Leading the Way in Life Science Technologies

GEN Exclusives

More »

Feature Articles

More »
May 1, 2010 (Vol. 30, No. 9)

Nucleic Acid Sample Prep

Faster, Highly Automated Methods Are Being Developed

  • Electrophoresis with a Twist

    Among the challenges Boreal Genomics sought to overcome in its development of a method for purifying and concentrating nucleic acids are the need to remove contaminants that can interfere with PCR-based amplification, the difficulty in extracting and enriching for low-abundance nucleic acids, the recovery of nucleic acids from dilute samples, and the need to accommodate a variety of sample types, including complex mixtures, viscous samples, and samples containing particulate matter such as soil and sand.

    At the heart of Boreal’s approach to nucleic acid sample prep is the synchronous coefficient of drag alteration (SCODA) electrophoretic concentration technology. Traditional electrophoresis separates samples linearly in a gel across which voltage is applied. The distance each molecular species travels through the gel depends on differences in their physical properties such as size, charge, linear charge density, and stiffness/conformational entropy.

    SCODA takes advantage of the strong charge of nucleic acids and of their non-linear electrophoretic behavior when exposed to a rotating electric field. The drag on nucleic acids that causes them to move in a spiral pattern rather than a circular orbit and to travel toward the center of the electric field relates to their long length and variable conformation.

    With the SCODA technique, injection of up to 5 mL of a cell lysate or other type of sample directly into the gel and application of a rotating electric field, sends the charged molecules in the sample into periodic orbits. After a full cycle of rotation, the nucleic acids will begin to drift toward the center of the gel, whereas contaminants will maintain circular orbits and return to their initial location.

    Ultimately, the back-and-forth pattern of field strength will cause the nucleic acid component of the sample to converge and focus to a tightly packed spot from which they can be eluted into a centrally located well in 10–50 µL of buffer.

    This method offers “advantages over column- or bead-based methods that rely on chemical affinity to separate mixtures,” says Andre Marziali, Ph.D., president and CSO of Boreal and director of engineering physics at the University of British Columbia.

    As SCODA technology relies only on differences in physical properties, it can reject contaminants that have chemical properties similar to those of nucleic acids. Furthermore, “because we are just enriching the nucleic acids that are already there,” compared to PCR amplification-based purification strategies, “we are not introducing any background noise.”

    He believes that SCODA will be beneficial in applications ranging from genetic biomarker detection, extraction of pathogen-derived nucleic acids from medical samples, isolation of fetal DNA from maternal blood, forensic studies, and nucleic acid analysis of plant material and soil samples.

    In his presentation, Dr. Marziali will describe the company’s second-tier technology that makes the focusing process sequence-specific by attaching short, single-stranded DNA probe sequences to the gel. When electrophoresis is performed at a temperature near the probe-target melting point, the target nucleic acid sequences present in a sample will briefly hybridize with the complementary probes immobilized on the gel, hampering their progress until they are again propelled forward by the transient electric field.

    The more time a molecule spends bound to probes, the less circular its orbit becomes. The bottom line, says Dr. Marziali is that by defining the probe sequences, “we can extract focused, matched sequences with single nucleotide resolution.”

Related content

Be sure to take the GEN Poll

Cancer vs. Zika: What Worries You Most?

While Zika continues to garner a lot of news coverage, a Mayo Clinic survey reveals that Americans believe the country’s most significant healthcare challenge is cancer. Compared to other diseases, does the possibility of developing cancer worry you the most?

More »