Nucleic Acid Extraction & Purification & How We Save You Time & Money

Manual methods rely heavily on combining chemical compounds. The resulting process is extremely time-intensive and requires the undivided attention of the laboratory technician at every step. There is also an increased risk of cross-contamination. As well, most of these methods require repeated centrifugation steps, followed by removal of supernatants depending on the type of specimen and additional mechanical treatment.

In general, the basic extraction method consists of four steps:

1. Cell lysis, in order to disrupt cells/tissues
2. Denaturation of nucleoprotein complexes
3. Inactivation of RNA/DNA nucleases (RNase for RNA extraction and DNase for DNA extraction)
4. Avoidance of contamination

One common method is to use guanidinium thiocyanate, phenol, chloroform, and ethanol in combination to separate and extract target nucleic acids, removing the impurities of proteins and salt from the nucleic acids. This adds multiple separation and purification steps to the overall process, such as repeated centrifugation.

Another method involves alkaline lysis, which can be used to isolate plasmid DNA. This works well with bacterial cultural (E. Coli, for example) and uses Sodium Dodecyl Sulfate (SDS) to induce alkaline denaturation of DNA by coating it with the sulfate. Plasmid DNA is then recovered after centrifugation.

Traditional extraction protocols and methods are often too difficult to use on a regular basis. And because extraction and purification are such an important part of biological research and clinical diagnostics, the methods used have needed to become simplified.

In fact, the need to simplify has led to the development of multiple, semi-automated methods, such as solid-phase nucleic acid purification, which is available as an extraction kit.

This method—it includes spin column-based nucleic acid purification, a method which quickly purifies nucleic acids silica as a solid phase—and the purification kits which are available through numerous manufacturers, have enabled scientists to speed up the purification process, resulting in a more efficient extraction procedure. It involves four similar yet simplified steps:

1. Cell lysis to disrupt cells/tissues
2. Nucleic acid absorption
3. Washing while avoiding contamination
4. Elution

This semi-automated method uses various substances for purification, the most commonly used being silica matrices (these materials include silica and glass particles as well glass powders and fibers), magnetic beads, and anion-exchange resin.

In fact, the use of magnetic beads essentially groups filtration, centrifugation, and separation into a single purification step.

Magnetic bead based nucleic acid purification, also referred to as particle-based purification, is a bind-wash-elute process, one in which the target nucleic acid is bound to the beads and washed, and finally separated from the other nucleic acids or impurities present.

This is performed using a magnetic field applied by a magnetic separation rack, which pulls the bound nucleic acids away from the remaining contaminants present in the aqueous solution. It is similar to silica based methods which also utilize a bind-wash-elute process.

Purification aside, these semi-automated extraction processes and commercial kits often follow a complicated series of steps, requiring a similar amount of work for a lesser yield, as is common with manual methods).

Because of this, automated, high-throughput systems have been developed. It’s important to consider whether or not your lab could benefit from such a system.

Extraction of DNA, and DNA isolation in general, has been sped up thanks to the development of automated extraction systems offered by manufacturers such as Thermo Fisher Scientific and Qiagen.

These systems greatly simplify the entire extraction process and allow for high-throughput without the need for specialized training. In fact, the ease-of-use offered by these automated systems means less training for you and your team.

Not only do automated systems decrease the working time and labor costs needed, they also increase the safety, quality, and reliably high yield of nucleic acid extraction and purification.

However, to compete with manual extraction methods, automated systems must meet several requirements: reproducible results, minimal cross-contamination, ease of use, and high quality output for downstream testing, along with a significant decrease in the amount of time a technician must spend with the instrument.

There are three major steps involved when using an automated system:

1. Add reagents to liquid samples
2. Place cartridges used by the kit in machine
3. Start your process

Automated instruments tend to utilize the same kits used in solid-phase nucleic acid extraction, but drastically reduce the time and effort required to complete the process. Compared to manual methods of the past, scientists agree that automation systems have made nucleic acid extraction and purification much simpler, faster, and accurate.

The most successful examples of automated systems are automated liquid handlers, which are routinely used in many life science and clinical analysis laboratories for dispensing precise amount of sample, reagents, or other liquids to designated containers.

Fully automated nucleic acid extraction protocols have been developed for such equipment, using either solid-phase or magnetic beads methods, however, high-throughput can, in some situations, compromise overall sensitivity, as degraded nucleic acid targets might be lost.

Besides the differences between manual and automated nucleic acid extraction methods, there are also some other important distinctions to make. Specifically, the distinctions between genomic DNA isolation and plasmid DNA isolation.

Genomic DNA isolation requires strong lysis in order to release the genomic DNA into a solution. This means either enzymatic or mechanical breakdown of the cell membranes is performed. If cell disruption is being performed using enzymes, it is typical to use proteinase K and lysozymes.

Mechanical breakdown typically involves the use of bead beating on a vortex. Afterwards, DNA purification is achieved using a method such as chloroform extraction, where phenol/chloroform is used for cleaning lipid bilayer, proteins, and other cell debris.

Plasmid DNA isolation, in comparison, typically requires mild alkaline lysis in order to get plasmid DNA into the solution, along with the genomic DNA.

Additionally, during genomic DNA isolation and following the cell lysis, genomic DNA can be purified from lipid membrane and proteins. On the other hand, during plasmid DNA isolation, neutralization with potassium acetate will separate plasmid DNA from genomic DNA.

Whole blood sample types are one of the main sources used to obtain DNA, and there are many different protocols available to perform nucleic acid extraction on such samples.