A rapid automated SSCP multiplex capillary electrophoresis protocol that detects the two common mutations implicated in hereditary hemochromatosis (HH).

Currently two mutations in the HFE gene are known to be associated with the manifestation of the autosomal recessive disorder hereditary hemochromatosis (HH). A single-base mutation resulting in Cys282Tyr appears to have a causative role in the development of the disease, and a point mutation resulting in His63Asp may also be involved. Recent observations with a fully automated capillary electrophoresis (CE) system (ABI Prism 310) suggested that this instrument could be used for the precise identification of known mutations based on single-strand conformation polymorphism (SSCP). Two DNA fragments, each specific for one of the HFE mutation sites and labeled with a different fluorophor, were coamplified and without further manipulation simultaneously analyzed by CE-SSCP. Wild-type samples showed a mobility pattern that was clearly distinguishable from homozygous Cys282Tyr, homozygous His63Asp, or a compound heterozygous sample. To evaluate the reliability of this system for the detection of both mutations, 20 samples were analyzed blind. All genotypes, which were called automatically, were in concordance with those obtained by a previously validated restriction fragment length polymorphism method. Thus, SSCP in combination with CE provides a fast and precise research tool for the simultaneous identification of the two common mutations implicated in HH.

Identification of known p53 point mutations by capillary electrophoresis using unique mobility profiles in a blinded study.

This study is part of an ongoing project at the National Institute of Standards and Technology (NIST) that generates a panel of DNA clones containing the most common mutations found in the human p53 tumor suppressor gene. This panel will be made available as a reference source for evaluation and testing for p53 mutations. Single strand conformation polymorphism (SSCP) analysis has found widespread acceptance as a tool for simply and rapidly screening for mutations, albeit with a detection rate that can be below 100%. We have begun to analyze mutations found in exon 7 of the p53 gene by SSCP using laser induced fluorescence capillary electrophoresis (LIF-CE). PCR fragments, containing single point mutations, were amplified from genomic DNA isolated from cell lines using primers labeled with two different fluorophores. This dual labeling approach allowed better traceability of mobility shifts as a function of the experimental conditions. While analyzing the clones H596, Colo320, Namalwa and wild type (reference samples) at different temperatures, ranging from 25 to 45 degrees C, it was observed that each mutation responded in a unique way to changes in temperature both in magnitude and direction of shifts relative to the wild type sample. In a blinded study, ten p53 exon 7 samples were matched automatically, using ABI PRISM Genotyper software, against the four reference samples. From these 10 samples, six were correctly identified as containing one of the reference mutations, two corresponded to wild type, and two were correctly identified as non-reference mutations. This approach should prove helpful in the rapid screening of target sequences that are known to bear a limited number of mutations.

Development of standard reference materials for diagnosis of p53 mutations: analysis by slab gel single strand conformation polymorphism.

We have amplified by polymerase chain reaction (PCR) a 2.0 kbp region of the p53 gene containing exons 5--9 from seven cell lines reported in the literature to contain the majority of mutations reported for this gene. Sequence analysis of these products show that all seven cell lines contain mutations within the mutational hot spots of the p53 gene. Six of the seven clones have single base substitutions and the seventh has a single base deletion. We have analyzed the seven p53 single point mutations by single strand conformation polymorphism (SSCP) analysis using fluorescence slab gel electrophoresis (SG-SSCP). Fluorescent-labeled PCR primers were used for amplification of specific exons for mutation detection. SG-SSCP was conducted using Model 373 and Model 377 DNA sequencers with GeneScan Software (Perkin Elmer, Applied Biosystem Division). Nine different gel systems were first tested for their ability to resolve the p53 mutations using the Model 373 instrument. Two gel systems were capable of resolving all of the mutations that were screened. Optimal results were obtained with 12% w/v acrylamide 50:1 plus 10% v/v glycerol. This gel system was used to evaluate the effect of temperature on the ability to resolve the mutations. The separation with respect to wild type varied for each mutation examined. Subambient temperature (20 degrees C) was preferable overall for discrimination of these mutations as a group. We intend to use this system to examine a much larger panel of p53 mutation standards that are now under development.

Detection of p53 point mutations by single strand conformation polymorphism: analysis by capillary electrophoresis.

