Sandwich hybridization DNA/RNA capture using magnetic beads

The vision of a cheese sandwich is actually quite useful when trying to understand the sandwich hybridization technique. In this analogy the cheese represents the single strand (ss) target DNA or RNA. The bread represents the oligonucleotide probe. For each target there are two oligonucleotide probes. The probes are synthesized to be perfectly complementary to the ends of the target ssDNA or RNA. Therefore, during an incubation period each oligonucleotide probe will bind to its specific end of the target DNA: one at the 5’ end and one at the 3’ end.

One probe is conjugated to a magnetic bead

Sandwich hybridization of DNA or RNA is not very useful unless there is a way to detect that it has occurred. This is where magnetic nanoparticles (MNPs) come in. Oligonucleotide probes are conjugated to the surface of a polymer coated magnetic particle by taking advantage of free amine groups and electrostatic interactions.

The magnetic nanoparticles, once hybridized with the target DNA, can be collected by magnetic separation, but can also be detected by a μNMR device. However, detection based on the magnetic particle alone is limited. The other oligonucleotide probe can be used to improve quantitative detection of hybridization 

The other probe is used to improve quantitative detection

The other oligonucleotide probe can be conjugated to a variety of objects: polymer nanoparticles, fluorescent probes, quantum dots, nanorattles. The object used depends on the goal of the assay.

The benefit of a polymer nanoparticle is that many probes can be conjugated to it at once. This means that multiple target DNA’s can bind to a single particle and become more concentrated, which is useful when characterizing a diluted or fragile sample.

The fluorescent tags, quantum dots, and nanorattles (raman spectroscopy) are all different techniques for achieving real time quantitative measurements of the hybridization process. This is extremely useful for designing point of care (POC) in vitro diagnostic (IVD) systems for the detection disease or infection.  

The workflow for a POC diagnostic would proceed as follows:

1. Design oligonucleotide probes to match target DNA.

2. Conjugate one probe to magnetic nanoparticles.

3. Conjugate the other probe to a fluorescent tag.

4. Incubate the probes with the sample. If the target DNA is present then hybridization will occur.

5. Use magnetic separation to collect the magnetic particles in a location where fluorescence can be measured.
6. A fluorescent signal will be detected if the target DNA is present in the sample. If there is no target DNA in the sample then no fluorescent signal will be detected.