OBJECTIVE: To develop a gold nanoparticle-based visual DNA microarray for simple and rapid screening of EGFR gene mutations. METHODS: The DNA fragments contain epidermal growth factor receptor (EGFR) exons 18, 19, 20 and 21 were amplified by polymerase chain reaction (PCR) using biotin-modified primers. The amino-modified oligonucleotides were immobilized on glass surface, which were used as the capturing probes to bind the complement biotinylated target DNA. After the PCR product has been hybridized to the immobilized capture probe DNA on the glass slides, the Streptavidin-conjugated gold nanoparticles were introduced to the microarray via specific binding to 5'-end biotin of the PCR products. The hybridization signal on array spots was enhanced and visualized by silver amplification. The EGFR mutation in 286 clinical samples from cancer patients were tested using the gold nanoparticle-based microarray and verified with Sanger DNA sequencing method. RESULTS: A novel visual DNA microarray has been developed to detect EGFR mutations in tumor tissue specimens rapidly; its limit of detection (LOD) is up to 10(-9) mol/L and distinguishes power to detect 5% gene mutation in the mixture samples. CONCLUSION: For its high specificity and sensitivity, simplicity, lower price and higher speed, the present visual mutation detecting technique has potential application in clinical fields.
OBJECTIVE: To develop a gold nanoparticle-based visual DNA microarray for simple and rapid screening of EGFR gene mutations. METHODS: The DNA fragments contain epidermal growth factor receptor (EGFR) exons 18, 19, 20 and 21 were amplified by polymerase chain reaction (PCR) using biotin-modified primers. The amino-modified oligonucleotides were immobilized on glass surface, which were used as the capturing probes to bind the complement biotinylated target DNA. After the PCR product has been hybridized to the immobilized capture probe DNA on the glass slides, the Streptavidin-conjugated gold nanoparticles were introduced to the microarray via specific binding to 5'-end biotin of the PCR products. The hybridization signal on array spots was enhanced and visualized by silver amplification. The EGFR mutation in 286 clinical samples from cancerpatients were tested using the gold nanoparticle-based microarray and verified with Sanger DNA sequencing method. RESULTS: A novel visual DNA microarray has been developed to detect EGFR mutations in tumor tissue specimens rapidly; its limit of detection (LOD) is up to 10(-9) mol/L and distinguishes power to detect 5% gene mutation in the mixture samples. CONCLUSION: For its high specificity and sensitivity, simplicity, lower price and higher speed, the present visual mutation detecting technique has potential application in clinical fields.
Entities:
Keywords:
DNA microarrays; Receptor; epidermal growth factor; nanoparticles; pharmacogenetics
Authors: Vidya Srinivasan; Robert D Stedtfeld; Dieter M Tourlousse; Samuel W Baushke; Yu Xin; Sarah M Miller; Trinh Pham; Jean-Marie Rouillard; Erdogan Gulari; James M Tiedje; Syed A Hashsham Journal: J Microbiol Methods Date: 2017-04-23 Impact factor: 2.363