The Problem

Overview

Column, bead, and precipitation-based kits used for extracting nucleic acids all follow the same fundamental workflow.



Cellular lipid membranes are broken down through the use of detergents, releasing proteins and nucleic acids into a solution.(1)
DNA and other polar molecules dissolve in water.(2)
Water's hydrogen molecules have a partial positive charge, and the oxygen molecule has a partial negative charge. The oxygen and nitrogen atoms on the phosphodiester backbone make DNA polar.(3, 4, 5)
Hydration shells are formed around polar solutes through dipole-dipole interactions.(6)
Proteases cleave proteins and strong salts, termed chaotropic salts, will further denature them along with dehydrating molecules.(7, 8)
Non-chaotropic salts (e.g. sodium chloride) cannot readily interact with the negatively charged phosphodiester backbone of DNA due to the high permittivity of these hydration shells. Chaotropic salts (e.g. guanidinium chloride) destabilize hydrogen bonding and form ion-dipole interactions between guanidinium and the phosphodiester backbone.(9)
The relative permittivity of a solvent (e.g. water, EtOH) is a measure of its polarity and ability to insulate solute charges (e.g. salt, DNA) from each other. Water has a high relative permittivity (80.1 at 20oC). When you swap water for EtOH, the lower permittivity (24.5 at 20oC) allows for cation-backbone interactions from non-chaotropic salts.(10, 11)


The cation-backbone interaction neutralizes DNA’s negative charge, and causes it to precipitate out of solution. The addition of EtOH also promotes aggregation due to inter helical interactions from diminished phosphate repulsion.(11-13)


After, DNA needs to be separated from the other cellular components in solution. Column-based kits will use silica to bind nucleic acids.
Inside the column is a porous monolythic substrate made of silica. This hydrophilic silica gel matrix has a positive charge.
Columns are typically made of silica particles, but can also be made of glass particles, glass powder, and glass microfibers.(14)


Polysaccharides and proteins do not bind to the column, and neither will DNA in a low salt solution. Adding a chaotropic salt will break the positive hydrogen bonds between water and negative oxygen molecules in silica. It will also dehydrate DNA, promoting bond formation between DNA and silica.(14-18)

EtOH-based wash solutions are added to the column to remove salt from DNA and wash out any trapped cellular components. Because DNA is bonded to a silica matrix within a tube, an external force needs to be applied to move liquid through the column efficiently. Typically this is done through the use of a centrifuge. Vacuum manifolds can also be employed.

Adding a low or no salt elution buffer releases DNA from the silica gel matrix. Typically around 80% of DNA is recovered due to imperfect binding and residual volume left in the column.(19, 20)

Adding a low or no salt elution buffer releases DNA from the silica gel matrix.(15)
Column-based extractions typically yield DNA with high purity but may be fragmented due to the workflow process. Overloading the column will result in a decreased yield and purity due to the potential for incomplete lysis and binding interference from debris. Decreasing wash volume or not drying the column after the final wash will promote residual salt and/or EtOH in the elution buffer.(19)
Bead-based kits commonly use carboxyl.
Beads across kits may vary in composition but they all have the same fundamental properties. They are only magnetic within a magnetic field, and reversibly bind DNA. Typically, they are coated in carboxyl groups (e.g. SPRI beads), which are negatively charged.(22)
Variations on bead construction require a tight match of buffer and bead surface chemistry. Beads can be coated with functional groups (e.g. synthetic polymers) or uncoated (e.g. porous glass).(23)

Anatomy of a Bead




Carboxyl molecules are very efficient at binding DNA, and beads have a larger binding capacity than columns. Because both the carboxyl groups and DNA are negatively charged, there is a slight repulsion between them when hydrated. Salt is needed to neutralize the DNA backbone, and the strength of the DNA-carboxyl bond is based on the salt concentration.(24)
The slight repulsion between DNA and the carboxyl groups allow for controlled, reversible binding.


High salt and PEG are needed for efficient DNA binding. A chaotropic salt dehydrates molecules and allows for DNA to transition to a clumping, flocculated state. PEG also excludes water volume to DNA and acts as a crowding agent. Together, DNA precipitates out of solution and binds beads.(24, 25)

EtOH is added to the magnetized pellet, and removes salt from the DNA. A low or no salt elution buffer releases DNA from the beads.

