Nucleic Acid Isolation And Purification

Nucleic acid isolation and purification is a complex technology with numerous benefits. Many applications need purified DNA and RNA and is an essential requirement in all aspects of molecular biology.

The nucleic acids are large polymeric molecules which are essential for life and just to reiterate are DNA and RNA. DNA was first isolated in 1869 by the Swede, Friedrich Miescher.

The purity and amount of DNA required and the process used depends on the intended application. Typical applications are:-

• detection of pathogens in foods or body tissues
• tissue typing for organ transplants
• human identity testing
• genetic research

Abbreviations used include dsDNA (double-stranded deoxyribonucleic acid), ssDNA (single-strand deoxyribonucleic acid) and RNA (ribonucleic acid).

What Are The Challenges In DNA Purification

DNA must be separated from other cellular components such as proteins, RNA, lipids, etc. 

Fragmentation of long DNA molecules is minimized by reduced levels of mechanical shearing or preventing the action of endogeneous nucleases which catalyse hydrolysis of the DNA.

The Quality Of The DNA Or RNA Is Extremely Important

The best yields of nucleic acid are obtained from fresh and frozen materials. Samples such as blood and body tissues must be processed correctly to minimize destruction of DNA by endogenous nucleases. When endogenous nucleases are active then the DNA yield is severely reduced. In this case rapid and prompt freezing is required followed by immediate processing or treatment with chelating agents such as EDTA to minimize any nuclease damage.

The overall process in nucleic acid purification is:-

1. Effective disruption of cells and tissues by using a detergent.
2. Denature proteins and nucleoproteins complexes using a protease and a denaturant.
3. Inactivate endogenous nucleases using chelating agents
4. Purify nucleic acid target away from other nucleic acids and protein. This might involve RNases, proteases, a selective matrix and some alcohol precipitations.

The time length for a typical DNA purification might be well over 3 days but new methods such as the RecoverEase technique have reduced that time to an eye-popping 17 hours.

Disruption of cells and tissues to release nucleic acids

Most purification methods disrupt cell membranes to release their contents using a lysis buffer. The DNA isolation method is based most often on a liquid phase nonorganic salt precipitation technique. These  mixtures contain:

• Detergent to disrupt the lipid bilayer of the cell membrane. A typical detergent is SDS (sodium dodecyl sulphate)
• Denaturants are added to release chromosomal DNA and denature proteins to stop further enzymic reactions.
• Proteins are precipitated with a salt solution
• The DNA is precipitated with alcohol and rehydrated.

The advantage of such a method is its easy and rapid. It generally requires no toxic materials so fume hood or hazardous waste disposal is needed. A high-quality DNA fraction is generated.

In some cases additional enzymes are needed for lysis of particular cell types:

• Gram-positive bacteria require lysozyne to disrupt the bacterial cell wall.
• Yeasts require addition of lyticase to breakdowen the cell wall
• Plant cells sometimes needs a cellulase pre-treatment.

Protein Denaturation

Protein denaturation is an essential step in releasing DNA. It involves changing the conformation of a protein in such a way that it unfolds. This disrupts its secondary structure but not the peptide bonds between amino acids. 

The change in protein conformation means the solubility of the proteins drops along with loss of biological activity. It also improves digestion by added proteases. In the process chromosomal DNA from nucleoprotein complexes is released as it unwinds from its associated histones.

Alkaline Extraction Method

A straightforward method of alkaline denaturation which destroys high molecular weight chromosomal DNA. It is ideal for isolating plasmid DNA. Centrifugation removes a pellet of denatured DNA and proteins.

Separation By Organic Extraction

DNA is polar and therefore insoluble in organic solvents. The earliest method was a straightforward ethanol precipitation. This involves ice-cold ethanol and/or isopropanol. DNA is insoluble in such alcohols so it aggregates forming a pellet upon centrifugation. The precipitation of DNA is improved by increasing the ionic strength of the surrounding solution using a buffer such as sodium acetate. Nowadays ethanol precipitation has been superseded by newer solvent systems.

In recent years, phenol:chloroform is used to extract DNA which takes about 35 minutes.

When phenol is mixed with the cell lysate, two phases form. DNA partitions to the upper aqueous phase, whilst denatured proteins partition to the lower organic phase. Phenol denatures the proteins.

