45 - 1 Ramsey Road, Shirley, NY 11967, USA Phone: 1 - 631 - 633 - 6938 ( USA) 44 - 161 - 818 - 6441 (Europe) Fax : 1 - 631 - 938 - 8221 Email : info@cd - bioparticles.com www.cd - bioparticles.com Magnetic Particles for Genotyping Based on Hybridization Probes March 2023 2 Single nucleotide polymorphism (SNP) is a genetic mutation that occurs at a frequency of no less than 1% when a single base variant is found in an organism. It is associated with an organism’s susceptibility to disease and tolerance to drugs. With the completion of the Human Genome Project (HGP), the focus of research on human genetic information has shifted over time from the study of DNA sequences to the study of biological functions at the molecular level in order to provide better protection for human life. It is imperative to create a nucleic acid detection platform suitable for clinical use that is efficient, rapid, capable of handling multiple samples and multiple polymorphous loci, and high throughput. Various detection techniques have been developed to identify SNPs, although few have been widely accepted. High purity and quality nucleic acid molecules are most often obtained prior to SNP identification. Many methods, including Chlorophenol , high - intensity Salt Precipitation, Silica Gel Column, and the increasingly popular magnetic particle method can be used to extract and purify nucleic acid molecules. The magnetic particle method has become a good choice for extraction steps due to the following advantages : High purity of target nucleic acids Consistent quality of resulting products Absence of toxic reagents and centrifugal steps Low technical requirements Short operation time Automatic extraction capacity Magnetic Particles for Genotyping They are generally composed of magnetic cores encapsulated into the polymer matrix and commonly modified with a layer of silica or gold coating with terminal functionalized groups. The enrichment of target nucleic acids by magnetic beads can improve the specificity and sensitivity of the molecule prior to hybridization, allowing for more accurate detection of target nucleic acids of specific lengths in complex samples. The streptavidin (SA) magnetic particles can be used to immobilize biotinylated PCR products. After dual - color hybridization, the analysis of fluorescence signals from the surface of the magnetic particles reveals the results of genotyping. 3 Several SNP genotyping methods for disease - associated studies requiring large sample sizes have been developed, such as TaqMan probe, molecular beacons, and dynamic allele - specific hybridization. Taqman Probe Figure 2. The flow diagram of nucleic acid extraction by magnetic particle method The TaqMan probe technique is a commonly used method to detect known SNPs. When a mixture of nucleic acids and protein impurities is obtained from a biological sample, magnetic particles are added for extraction. After binding to the DNA in the mixture, an external magnetic field draws the particles to the outer edge of the containment tube, immobilizing them. When the particles are immobilized, the DNA bound with the particles is retained during the washing step. An eluting buffer is added and the magnetic field is removed, after which the DNA is released as a purified sample to be used as a test sample for the TaqMan probe. The TaqMan probe technique uses fluorescence resonance energy transfer (FRET) to detect DNA sequences. Fluorophore is modified at the 5’end of the probe and the quenching group is modified at the 3’end. When the probe is integrated with the target DNA, the fluorophore is close to the Figure 1. SNP genotyping mechanism. 4 quenching group, so FRET quenches the fluorescence. When the probe is cut off by polymerase, the fluorophore is separated from the quenching group and the fluorescence recovers. SNPs can be identified by checking the fluorescence intensity. The advantage of this method is that it is simple to use and allows for a one step PCR reaction, without the need for purification and pretreatment. Additionally, the results are reliable. However, it is only suitable for the detection of a few sites and high sample flux. A pair of probes is required for each locus, increasing the cost of probe synthesis. Figure 3. Schematic diagram of TaqMan probe working principle Molecular Beacons Molecular Beacon is a tool similar to the TaqMan probe. It consists of a stem and a loop molecular The 5’ end contains a fluorescent group and the 3’ end has a quenching group. When the Molecular Beacon does not match the target DNA, the fluorophore and quencher remain close together, preventing the occurrence of fluorescence. This high specificity allows the detection of SNPs with different colored fluorophores. Figure 4. Schematic diagram of the working principle of molecular beacons 5 Dynamic Allele - Specific Hybridization (DASH) DASH is mainly used to identify SNPs when the denaturation temperature of the probe and target binding is higher than that of the incomplete binding. It measures the denaturation temperature of the double strand by recording the decrease in fluorescence signal when the temperature reaches the denaturation point. This method is simple to operate, but it does not accurately determine the type of SNPs. Related products: CD Bioparticles provides Silica Magnetic Particles and Purification kits designed for Genotyping Silica Magnetic Particles Gold Coated Magnetic Nanoparticles Carboxyl , Streptavidin , Hydroxyl, Amine, and Heparin functionalization are available • MagIso ™ DNA & RNA Magnetic Silica Particles • Oligo(dT) Magnetic Particles • Carboxyl Magnetic Particles • Streptavidin Silica Magnetic Particles Bulk purchasing options are available — Please contact us if you are interested Catalog # Product Name WHK-CG01 MagIso ™ Genomic DNA Purification Kit, Blood WHK-CG02 MagIso ™ Blood & Tissue DNA Kit WHK-CG03 MagIso ™ Genomic DNA Purification Kit, Dried Blood Spot (DBS) WHK-CG15 MagIso ™ Plasmid DNA Purification Kit WHK-CG16 MagIso ™ Bulk Plasma cfDNA Extraction Kit WHK-CG19 MagIso ™ DNA Select Isolation Kit WHK-CG30 MagIso ™ DNA Reverse Purification Kit DNA Extraction and Purification Kit