In the field of molecular biology and biomedical research, fluorescent labeling is an indispensable tool for high-sensitivity detection and analysis of biomolecules. FAM Phosphoramidite (5'-Fluorescein-CE Phosphoramidite) is a crucial chemical reagent widely used for synthesizing fluorescently labeled oligonucleotides and other biomolecules. This article provides a detailed introduction to the structure, synthesis process, applications, and significance of FAM Phosphoramidite in research.

Structure and Characteristics of FAM Phosphoramidite

The core structure of FAM Phosphoramidite consists of a fluorescein (FAM) group linked to a nucleotide via a phosphoramidite bond. FAM is a commonly used green fluorescent dye with the following features:

• Excitation and Emission Spectra: FAM emits strong green fluorescence at approximately 520 nm when excited by blue light around 495 nm.
• Chemical Stability: The FAM group exhibits high chemical stability during synthesis and storage, making it resistant to degradation or inactivation.

Synthesis Process

In the laboratory, FAM Phosphoramidite is integrated with other nucleotides using an automated DNA synthesizer, typically added to the 5' or 3' end of the oligonucleotide. The synthesis steps are as follows:

1. Phosphoramidite Chemistry: Utilizing phosphoramidite chemistry, nucleotide units are sequentially linked. FAM Phosphoramidite is added as the final unit in the reaction.
2. Deprotection: After synthesis, a deprotection step removes protecting groups, releasing the complete oligonucleotide with the FAM label.
3. Purification: The synthesized oligonucleotide is purified using high-performance liquid chromatography (HPLC) to remove unreacted reagents and by-products.

Application Areas

Quantitative PCR (qPCR):

• Principle: In qPCR, FAM-labeled probes or primers are used to monitor the accumulation of PCR products in real-time. The fluorescence intensity of FAM correlates with the amount of target DNA, enabling quantitative analysis.
• Advantages: High sensitivity and specificity, suitable for gene expression analysis, pathogen detection, and mutation screening.

Fluorescence In Situ Hybridization (FISH):

• Principle: FISH uses FAM-labeled probes to hybridize with target nucleic acid sequences, observed under a fluorescence microscope to determine the location and distribution of specific genes in cells or tissues.
• Applications: Used in genomic research, cancer diagnostics, genetic disorder detection, and cell typing.

DNA Sequencing:

• Principle: In Sanger sequencing or next-generation sequencing (NGS), FAM-labeled nucleotides mark amplified fragments. Fluorescence signals are detected to determine nucleotide sequences.
• Advantages: High throughput and accuracy, widely used in genome sequencing, variant detection, and functional gene studies.

Molecular Biology Research:

• Fluorescence Resonance Energy Transfer (FRET): FAM labeling is used in FRET experiments to study molecular interactions by monitoring energy transfer between donor and acceptor molecules, such as protein-protein or protein-DNA interactions.
• Cell Imaging: FAM-labeled oligonucleotides are used for cell imaging to study gene expression and intracellular molecular dynamics.

Considerations

While FAM Phosphoramidite excels in various applications, certain precautions are necessary during use and storage:

• Light Sensitivity: FAM-labeled oligonucleotides are light-sensitive and should be handled and stored in dark conditions to prevent fluorescence signal degradation.
• Stability: FAM Phosphoramidite and its labeled oligonucleotides are stable under appropriate conditions but should avoid extreme pH and temperatures to maintain activity and performance.
• Purity: Ensuring high purity of synthesized FAM-labeled oligonucleotides through rigorous purification is crucial for accurate experimental results.

Conclusion

FAM Phosphoramidite, as a key fluorescent labeling reagent, provides a powerful tool for molecular biology and biomedical research. Its extensive applications in quantitative PCR, fluorescence in situ hybridization, DNA sequencing, and molecular interaction studies enable scientists to conduct high-sensitivity and high-specificity detection and analysis, driving advances in genomics, diagnostics, and drug development. With ongoing technological progress, FAM Phosphoramidite and related techniques will continue to bring innovations and breakthroughs to life science research.

High-Quality Fluorescently Labeled Oligonucleotide Synthesis: Choose SBS Genetech

When selecting reagents for fluorescently labeled oligonucleotide synthesis, quality and reliability are paramount. As an industry-leading biotechnology company, SBS Genetech is committed to providing researchers with high-quality fluorescently labeled DNA Synthesis Products. We utilize advanced synthesis technology and rigorous quality control systems to ensure that each batch of products boasts exceptional purity and stability.

Our Advantages Include:

• Strict Quality Control: Each batch undergoes rigorous quality testing, including spectroscopic analysis and mass spectrometry verification, to ensure its fluorescent performance and chemical stability.
• Custom Services: We offer custom synthesis services tailored to customer needs, meeting various experimental requirements, including special modifications, specific sequences, and labels.
• Technical Support: Our expert team provides comprehensive technical support and consultation services to help customers resolve various issues encountered during experiments.

Extensive Application Areas

Our labeled oligonucleotides are widely used in the following research areas:

• Gene Expression Analysis: Highly sensitive qPCR for precise measurement of gene expression levels.
• Pathogen Detection: Rapid and accurate detection of various pathogens, including viruses, bacteria, and fungi.
• Genomic Research: Used in genome sequencing, SNP detection, and mutation analysis, driving advancements in genomics research.
• Molecular Interaction Studies: Utilizing FRET technology to study molecular interactions, revealing dynamic mechanisms of biomolecules.

Welcome to Order

At SBS Genetech, we are dedicated to advancing the frontiers of scientific research. We understand the complexity and rigor of scientific work, and it is our responsibility and mission to provide high-quality reagents and excellent service. If you need high-quality fluorescent labeling reagents, please feel free to contact us. We are committed to providing you with superior products and professional support, helping you achieve greater success in your research endeavors.

Choosing SBS Genetech means choosing excellence and reliability. Let us work together to contribute to the bright future of scientific research.