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Objective To research a simple and sensitive K-ras gene mutations detection method in order to be suitable for the routine mutation detection.Methods The corresponding detection locus oligonucleotide probe was designed.By the connection,amplification,labeling and ELISA reaction in probe,the mutation locus genotype was determined by the ELISA reaction detection value.With the six point mutations of G12S,G12R,G12C,G12D,G12A and G12V in 12 codons of K-ras gene as the detection objects,the plasma circulation DNA sample in 72 cases of lung cancer was detected,then the results were compared with those obtained by the direct sequencing.Results Three samples were identified as the G12S,G12R and G12A mutatins by the established method.But no K-ras mutations were detected in the samples by using the direct sequencing,indicating that the direct sequencing had lower sensitivity and was not suitable for the mutation detection of heterogeneous samples such as circulating DNA.Conclusion The simple and sensitive K-ras gene mutation detection method is established and can conduct the routine mutation detection for the heterogeneous samples.
RESUMO
Objective To establish a single nucleotide polymorphisms genotyping (SNP) method for a convenient, accurate, and routine analysis of clinical samples. Methods Based on the design of oligonucleotide probe, the assay was performed through three steps:the conjunction of the detection probe, universal amplification, labeling and ELISA reaction. The genotype of each SNP was revealed by reading signals of each set of reaction tubes. This assay was applied to detect sixty-two plasma samples of lung cancer for circulating DNA for three SNPs of EGFR, c.2573T＞G(L858R), EGFR, c.2582T＞G＞T(G719C). Results were compared with those obtained by direct sequencing. Results The heterozygote mutation was identified for L858R by both methods, although no mutation was detected for L861Q and G719C. Six samples were identified as heterozygotes with the new method, and only two samples were unambiguously identified as heterozygotes by the direct sequencing. Two additional samples could not be identified as heterozygotes because the peak of mutant allele was very low compared with that of wild allele. Conclusion The developed method enabled accurate identification of SNP in a convenient manner, and which is adapted to routine analysis from heterogeneous samples unambiguously.
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Objective To establish a simple,rapid and sensitive nucleotide polymorphisms genotyping method in order to conduct the routine clinical detections under the simple laboratory condition by this method.Methods Based on the ligase-agarose gel electrophoresis,the oligonucleotide detection probes of mutational sites was designed.The detection underwent the detection probe connecting,purification and universal amplification,finally the mutation genotypes of detection sites were judged by the ap-peared bands in the agarose gel electrophoresis(AGE).With the 3 SNP sites EGFR,c.2573T>G(L858R),EGFR,c.2582T> A (L861Q)and EGFR,c.2155 G>T(G719C)in epidermal growth factor receptor(EGFR)gene as the detection objects,the plasmid template and plasma circulating DNA sample in lung cancer were performed the detection.Results The established method was easy to operate with higher specificity and sensitivity.After 20-30 cycles of PCR amplification,the genotype of detection sites was clearly estimated according to the amplification band.When detecting the mixed alleles in the heterogeneous sample,minimal 2.5%mutation alleles could be detected out.This method and the direct sequencing method could respectively detect 6 cases and 2 cases of heterozygotes mutation in the SNP site of L858R among 62 samples of lung cancer.Conclusion The established detection method for SNP genotyping is suitable to the routine mutation detection on the heterogeneous samples under the simple laboratory condi-tion.