Effective use of knowledge of human genome sequences in studies of hereditary diseases or cancer heavily depends on efficient methods for detection of mutations in individual samples. We describe here a simple and efficient mutation scanning system in which PCR products are post-labeled with two different fluorescent dyes in one tube, and analyzed by an automated capillary electrophoresis system using single-strand conformation polymorphism (SSCP) conditions (PLACE-SSCP). With the appropriate use of an internal control DNA, differences in electrophoretic mobilities between a reference and samples are precisely evaluated, then the presence of mutations is statistically judged. Thirty-three of 34 known mutations in fragments of three unrelated sequence contexts up to 741 bp were detected using one electrophoresis condition at the confidence level of <0.3% false positive. All the mutations were detected by analyzing at two temperatures. The described system has the advantage of little human intervention, short analysis time, high sensitivity, and objectivity of data interpretation.PCR-single-strand conformation polymorphism (SSCP) analysis is a widely used method for the detection of sequence differences in PCR products (Hayashi and Yandell 1993). In the original method, radioactively labeled PCR products amplified from reference and sample DNAs are analyzed by nondenaturing polyacrylamide slab gel electrophoresis in a single-stranded state (Orita et al. 1989). Mutations are detected as differences in mobility between each strand of a reference and test samples, because the sample strands have different conformations if there are sequence changes, and therefore migrate differently during electrophoresis. PCR-SSCP requires minimal sample handling before separation, but the use of radioisotopes and dependence on gel electrophoresis represent disadvantages in largescale mutation screening. Other limitations of the conventional SSCP technique are that its high sensitivity is assured only for short (<300 bp) DNA fragments, and that the data interpretation involves visual examination of band shifts, a process that requires experience and tends to be subjective.Fluorescence-based PCR-SSCP (F-SSCP) has been developed in which fluorescently labeled PCR products are electrophoresed and detected by automated DNA sequencer (Makino et al. 1992). This method is nonradioactive, is highly sensitive, and has the advantage of direct data storage and processing by the computer of the automated DNA sequencer. In this system, however, peak detection depended on a single fluorescent dye, and normalization of lane-to-lane mobility variations is difficult. Subsequently, SSCP analysis using gel-based DNA sequencer with multicolor fluorescence detection has been developed (Ellison et al. 1993;Iwahana et al. 1994Iwahana et al. , 1995. The multiple fluorescent detection system permitted correction of lane-tolane variations by coelectrophoresis of an internal size standard DNA labeled with a dye different from that of the sample DNA. The relat...
In some patients with RA, dysfunction of p53 might play a role in the proliferation of the synovial tissue. G245D mutation might especially need further study as it is the first recurrently identified p53 mutation in RA and is also one of the frequently identified mutations in human cancers.
Mcl-1 and Bcl-xL are crucial regulators of apoptosis, therefore dual inhibitors of both proteins could serve as promising new anticancer drugs. To design Mcl-1/Bcl-xL dual inhibitors, we performed structure-guided analyses of the corresponding selective Mcl-1 and Bcl-xL inhibitors. A cocrystal structure of a pyrazolo[1,5-a]pyridine derivative with Mcl-1 protein was successfully determined and revealed the protein-ligand binding mode. The key structure for Bcl-xL inhibition was further confirmed through the substructural analysis of ABT-263, a representative Bcl-xL/Bcl-2/Bcl-w inhibitor developed by Abbott Laboratories. On the basis of the structural data from this analysis, we designed hybrid compounds by tethering the Mcl-1 and Bcl-xL inhibitors together. The results of X-ray crystallographic analysis of hybrid compound 10 in complexes with both Mcl-1 and Bcl-xL demonstrated its binding mode with each protein. Following further optimization, compound 11 showed potent Mcl-1/Bcl-xL dual inhibitory activity (Mcl-1, IC50 = 0.088 μM; and Bcl-xL, IC50 = 0.0037 μM).
A method for fluorescent postlabeling of PCR products has been developed. The method uses Klenow fragment of DNA polymerase I that exchanges the 3'-terminal residue of PCR-amplified DNA fragment for fluorescent nucleotides. All reactions, including PCR, are performed in one tube simply by successive addition of reagents. The products can be applied directly to fluorescence-based automated DNA sequencers without purification for either length determination in denaturing electrophoresis or mutation detection in SSCP electrophoresis.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.