Therefore, determining the genotype according to direct sequencing results alone is difficult. These results confirm that the proposed method is superior to direct sequencing in terms of its sensitivity and applicability for detecting inhomogeneous samples. Biosci Biotech Bioch The proposed detection method can detect six kinds of mutation behaviors of codon 12 through eight reactions, as well as the mutation behaviors of codons 13 and 16 using the same principle.
This method is simpler and does not need expensive experimental instruments or reagents, such as fluorescent probes and high-quality detection samples, when compared to methods like fluorescent quantitative PCR, mass spectrometry, or DNA direct sequencing. Compared with the RFLP method, ours has no limitations on the enzyme cutting site and is more applicable for mutation detection under simple and conventional experimental conditions.
The high specificity of the proposed method can be maintained by: 1 using one pair of probes and four similar reaction systems, when detecting one single-base mutation site. Each reaction system was added with one dNTP to ensure that the PC reaction could be accomplished only upon complementation between the added dNTP and the base at the mutation site of the detection template.
In the four reaction systems, the PCR amplification results of two systems could be used to determine the genotype of the mutation site, and the remaining reaction systems could be used as a control group to avoid non-specific results. Therefore, the proposed method can be used to detect mutations in inhomogeneous samples, design Tag1 and Tag2 on the probe sequence, and implement the secondary amplification of products after PC reaction in order to increase their numbers, resulting in a fast detection under the AGE level.
Gene Anal Biochem In the present study, KRAS mutations in the samples could not be judged based on the sequencing spectra alone, because the sequencing peaks that corresponded to the mutation sites in the direct sequencing spectra could not be separated from the background signal.
Mol Cell Probes Hence, these techniques cannot be applied under conventional laboratory conditions. This method can be easily operated, and is applicable for inhomogeneous samples under simple experimental conditions e. Furthermore, this method does not need expensive experimental apparatuses and reagents.
However, it cannot detect unknown mutation sites, and the detection sensitivity of AGE is limited. The proposed method has limited detection capabilities. Abrir menu Brasil. Genetics and Molecular Biology. Abrir menu. E-mail: tangyitong Abstract This study aimed to develop a simple and rapid method to detect KRAS gene mutations for conventional clinical applications under laboratory conditions. Figure 1 Design of the oligonucleotide probes. The open circle is the symbol of the biotin label.
H1, H2, Tag1, and Tag2 represent the segments of the oligonucleotide probes. Figure 2 Detection assay for codon 12 of K-ras. Open circles represent the biotin label; closed circles are the symbol of streptavidin-coupled magnetic beads; N1 and N2 represent the bases of G, A, C, and T; G1, A1, C1, T1, and G2, A2, C2, T2 represent the four reactions corresponding to each pair of detection probes. Figure 3 Optimization of hybridization temperature.
G, A, C and T represent the four reactions corresponding to each pair of detection probes. Figure 4 Specificity detection with the PCR cycle numbers. Figure 5 Sensitivity detection with agarose gel electrophoresis. Figure 6 Mutation detection of the clinical samples. A The results of agarose gel electrophoresis for codon 12 of the K-ras gene. B Results of the direct sequencing. Associate Editor: Luis F. Previous work suggested that mutations are detected if they are in a DNA's first melting domain, and the melting domain is well separated from final strand dissociation.
Two criteria from the DNA melting theory were established to determine when both of these conditions are met. The criteria involve calculating the derivative melting curve as well as the melting map of a DNA sequence.
The approach was applied to the cDNA sequence of the human p53 gene. Mutations in the p53 gene are common in human cancers and are generally located in four 'hot spot' regions.
The technique, first described by Fischer and Lerman 1 , … more. Citations 3 Recent citations: R. Mathew, , Genetic Predisposition to Cancer. Related articles Based on techniques. Dlouhy et al. See more.
Green et al. Thompson , , Springer Protocols. References Fischer, S. Cell 16 , — Myers, R. M, Maniaus, T, and Lerman, L. Methods Enzymol , — Lerman, L S. Methods Enzymol , — Lerman, L.
S, Silverstein, K. Cold Spring Harbor Symp. Quant Biol. Acid Sci. USA 80 , —
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