Nucleic Acid Extraction and Concentration

The use of cationic colloidal supports (i.e., positively charged particles) in nucleic acid adsorption (i.e., nucleotide multimers) according to size has been explored in numerous studies. The interaction between cationic particles and such negatively charged polyelectrolytes is believed to be based on electrostatic

FIG. 4 Selective purification of mRNA with magnetic beads bearing dT oligonucleotide.

Poly-dT

Poly-dT

FIG. 4 Selective purification of mRNA with magnetic beads bearing dT oligonucleotide.

Poly-dA

Poly-dA

attractive forces. After nucleic acid adsorption and magnetic separation of the colloidal particles, conditions such as pH, salinity, and temperature are adjusted to release the nucleic acids from the support for amplification or direct analysis as schematized in Fig. 5.

For the Human Genome Project the Whitehead Institute at MIT developed a method called solid-phase reversible immobilization (SPRI) wherein DNA is captured onto carboxyl-modified encapsulated superparamagnetic microspheres. After the DNA is bound, the beads are washed with ethanol. The DNA is then eluted from the beads in a low ionic strength solution. This method leads to high-quality DNA template purification and can be used with major templates and enzymes responsible for sequencing [47]. It can also be applied to detecting hybridization between complementary nucleic acid sequences. A long poly-nucleotide that is partially complementary to a short oligonucleotide is chemically grafted onto the magnetic particles. The short probe is used as a spacer arm and does not bind directly to the particles but rather to the first bonded single-stranded nucleic acid. The specific capture of any nucleic acid sequence can then be performed by a hybridization process on the second, short oligonu-cleotide, as schematically presented in Fig. 6.

The hybridization can also be performed first in solution. The hybrids are separated from the unbounded probes by magnetic particles bearing a short oli-gonucleotide (with a specific sequence complementary to the target) to capture

FIG. 5 Illustration of nucleic acids adsorption, extraction, concentration, and amplification.

ODN-2

ODN-1

ODN-2

Target

FIG. 6 Schematic illustration of specific capture of nucleic acid fragment by a double-hybridization process onto magnetic polymer particles. ODN-1 oligonucleotide (of a given sequence); ODN-2 oligonucleotides act as a spacer arm and specific to a given part of ODN-1.

Target

ODN-1

FIG. 6 Schematic illustration of specific capture of nucleic acid fragment by a double-hybridization process onto magnetic polymer particles. ODN-1 oligonucleotide (of a given sequence); ODN-2 oligonucleotides act as a spacer arm and specific to a given part of ODN-1.

the hybrids. The hybridization yield is 20% in solution and only 5% after binding to the beads.

Another method for capturing free nucleic acids in any biological sample uses positively charged magnetic microspheres for immobilization (via electrostatic interaction) of rRNA as well as DNA molecules. The nucleic acids released are not damaged after the capture of rRNA or DNA and can be amplified via a hybridization process with RT-PCR or PCR. The immobilization of rRNA is favored in the presence of urine combined with acetic acid. Efficient rRNA hybridization is obtained with the proper reagents. RNA/DNA hybrids are recovered from the buffer solution and the unhybridized probes left in solution. Captured polynucleotide probes can then be eluted from the beads with the appropriate conditions. Accordingly, nucleic acids can be purified from such biological samples as cell lysate and sputum. For these applications, various cationic magnetic particles are available; these include quaternary ammonium containing magnetic microsphere and poly-D-lysine-functionalized microspheres. In addition, magnetic particles with amine microspheres have been demonstrated to be compatible with a chemiluminescent nonisotopic assay [31] or electro-chemiluminescence [48] with streptavidin-bearing magnetic particles.

We see from this description of the use of magnetic beads in molecular biology for various applications that various types of magnetic beads appear to be compatible with many enzyme reactions, even PCR or sequencing. Specific biomolecule separation is the application that has been most widely studied, probably because the major advantage of magnetic particles is their easy removability from any liquid simply, just under a magnetic field, even in an unclear and complex solution.

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