DNA being a polar molecule has a negatively charged phosphate backbone. The polarity means it is more soluble in the polar aqueous phase. Again centrifugation leaves denatured proteins in the organic phase which is subsequently removed whilst the aqueous phase contains the nucleic acid. This fraction is mixed with chloroform which removes phenol residues from solution.

To encourage further purification, an ethanol wash is needed which takes 10 minutes per sample and leaves the DNA in a pelleted form.

The Agilent RecoverEase Method For Nucleic Acid Purification

Agilent have developed a method termed the RecoverEase which reduces the phenol-chloroform ethanol method from at least 3 days to about 17 hours. The technique can isolate high molecular weight genomic DNA from a range of tissues without the need for ethanol precipitation or organic solvent extraction.

The process requires physical disaggregation of the tissue, a course filtration and then cell nuclei isolation using a short centrifugation. The pellet is incubated with a very active protease solution containing proteinase K to predigest the cellular proteins.

Digestion continues and the DNA extract is transferred to a free-floating dialysis cup which permits overnight dialysis of the digested peptides over a semi-permeable membrane. The DNA obtained apparently measures between 100 and 500 kilobytes in length. The nucleic acid purification technique means that it can be then used for other analyses including PCR, blot analysis and so on.

The main benefit of the method is that it takes just 17 hours to generate a DNA prep. suited to further analysis. These DNA isolation kits are more convenient, do away with toxic chemicals such as phenol and chloroform. There are also fewer shearing forces exerted because the number of steps are reduced. The DNA is also fully hydrated following dialysis.

The manufacturer Agilent warn that particular tissues like bladder and skin are not suitable for the application of their isolation kit.

Cesium Chloride (CsCl) Density Gradient Centrifugation

A method of preparative density-gradient ultracentrifugation for DNA. It is ideal for isolating plasmid DNA. The purified nucleic acid must be re-precipitated with alcohol. 

Separation By Binding To a Solid Support as In Solid Phase Extraction

Most modern DNA purification methods are based on purification of DNA from crude cell lysates by selective binding to a support material. Support materials include silica and anion-exchange resins.

The advantages are speed and convenience with no organic solvents.

It is amenable to both automation and miniaturization.

Commercial nucleic acid extraction methods have largely superseded the more time-consuming precipitation techniques. Nowadays, solid-phase extraction is much more popular, especially the spin-column types.

(1) The Spin Column-Based Nucleic Acid Purification Method

The Boom method as it is also sometimes known is probably the most recognised solid phase extraction method and was developed by the eponymous author Willem R. Boom around 1990. This method uses silica particles. The silica beads are critical as nucleic acids bind to silica in the presence of a chaotropic substance. The binding of nucleic acid to silica was well-known even before purification methods were formally developed.

Chaotropes are compounds such as sodium iodide and sodium perchlorate. Binding agents like these have been superseded by compounds such as guanidinium thiocyanate or hydrochloride.

In the early years DNA for example was extracted by binding to glass beads or even diatomaceous earth which is still used to clear fruit juice for example. Nowadays, various types of silica are used. These include amorphous silicon dioxide and glass powder, alkyl silica, aluminium silicate (zeolite) or activated silica with -NH2 (amino) which are suitable as nucleic acid binding materials. Glass powder and beads have largely been superseded by silica gel.

Nucleic Acid Purification – The Procedure

As with all nucleic acid procedures, cells are lysed, the nucleic acid is bound to the silica which is usually ion the form of a gel membrane, this is washed and then the nucleic acid is eluted. Lysis follows a typical routine.

The lysate is buffered and ethanol or isopropanol is added to form a binding solution. The whole mix is transferred to the spin column which is placed in a centrifuge. Centrifugation forces the binding solution through the silica gel membrane inside the spin column. When the pH and salt concentration of the binding solution are optimal, the nucleic acid binds to the silica gel membrane as the solution passes through.

The gel membrane is washed with buffer with any flow-through removed. The column is placed in a centrifuge again which forces more wash buffer through the membrane. Any remaining impurities on the membrane are removed and only nucleic acid is left on the silica gel.

Elution occurs by adding more wash buffer and then an elution buffer which may be just water. The column is placed in the centrifuge again which forces the elution buffer through the membrane. The elution buffer removes the nucleic acid from the membrane and it is collected from the bottom of the column.

