The Solution

Overview

Charge is a fundamental attribute of any biological molecule; in solution, it can be used not only to separate molecules, but also to concentrate and focus them.

DNA is negatively charged because of the phosphate groups present on every nucleotide. Phosphate (PO4) has a -3 formal charge.(1) Two ester bonds are formed between the 3' carbon and 5’ carbon atoms of two sugar molecules, leaving a residual charge of -1.(2) In an aqueous solution near neutral pH, the phosphase groups on the DNA backbone have an ionization of the form H2PO4-

Electric fields applied to a solution will create a force on the ionic molecules which pushes them in a direction. Cations migrate to a negatively charged electrode and anions migrate to a positively charged electrode. The greater the charge of a molecule, the greater the force it experiences. The larger the molecule, the more friction it generates. Therefore mobility depends on the charge to mass ratio, or electrophoretic mobility.(3)

Differences in electrophoretic mobility can be used to separate molecules from one another. Isotachophoresis (ITP) employs non-uniform electric fields to not only separate molecules, but to stack and preconcentrate them.


Standard separation techniques commonly rely on multiple analyte properties, such as differences in charge, size, solubility, and/or binding affinity, in addition to a solid surface.

Chromatography uses two phases, a stationary phase and mobile phase, to separate molecules based on their affinity to the stationary phase. The stationary phase can be a solid or liquid, whereas the mobile phase is a liquid or gas. Ionic, affinity, size exclusion, hydrophobic interaction, and multimodal separation techniques are all common in chromatography.(4, 5)

Ion exchange chromatography separates molecules by charge, which can be adjusted based on the buffer pH. The solid, stationary phase is a resin column functionalized with ligands containing the opposite charge of the molecules of interest. The mobile phase has low to medium conductivity, and adsorption is driven by ionic interactions. Increasing salt will progressively elute weaker to stronger interactions. pH can also be used to affect separation. In a higher pH, positively charged ions will become less protonated, thus less charged whereas negatively charged ions will become more protonated, thus more positively charged (less negatively charged) in lower pHs. In both scenarios, ionic interactivity is decreased thereby promoting elution.The column contains the opposite charge of the molecules of interest, effectively binding them.(6, 7)

Affnity chromatography uses binding interactions to separate molecules of interest. A ligand is bound to the stationary phase, and the binding partner in the mobile phase binds to the ligand and is separated.(8)
Size exclusion chromatography uses spherical beads with a set pore size to include or exclude molecules smaller or larger than the pores respectively. Hydrophobic interaction chromatography uses aliphatic compounds on the stationary phase to bind hydrophobic amino acids by Van Der Waals interactions.(9)

Multimodal chromatography leverages ligands that can interact with molecules of interest using multiple properties (e.g. affinity, ionic, etc.).(10)

Electrophoresis uses an electric field and porous gel matrix to separate molecules based on charge, size, and shape. The gel is a physical structure for containing molecules. Once submerged in buffer, the flow of an electric current through the gel will separate molecules by size. Smaller molecules will experience less resistance migrating though the pore matrix and migrate faster than larger molecules.(11, 12, 13)

Stacking and gradient gel electrophoresis further increase protein resolution by employing high mobility and high concentration Cl- ions in the gel buffer and low mobility low concentration Gly- in the cathode buffer to compress and stack proteins. This stack progressively moves through a gel of continuously decreasing pore size, separating proteins based on frictional resistance.(14)

Ultracentrifugation separates molecules suspended in liquid based on physical properties such as mass, density, and shape using g-forces produced from a high powered centrifuge. Larger and denser molecules pellet to the bottom of a tube, followed by particles of decreasing sediment. Rate-zonal centrifugation employs a density gradient to separate particles into zones based on size and mass, not density, whereas isopycnic centrifugation uses a gradient to separate particles based on density.(15)

Isotachophoresis separates and focuses charged molecules in solution solely based on their ionic mobility.(16)

To separate and concentrate a molecule of interest such as DNA, two solutions with different ionic mobilities are used. The leading electrolyte (LE) contains high conductivity, high mobility negatively charged ions. The trailing electrolyte (TE) contains low conductivity, low mobility negatively charged co-ions.

