Nucleic Acid Extraction & Purification & How We Save You Time & Money
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Nucleic acid extraction and purification are basic yet key methods used in research across molecular biology, biopharmaceuticals, biotechnology, and various medical sciences.
Nucleic acid sample preparation includes a wide range of extraction and purification techniques that are used to transform a sample from one that can’t be directly analyzed into one that can, thus fitting the specific requirements of the analytical technique being performed downstream. It is highly important to purify and extract your samples when planning on performing quantification and quantitation of nucleic acids.
That’s because purity and yield of the extracted nucleic acids is critical to performance in those downstream applications, which include polymerase chain reaction (PCR)—more specifically, real-time PCR (qPCR)—as well as reverse transcription PCR (RT-PCR) and sequencing. In clinical and research laboratories today, RNA, DNA, and total nucleic acids (total RNA and DNA) are isolated and extracted through both manual and automated techniques.
Manual extraction requires multiple steps, increasing the risk of contamination. It is also considered highly complex, so not every laboratory technician is capable of performing extraction and purification manually.
That being said, another downside to manual extraction is that the entire process is extremely time consuming and labor-intensive, requiring the undivided attention of the technician.
Automated extraction, on the other hand, has many benefits over more traditional manual methods. The most important being the consistency of the isolated nucleic acid. Sample manipulation and reagent use is reduced, significantly decreasing the chance of cross-contamination.
Additionally, automated instruments and systems reduce the overall complexity of extraction and purification, making testing easier to perform by a larger number of scientists.
And while automated extraction instruments have many benefits over manual methods, the cost per test can be drastically higher than manual methods. Automation also only typically makes sense when a high throughput of samples is required.
Knowing your and your team’s exact sample preparation needs is an important step in deciding which method—whether it is manual or automated—as well as which instrument, is right for your lab.
Below we will cover some of the techniques and characterization methods available, as well as the basics of DNA purification.
Extraction & Purification Methods
Nucleic acid extraction and purification are the essential first steps for countless downstream applications, as high quality, highly pure DNA and RNA is important in a wide variety of research and clinical applications. Extraction and purification can be performed using either manual or automated methods. We will cover the basic steps of these methods below.
Manual Nucleic Acid Isolation
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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:
- Cell lysis, in order to disrupt cells/tissues
- Denaturation of nucleoprotein complexes
- Inactivation of RNA/DNA nucleases (RNase for RNA extraction and DNase for DNA extraction)
- 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:
- Cell lysis to disrupt cells/tissues
- Nucleic acid absorption
- Washing while avoiding contamination
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.
Automated Nucleic Acid Isolation
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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:
- Add reagents to liquid samples
- Place cartridges used by the kit in machine
- 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.
Genomic vs. Plasmid DNA Isolation
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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.
Automated DNA & RNA Extraction Instrument Leases to Fit Every Need
The need to increase accuracy and speed has pushed the importance of automation to the forefront of the life sciences and molecular biology in particular.
Minimizing human intervention and increasing efficiencies in the lab has escalated as a result of events like the COVID-19 pandemic, with laboratories striving hard to develop diagnostics and therapeutics to fight the virus while having to do so with a limited number of scientists and technicians.
Finding ways to automate nucleic acid purification to increase throughput, reproducibility, and accuracy, while also processing samples with a high degree of purity, has been crucial in this effort.
Connect with us at +1 888-927-3802 or complete our contact form and we can discuss all your extraction and purification needs in further detail.
This off-balance sheet financing structure provides three options at the end of the term. The lessee has the option to return the equipment to the lessor, renew at a discounted rate, or purchase the instrument for the fair market value. Monthly payments are also 100% tax deductible which yields additional monetary savings.
If you recently bought equipment, Excedr can offer you cash for your device and convert your purchase into a long-term rental. This is called a sale leaseback. If you’ve paid for equipment within the last ninety days, we can help you recoup your investment and allow you to make low monthly payments. This also frees up money in your budget rather than tying it down to a fixed asset.
Extraction & Purification System Manufacturers & Models
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Thermo Fisher Scientific:
KingFisher Purification Systems, KingFisher Duo Prime, KingFisher Flex, KingFisher Presto
DNA Extraction Systems (DNA, RNA, and Protein)
Freedom Evo, Freedom EVO NAP Workstation, Freedom EVO 75 configuration, Freedom EVO HSM Workstation
chemagic systems, chemagic 360, chemagic Prime, chemagic Prime Junior, chemagic Prepito, LabChip GX Touch 24, LabChip GX Touch HT, LabChip GXII Touch HT
Maxwell 16 System, Maxwell RSC Instrument, Maxwell RSC 48 Instrument, Maxwell CSC Instrument, Maxwell CSC 48 Instrument, Maxwell FSC Instrument, Maxwell 16 Instrument, Maxwell 16 Forensic Instruments
iQ-Check Prep Solution
MagCore System, MagCore HF16, MagCore HF16 Plus, MagCore HF48, MagCore Super
Perfinity Workstation, Perfinity iDP
taco, taco mini, POCKIT, POCKIT Xpress, POCKIT combo, POCKIT Micro Series, POCKIT Micro, POCKIT Micro Plus, POCKIT Micro Duo, POCKIT Central
QuickGene, QuickGene-Auto 12S, QuickGene-Mini 8L, QuickGene 810, QuickGene 610L, QuickGene-Mini80, FlexSTAR+, FLEX STAR, GENE PREP, AUTOPURE
VERITY, VERITY PLC, VERITY PLC-MS System, VERITY Oligo, VERITY GPC Clean-up System
Roche Life Science:
MagNA Pure system, MagNA Pure 96 System, MagNA Pure 24 System, MagNA Pure LC 2.0 System, MagNA Pure Compact System
LabTurbo 96XC Compact System, LabTurbo 48 Compact, LabTurbo 24 Compact System, LabTurbo 24 AIO SP-qPCR Automation System, VacEZor