How does fluorescently labeled dCTP illuminate the invisible DNA world?

Its working principle can be understood as a clever "substitution" during DNA replication.

In molecular biology experiments such as PCR and probe labeling, DNA polymerase uses the template strand to sequentially link deoxynucleotides (dNTPs) to form a new DNA strand.

Essentially, a special dCTP with a Cy-series fluorophore (such as Cy3 or Cy5) attached to the cytosine base undergoes three key steps after being added to the reaction system:

1. Precise Incorporation: DNA polymerase recognizes it as a regular dCTP and efficiently integrates it into the corresponding position of the newly synthesized DNA strand;

2. Permanent Labeling: After incorporation, the Cy3 (orange-red/orange-yellow fluorescence) or Cy5 (red fluorescence) group acts as a "fluorescent beacon," firmly attached to the DNA molecule;

3. Signal Release: Under specific excitation light (Cy3: 550 nm, Cy5: 649 nm), these "beacons" emit intense and stable fluorescence, making otherwise invisible DNA molecules clearly visible for subsequent detection and analysis;

The entire process ensures labeling specificity and high efficiency, forming a core cornerstone for downstream applications such as DNA microarrays, Sanger sequencing, and NGS.

1. Two-color microarray gene chip analysis

Cy3-dCTP and Cy5-dCTP are used to label cDNA from different samples, enabling visualization and precise quantification of gene expression differences through dual-color fluorescence signal overlay and comparison;

2. Sanger and NGS sequencing

In Sanger sequencing or NGS library construction, Cy3/Cy5-dCTP can be flexibly selected for differential fluorescent labeling, facilitating sample pooling and multiplex detection with visual quality control;

3. qPCR quantitative analysis

Compatible with instrument fluorescence channels, Cy3 or Cy5 labeled probes can be easily adapted for singleplex or duplex qPCR quantitative analysis, increasing detection throughput and efficiency;

4. Environmental microorganism detection

Combined with specific probes, Cy3/Cy5-dCTP labeling of target microbial DNA in complex samples such as soil and water aids microbial community structure analysis and rapid source tracing;

✅ High Sensitivity & Precision: Cy3/Cy5-dCTP offers superior sensitivity and specificity, accurately capturing target DNA information for rapid and reliable detection;

✅Rapid Penetration: Unique sulfonation groups provide excellent cell membrane permeability, enabling rapid labeling of live or fixed cells;

✅ Strong Fluorescence: Cy3-dCTP (orange-red) and Cy5-dCTP (red) provide strong, stable fluorescent signals, clearly presenting target locations and reducing observation difficulty;

✅ Flexible Application: Integrates fluorescent reporter groups with biochemical activity, serving as both imaging probes and direct participants in DNA synthesis, compatible with diverse molecular biology applications;

✅ Dual-Color Compatibility: Cy3 and Cy5 dye emission spectra are clearly distinguishable, allowing adaptation to multicolor detection systems and simultaneous analysis of multiple targets in the same reaction, greatly enhancing experimental efficiency;