The method has also been adapted further into a magnetic-bead process. It is also described as the magnetic beads process because magnetic materials are incorporated into the silica particles.

The use of magnetic beads to isolate and purify nucleic acids from complex samples has been often patented. Two patents prepared by Amersham International Plc., US Pat. Nos. 5,665,554 and 5,523,231 describe the use of magnetic beads to isolate total DNA by precipitation.

Commercial kits include the DNeasy® Blood and Tissue Kit and the DNeasy® Plant Mini Kit (Qiagen), as well as the NucleoSpin® Plant Kit II (Macherey‐Nagel).

(2) Chelex Extraction

This involves adding the Chelex resin to the sample, boiling the solution, then vortexing and centrifuging it.

Chelex is a chelating material made by Bio-Rad. It is used to purify other compounds via ion-exchange. the resin is used for DNA purification in preparation for the polymerase chain reaction by binding cations including Mg2+ ions. Magnesium ions are cofactors for DNases. Chelex protects the sample from DNases that might remain active after the boiling and could subsequently degrade the DNA, rendering it unsuitable for PCR. After boiling, the Chelex-DNA preparation is stable and can be stored at 4°C for 3–4 months. Polar resin beads bind polar cellular components after breaking open cells, while DNA and RNA remain in water solution above chelex (Walsh et al., 1991).

The cellular materials bind to the Chelex beads, while the DNA is available in the supernatant. The Chelex method is much faster and simpler than organic extraction, and it only requires one tube, which decreases the risk of DNA contamination. Unfortunately, Chelex extraction does not yield as much quantity and the DNA yielded is single-stranded, which means it can only be used for PCR-based analyses and not for RFLP.

(3) Anion-Exchange Columns

The exchange columns is based on interaction between negatively charged phosphates in DNA and positively charged particles.

1. The DNA binds under low-salt conditions.
2. The protein and RNA are washed away using higher salt buffers.
3. DNA is eluted with high salt which neutralizes negative charge on the DNA.
4. The eluted DNA is recovered by ethanol precipitation.

The advantages here are no organic solvents and it is generally faster and more hands on than using the silica based method which doesn’t require ethanol.

DNA Quantitation

Once DNA is purified, it is usually quantified. Typical quantities are usually in the milligram to picogram range which is quite a range. Two assays are used: absorbance or fluorescence to measure nucleic acid concentration. Most laboratories use as adapted single cuvette protocol usinga 96- and 384-well microplate-based format. These are standardized formats which are used in conjunction with various pieces of instrumentation that allow for rapid quantification of large number of samples. 

Quantification Methods In Nucleic Acid Purification Spectrophotometry

The method uses light absorbance to measure concentration. Given most biological methods absorb light this method is ideal as absorbance of light occurs at specific wavelengths.

• Ideal for determining dsDNA, ssDNA, RNA at a wavelength of 260nm

Fluorometric Methods

Relies on fluorescence and is ideal with dsDNA. Fluorescent dyes include PicoGreen. The level of purity is based on the ratio of absorbance at 260nm to 280nm.

RNA Extraction And Isolation Method

RNA is less straightforward to extract than DNA but the methodology is more or less similar. Eliminating inhibitory materials and RNAses can be especially difficult. RNases, similarly to DNases are highly active once released into the milieu. Typically knocking out endogenous RNAses relies on guanidine isothiocyanate in a lysis solution.

Commercial manufacturers with suitable technologies for general RNA purification include a Qiagen silica column, the Ambion magnetic bead approach and Trizol which is a chloroform-phenol solution. The Trizol approach is now increasingly becoming one of the most popular approaches.

Obtaining a high quality RNA is the most important step of any operation using this nucleic acid. Scrupulous handling of samples along with RNase free materials reduces the risk of RNA degradation to a minimum and prevents introducing new RNases into the mix.

RNase contamination occurs by a number of routes such as from body fluids including perspiration, from cross-contamination via pipettes and test tubes, from lab surfaces and even air-borne organisms, the water and buffers must be filtered well. One of the most expensive aspects of the purification process is using certified RNase-free tips and tubes, excluding dust and bacteria, using RNase free water and always using best practice in the laboratory.

The isolated RNA is stored in an RNase free solution. If it is used within 24 hours it can stored at fridge temperatures otherwise anything longer means storage at -70 Centigrade.

