A Streamlined Mutation Detection System: Multicolor Post-PCR Fluorescence Labeling and Single-Strand Conformational Polymorphism Analysis by Capillary Electrophoresis

Abstract

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...

Narrowing of the regions of allelic losses of chromosome 1p36 in meningioma tissues by an improved SSCP analysis

“…Oligonucleotide primer pairs (custom synthesized at SIGMA Genosys, Hokkaido, Japan) were made to carry either a 5 0 -ATT or 5 0 -GTT for postlabeling purposes, as described previously. 19 Polymerase chain reaction PCR was performed in a total volume of 5 ll containing 25 ng of template DNA, 0.25 lM of each primer, 0.2 mM of each nucleotide, 0.125 U of AmpliTaq 1 DNA polymerase (Applied Biosystems, Foster City, CA), 27.5 ng of TaqStart TM antibody (Clontech Laboratories, Palo Alto, CA), 2 mM MgCl 2 , 10 mM Tris-HCl, pH 8.3, 50 mM KCl and 5% DMSO. The thermal cycle profile was 1 min at 94°C for initial heating, followed by 40 cycles of 30 sec at 94°C, 30 sec at 60°C and 1 min at 72°C.…”

Comparison of fluorescent single-strand conformation polymorphism analysis and denaturing high-performance liquid chromatography for detection of EXT1 and EXT2 mutations in hereditary multiple exostoses

“…This initial cost, which can rise greatly if screening many different exons, can be circumvented by the use of an alternative protocol, post-label F-SSCP. 32 This technique involves amplification of the DNA region of interest using unlabelled primers with 5' ATT extensions, then performing a Klenow fragment nucleotide exchange reaction with fluorescently-labelled dUTPs. This has proved successful in our hands and should theoretically result in no loss of sensitivity, although this has not been thoroughly tested.…”

Development of a simple and highly sensitive mutation screening system by enzyme mismatch cleavage with optimized conditions for standard laboratories

“…It is now possible that labeling PCR products with fluorescent and adapted SSCP and HA to automated DNA sequencing machines either slab gel electrophoresis [24][25][26][27][28] or capillary electrophoresis (CE) [29][30][31][32][33]. PCR products can be labeled by fluorescent primers [24][25][26] or fluorescent deoxynucleotides either during PCR [27] or after PCR [34,35].…”