研究目的
Early detection of mutation carriers in predisposing genes such as BRCA1 plays an important role in disease prevention. This work developed a quantum dots-based (QDs-based) fluorescence resonance energy transfer (FRET) technique for the detection of single-base mismatch DNA in BRCA1 gene.
研究成果
The FRET-based technique using QDs and AgNCs allows for sensitive and selective detection of single-base mismatch DNA in the BRCA1 gene, with a low detection limit and potential for clinical SNP diagnosis. The method distinguishes between complementary and mismatch DNA based on FRET efficiency differences.
研究不足
The method may have limitations in terms of specificity in complex biological samples, potential interference from other biomolecules, and the need for optimization of experimental conditions such as pH and concentration ratios. It relies on synthetic DNA probes and may not be directly applicable to real clinical samples without further validation.
1：Experimental Design and Method Selection:
A FRET system was designed using CdTe quantum dots as donors and DNA-templated silver nanoclusters (AgNCs) as acceptors. The method relies on the interaction between QDs and dsDNA formed by hybridization of target DNA with AgNC-labeled ssDNA, enabling detection based on FRET efficiency differences between complementary and single-base mismatch DNA.
2：Sample Selection and Data Sources:
Synthetic oligonucleotide strands (DNA labeled to AgNC, wild type target, mutant target, non-complementary target) were used, purchased from Shanghai Generay Biotech Co. and purified by PAGE.
3：List of Experimental Equipment and Materials:
Apparatus included Perkin-Elmer lambda 25 spectrometer for absorption spectra, Perkin Elmer LS-45 fluorescence spectrometer for fluorescence measurements, and Zeiss EM10C transmission electron microscope for TEM imaging. Materials included cadmium chloride hydrate, tellurium powder, silver nitrate, sodium borohydride, thioglycolic acid, and deionized water from Millipore Milli-Q system.
4：Experimental Procedures and Operational Workflow:
DNA-AgNCs were synthesized by incubating AgNO3 with DNA, reducing with NaBH4, and aging. CdTe QDs were synthesized from CdCl2, TGA, NaHTe solution under argon flow. Hybridization involved incubating target DNA with DNA-AgNCs at 37°C for 1h, then adding QDs to form complexes for FRET measurement. Fluorescence spectra were recorded with excitation at 370 nm.
5：Data Analysis Methods:
Fluorescence intensity was measured and correlated to DNA concentration. Detection limit and linear range were determined from calibration curves. Selectivity was assessed by comparing responses to different DNA sequences.
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