The Guanidinium-Based Organic Isolation Method Or The Guanidinium Thiocyanate-Phenol-Chloroform Extraction

A phenol/guanidinium solution disrupts cells and solubilizes their cell components but is gentle enough to retain the integrity of the RNA polymer. The method involves adding chloroform which is mixed and centrifuged. The proteins and DNA remain at the interface. The RNA is removed with the aqueous top layer. This is precipitated with alcohol and then rehydrated. Its advantage is it is faster than CsCl method. The main disadvantage is it needs a fume hood and there are hazardous waste disposal issues

The method only takes 4 hours.

Filter-Based RNA Isolation

This method involves specialised filter-based, spin basket formats that use membranes seated at the bottom of a small plastic basket. 

The samples are lysed in a buffer containing RNAses inhibitors which are usually guanidine salts that are bound to the membrane by passing the lysate through the membrane using centrifugal force. The wash solutions are passed through the membrane and discraded. A appropriate elution solution is applied and the sample is collected into a tube by centrifugation.

The advantage of the spin basket approach is its convenience and ease of use. RNA can be isolated from DNA. Membranes can be manufactured of various dimensions. Drawbacks include a propensity for the membranes to clog up with particulates. Plenty of other nucleic acid can also bind to the membranes which reduces specificity of the technique.

Magnetic Bead RNA Extraction

Similar in many respects to DNA extraction, paramagnetic beads witha special nucleic acid binding surface will adsorb RNA following cell lysis. The bead with RNA is captured on a magnetic system whilst the supernatant containing the cell debris is washed away along with other contaminants in further washes.

A 96 well format means that throughout it high. The method has equivalent sensitivity to a Qiagen procedure for tracheal specimens. It is more sensitive for CL swab specimens.

Oligp(dT)-Cellulose Chromatography

Ideal for purification of RNA. essential for purifying large quantities of RNA from mammalian cells.

RNA Extraction From Yeast

Yeast are one of the most difficult cells to break and release RNA. The yeast are usually suspended in a sodium acetate and EDTA buffer which is saturated with phenol. Vortexing means thorough mixing to suspend the cell fragments. The phenol buffer causes cell lysis.

Tubes containing yeasts are centrifuged for 30 seconds.

The top layer is removed using an RNase free blue tip. The whole is resuspended in phenol buffer and centrifuged again as part of a complete hydrolysis. The top layer is taken off and added to a new tube.

A 1:1 phenol plus chloroform solution is added at room temperature. The samples are mixed for 20 seconds by vortexing. The layers are separated by centrifuging the tubes for a couple of minutes. The top aqueous layer is removed for further work.

A 3 M sodium acetate solution at pH 5.3 along with absolute ethanol is added. Wholesale precipitation occurs. The pellet is resuspended in DEPC-treated distilled water followed by further washing with absolute ethanol and then 70% ethanol. Further centrifugation means a pellet is created whilst the supernatant is discarded. The open tube is often dried for 5 minutes before redissolving in distilled water. Usually the RNA is diluted to 1 microgram/microlitre.

Extraction Of Plasmid DNA

When bacteria are lysed under alkaline conditions (pH 12.0–12.5) both chromosomal DNA and protein are denatured; the plasmid DNA however, remains stable. Some scientists reduce the concentration of NaOH used to 0.1M in order to reduce the occurrence of ssDNA. After the addition of acetate-containing neutralization buffer the large and less supercoiled chromosomal DNA and proteins precipitate, but the small bacterial DNA plasmids stay in solution.

Kits are available from various manufacturers to purify plasmid DNA depending on the yield size.  

References

US Patent 5523231A (1996-06-04). Method to isolate macromolecules using magnetically attractable beads which do not specifically bind the macromolecules. (Amersham Int. Plc.)

US Patent 5665554A (1997-09-09) Magnetic bead precipitation method. (Amersham Int. Plc.)

WO2010072834A1 (2010-07-01) Nucleic acid purification method (Qiagen GmbH)

WO2013045432A1 (2013-04-04) Rapid method for isolating extracellular nucleic acids (Qiagen GmbH)

Walsh, P.S., Metzger D.A., and Higuchi, R. (1991). Chelex 100 as a Medium for Simple Extraction of DNA for PCR-Based Typing from Forensic Material. BioTechniques. 10 (4): pp.506–513. PMID 1867860