DETECTION OF REPETITIVE OLIGONUCLEOTIDE SEQUENCES
Modern biomedical research extensively relies on detection and quantification of specific nucleic acid targets. The most widely used technique is based on Polymerase Chain Reaction (PCR), which allows amplification of the target molecule and its subsequent detection by gel electrophoresis or fluorescence (e.g., quantitative PCR). However, it is time consuming, laborious, and is restricted to cases where the tested sample is in reasonably good condition (e.g., the DNA/RNA is intact and no polymerase inhibitors are present).
To detect rapidly a repetitive DNA sequence, we offer two alternatives to the PCR method which are combined with our lab fluorescence-based bio-sensor system, the magnetic modulation bio-sensing system (MMB):
1. Partial PCR - using a double quenched fluorescent resonance energy transfer (FRET) based probe.
2. Non PCR - using a sandwich hybridization assay (SHA).
To detect rapidly a repetitive DNA sequence, we combined a modified double-quenched FRET-based probe with the MMB in our lab. We managed to determine chick sex in ova within 13 min, with 100% sensitivity and specificity. Compared to quantitative PCR, our assay is 4−9 times faster.
In June 8th, 2019, our paper on detection of repetitive nucleic acid sequences using double-quenched FRET-based probe in MMB system was published in the American Chemical Society journal.
The paper, entitled 'Rapid and Sensitive Detection of Repetitive Nucleic Acid Sequences Using Magnetically Modulated Biosensors', can be found in the Publications section, and at DOI: 10.1021/acsomega. 9b01071
An alternative technique to PCR is the sandwich hybridization assay (SHA). In SHA, the presence of the target DNA/RNA sequence is recognized by two oligonucleotide probes, complementary to specific sites on the target molecule. The specificity of a sandwich hybridization assay (SHA) and the high sensitivity of a magnetic modulation biosensing (MMB), have enabled detection of DNA fragments without enzymatic signal amplification. The calculated limit of detection (LoD) was 685 fM, which is at least 15 times better than other commonly used SHA detection techniques.
In August 9th, 2019, our paper on detection of repetitive nucleic acid sequences using SHA in MMB system was published in the Journal of Biophotonics.
The paper, entitled 'Detecting nucleic acid fragments in serum using a magnetically modulated sandwich assay', can be found in the Publications section, and at DOI: 10.1002/jbio.201900104.