We have analyzed five p53 single point mutations by single strand conformation polymorphism using capillary electrophoresis (CE-SSCP) and have compared these measurements to measurements obtained by slab gel electrophoresis (SG-SSCP). PCR primers were used for amplification of specific exons for mutation detection. 5' Primers were labeled with FAM (5-carboxyfluorescein) and 3' primers were labeled with JOE (2',7'-dimethoxy-4',5'-dichloro-6-carboxyfluorescein). CE-SSCP was performed using the Perkin Elmer ABI PRISM 310 Genetic Analyzer with GeneScan Software and the Beckman P/ACE 5510 CE equipped for laser-induced fluorescence detection. Although the shifts in migration times for the p53 mutations relative to the corresponding wild-type strands could be successfully detected by either SG or CE analysis, the individual electrophoresis run times were about tenfold faster and more automated with capillary electrophoresis. The CE-SSCP measurements were performed at temperatures ranging from 10 to 60 degrees C on a prototype instrument. For mutations measured at ambient temperature (25 degrees C), characteristic shifts in direction and magnitude were observed in the migration times of both strands of all mutations relative to the wild type. This demonstrated the ability of CE at ambient temperature to resolve these mutations. However, the magnitude and direction of shifts in migration time varied with temperature in a discrete pattern for each mutation and resulted in a temperature-specific profile for each mutation. This demonstrated that extended temperature control will be an important advantage in resolving single point mutations by CE-SSCP. In addition, by using CE, discrete intra-strand isoforms could be easily observed at different temperatures. The combination of mutation-specific temperature profiling and analysis of isoforms by CE-SSCP should be of help to the diagnostic community in the detection of genetic mutations.

Genotyping of forensic short tandem repeat (STR) systems based on sizing precision in a capillary electrophoresis instrument.

Automated fluorescence analysis of polymerase chain reaction (PCR)-amplified short tandem repeat (STR) systems by capillary electrophoresis (CE) is becoming an established tool both in forensic casework and in the implementation of both state and national convicted offender DNA databases. A new capillary electrophoresis instrument, the ABI Prism 310 Genetic Analyzer, along with the Performance Optimized Polymer 4 (POP-4) provides an automated and precise method for simultaneously analyzing ten fluorescently labeled STR loci from a single PCR amplification kit, which provides a power of discrimination of approximately one in five billion from a single PCR amplification. Data are presented on sizing precision, sizing accuracy, and resolution for the STR loci in the AmpFlSTR Profiler kit. Sizing accuracy is highly dependent on the electrophoresis system, and therefore the reporting of alleles based on the nucleotide size obtained from an electrophoresis system is not recommended for forensic work. The precision of the 310 capillary electrophoresis system, coupled with software developed for automated genotyping of alleles based on the use of an allelic ladder, allows for accurate genotyping of STR loci. Sizing precision of < or = 0.16 nucleotide standard deviation was obtained with this system, thus allowing for accurate genotyping of length variants that differ in length by a single nucleotide.

A streamlined mutation detection system: multicolor post-PCR fluorescence labeling and single-strand conformational polymorphism analysis by capillary electrophoresis.

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.

Capillary electrophoresis as a technique to analyze sequence-induced anomalously migrating DNA fragments.

Sequence-induced anomalous migration of double-stranded (ds) DNA in native gel electrophoresis is a well known phenomenon. The retardation of migration is more obvious in polyacrylamide compared with agarose gels, and is greatly affected by the concentration of the gel and the temperature. This anomalous migration results in a difference between calculated and actual sizes of the affected DNA fragments. A low viscosity polymer solution (DNA Fragment Analysis Reagent) under investigation for use in dsDNA analysis by capillary electrophoresis is shown to be useful for the visualization of anomalies in migration of dsDNA fragments. Comparable with traditional slab gel systems, the retardation effect, indicative of bent or curved DNA, is strongly dependent on polymer concentration and separation temperature. These dependencies have implications on the accurate sizing of dsDNA fragments with unknown sequences and secondary structures.

Reduced expression of AP27 protein, the product of a growth factor-repressible gene, is associated with diminished adipocyte differentiation.

We have recently characterized an adipocyte cDNA (clone 5) that is enhanced in expression by environmental and hormonal conditions favoring adipogenic differentiation. Moreover, certain agents including fibroblast growth factor and phorbol 12-myristate 13-acetate (but not epidermal growth factor) markedly inhibit clone 5 gene expression and prevent TA1 cell differentiation. These results led us to propose that a threshold level of the clone 5 gene product (AP27 protein) is required for triggering adipocyte differentiation. We have constructed vectors that direct the synthesis of clone 5 antisense RNA to reduce the levels of AP27 in adipogenic cell lines TA1 and 3T3-L1. We show here that when these cells express clone 5 antisense RNA, they fail to undergo morphological differentiation, whereas adipogenesis is unaffected in cells expressing antisense beta-actin or ferritin heavy-chain RNA. We further show that cells expressing clone 5 antisense RNA (but not the other antisense RNAs) are unable to induce the expression of characteristic "adipocyte-specific" mRNAs. The level of inhibition of differentiation by clone 5 antisense RNA correlates with decreased levels of AP27 protein. These results provide strong evidence that expression of AP27 is linked to adipogenic differentiation and that AP27 may be a component of an as-yet-uncharacterized signal-transduction pathway required for the triggering of adipocyte differentiation.