Typically, yield ranges from 80-90% as less volume is lost on beads. However yield can be significantly decreased if beads are overdried.(26) Purity is typically high as particles are resuspended during binding, and this helps remove contaminants. Residual EtOH and beads are the highest risk to purity. Residual EtOH present during bead resuspension and elution can interfere with downstream applications in addition to creating variable net volumes. Residual beads can interfere with downstream QC such as electeropherogram analysis on Agilent’s BioAnalyzer or cause light scatter and abnormal absorbance spectra.(27)

There are additional handling sensitivities. Many types of beads are stored at 4C and require temperature equilibration prior to use. For all bead types, there is a targeted volumetric ratio of beads to solution, therefore complete bead resuspension and careful pipetting is critical.(25, 26)

Solution-based kits do not employ a physical substrate. Instead, centrifugation along with selective precipitation separate nucleic acids.
Liquid Phase Extraction (LPE) is the oldest extraction method. It is used to isolate DNA from other cellular components through phase separation by relying on the affinity of DNA to another immiscible liquid phase. In organic extractions, proteins remain in the phenol/chloroform phase, whereas DNA is removed from the interface of the organic and aqueous phase.(28, 29)
Organic extraction uses Guanidinium Thio-Phenol-Chloroform. Phenol:chloroform is added, followed by EtOH precipitation. These solutions are highly toxic.


Alternative techniques to chloroform-based organic extractions include a salt precipitation to remove proteins followed by an EtOH DNA precipitation.(30) Also, there are single solutions that solubilize all cellular components and allow for EtOH DNA precipitation. In general, both salt and EtOH precipitate DNA, and lower temperatures encourage DNA to transition to a clumping, flocculated state.

In addition to being difficult to automate, there is a higher risk of decreased purity from DNA isolated at an interface due to residual organic solvents. There is also a risk of residual EtOH from incomplete drying, or decreased yield from overdrawing.


Salt and debris are washed away by ethanol.
Both water and EtOH are polar and can solvate ions in accordance with their relative permittivity; EtOH can only dissolve some salts. Because EtOH and water are miscible, changing the EtOH:water ratio can allow for selective ionic precipitation.(31)
DNA is rehydrated and resuspended in an aqueous solution.
DNA is dissolved in a solution that typically contains Tris, a buffering agent, and EDTA, a chemical that sequesters metal ions. The combination of these two agents provide DNA with an optimal pH and prevents it from degradation.(32)
DNase activity is prevented by EDTA because the hydrolytic cleavage of phosphodiester bonds requires magnesium ions.


The concentration of eluted DNA can be determined using intercalating dyes such as PicoGreen and Qubit dsDNA Reagent. They are highly specific to dsDNA, and because fluorescence is a readout, contaminants mostly will not affect it. However, concentration may be overestimated if there is contaminating RNA due to dye intercalating into secondary structure. PicoGreen is also known to be sensitive to CTAB, a detergent used in some DNA extractions which will reduce or quench the signal.(21, 33) Finally, all of these assays are destructive, and require sacrifice of part the sample.

Absorbance can be used to obtain a baseline assessment of DNA purity. Double-stranded DNA molecules have the highest absorbance of light at 260 nm due to nucleotide heterocyclic rings.(34) However, other molecules can also absorb at that wavelength, therefore assessing at least two wavelengths such as an A260/280 ratio can be used to inform DNA purity. A value of 1.8 is used as benchmarks for pure DNA; values between 1.8-2.0 are also considered pure.(27) Lower A260/280 ratios may be indicative of protein contamination due to absorbance from aromatic ring structures.(34)

A260/230 can also be used to assess purity. Sample values between 1.8 and 2.2 are considered pure. Lower A260/230 can be indicative of residual chaotropic salts or organic compounds, or blanking with water instead of TE.(27, 34)

It is possible for a DNA sample to have ratios that indicate purity with the presence of some contaminants. Contaminants can be detected by a shift in the wavelength trough, therefore the entire spectrum should be assessed.(27)

Other Molecules that Absorb at


A260 A230
Protein Guanidine HCl
RNA EDTA
Single stranded DNA Carbohydrates
Free nucleotides Urea
Phenol Phenolate ions
Other contaminants

Residual ethanol may be present in the eluate if DNA does not freeze at -20oC, floats out of the well when loading an agarose gel, and/or smells of ethanol.(21, 35) DNA-protein complexes will run slower than naked DNA in an agarose gel; a doublet band may also be detected. It may also appear as if DNA did not enter the gel, as the well may be stained. Commonly, if protein-DNA complexes are suspected the sample is treated with proteinase K and repurified.(21)



References

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