throughput nucleic acid extraction is not required for your applications and if you want a cheaper magnetic bead-based extraction tool, magnetic racks would be a great option for you.
Here is the working principle. After the nucleic acid binds to the magnetic beads, the magnetic rack comes into play. On placing the tubes into the magnetic rack, the beads along with the bound nucleic acid will cling magnetically to the side of the wall. This makes it easy to wash away contaminants using a pipette. In the elution step, the magnet holds the beads again so that high-quality nucleic acid can be separated and transferred to a clean tube.
Magnetic rack is an economical and fast way of extracting nucleic acid without the need of centrifuging.
For more information on the life science tools used in separation and purification of biomacromolecules like nucleic acids, please Contact Us.