Etymology of Isotachophoresis


Isos = equal
Takhos = speed
Phoresis = being carried
To maintain electric neutrality and pH within a zone, positively charged counterions (cations) are also added.

A biological sample is gently lysed and added to either the leading or trailing electrolyte.(17) After applying a current, both trailing and leading anions migrate toward the positive electrode. The differential conductivity of these two zones creates an electric field gradient with a high electric field in the low conductivity TE, and a low electric field in the high conductivity LE.

DNA, whose electrophoretic mobility is lower than the leading electrolyte or higher than the trailing electrolyte, will respectively fall behind or speed ahead until reaching the interface between the two solutions. It is in this location, termed the ITP zone, where DNA will focus and concentrate into a near Gaussian peak.(18)

TE and LE chemistries are precisely formulated to selectively focus only target nucleic acid molecules and leave behind unwanted species in the chip’s separation channel. In essence, lysate components will either migrate in the opposite direction (e.g. positively charged magnesium, iron, calcium, etc.), or stay in the trailing electrolyte (e.g. negatively charged serum proteins and surfactants).

To elute DNA, 10 mM Tris-HCl is added to the elution reservoir and DNA is gently electromigrated into it.

The end result is concentrated, purified DNA.



References

  1. "Phosphate" Wikipedia, the free encyclopedia. Wikipedia, the free encyclopedia, 26 April 2017. Web. 9 May 2017.
  2. "Phosphodiester bond" Wikipedia, the free encyclopedia. Wikipedia, the free encyclopedia, 4 January 2017. Web. 9 May 2017.
  3. Woodbury, N. "Electrophoresis" Arizona State University. Accessed 6.6.17.
  4. Giddings, "Chromatography" Encyclopedia Britannica. Web. 10 May 2017
  5. "Types of chromatography" Bio-Rad Laboratories. Web. 10 May 2017.
  6. "Principles of ion exchange chromatography" Tosoh Bioscience LLC. Tosoh Bioscience LLC. Web. 10 May 2017
  7. "Ion exchange chromatography" Bio-Rad Laboratories. Web. 10 May 2017.
  8. "Introduction to affinity chromatography" Bio-Rad Laboratories. Web. 10 May 2017.
  9. "Introduction to size exclusion chromatography" Bio-Rad Laboratories. Web. 10 May 2017.
  10. Multimodal or mixed-mode chromatography" Bio-Rad Laboratories. Web. 10 May 2017.
  11. Chen, C.W. and Thomas, C.A. Jr. "Recovery of DNA segments from agarose gels". Analytical Biochemistry 101 (1980), 339–41.
  12. "Gel electrophoresis of proteins" Wikipedia, the free encyclopedia. Wikipedia, the free encyclopedia, 14 March 2017. Web. 9 May 2017.
  13. "Nucleic acid electrophoresis" Bio-Rad Laboratories. Web. 10 May 2017.
  14. "How does the stacking gel increase resolution during SDS-PAGE?" Promega Corporation. Promega Corporation. Web. 10 May 2017.
  15. "Centrifugation Separations" Sigma-Aldrich. Web. 10 May 2017.
  16. Everaerts, F. M.; Beckers, J. L.; Verheggen, T. P. E. M. Isotachophoresis: theory, instrumentation, and applications; Elsevier Scientific Pub. Co.: Amsterdam and New York, 1976.
  17. Persat, A., Marshall, L.A., and Santiago, J. (2009) Purification of nucleic acids from whole blood using isotachophoresis). Anal Chem 81: 9507-95411
  18. Khurana, T. K.; Santiago, J. G. "Preconcentration, separation, and indirect detection of nonfluorescent analytes using fluorescent mobility markers" Anal. Chem. 2008, 80, 279–286