Single strand conformation polymorphism analysis of ras oncogene by capillary electrophoresis with laser-induced fluorescence detector

Single strand conformation polymorphism (SSCP) analysis of the N-ras oncogene was achieved by capillary electrophoresis with a laser-induced fluorescence detector (CE-LIF) using methylcellulose as a molecular sieving agent. The PCR-amplified N-ras oncogene, which is known to have a point mutation at codon 61 in the neuroblastoma, was investigated by CE-LIF combined with SSCP (SSCP-CE-LIF). A mixture of wild- and mutant-type single strand DNA fragments (103 bp) of the N-ras oncogene was separated by buffer solution containing 1.0% methylcellulose and 0.2 microM fluorescent dye (YO-PRO-1) at 25 degrees C. The SSCP-CE-LIF technique gave good resolution for wild- and mutant-type single strand DNA fragments with separation completed within 7 min. SSCP analysis using a CE system with a LIF detector was successfully applied to the detection of the one point mutation on the N-ras oncogene.     

Structural factors determining DNA length limitations in conformation-sensitive mutation detection methods

Numerous mutations and polymorphisms in human genes remain to be identified using reliable methods. Of the available mutation scanning methods those dependent on structural change-induced mobility shifts are highly effective. Their efficiency is, however, DNA length-sensitive and the reasons for that are poorly understood. In this study, we explain why scanning genes for mutations is less effective in longer DNA fragments, and reveal the factors which are behind this effect. We have performed a systematic analysis of the same sequence variants of exon 11 of the BRCA1 gene in DNA fragments of three different lengths using the combined single-strand conformation polymorphism (SSCP) and heteroduplex analysis (DA) by capillary electrophoresis (CE). There are two major structural factors responsible for the reduced mutation detection rate in long amplicons. The first is increased contribution from other secondary structure modules and domains in longer fragments, which mask the structural change induced by the mutation. The second is higher frequency of single-nucleotide polymorphisms (SNPs) including common polymorphisms in longer fragments. This makes it necessary to distinguish the structural effect of the mutation from that of each polymorphic variant, which is often difficult to achieve. Taking these factors into account, an efficient scanning of genes for sequence variants by conformation-sensitive methods may be performed.     

Evaluation of sieving matrices used to separate alleles by cycling temperature capillary electrophoresis

Denaturing CE (DCE) is a powerful tool for analysis of DNA variation. The development of commercial multi-CE instruments allows large-scale studies of DNA variation (many samples and many fragments). However, the cost of consumables like capillary arrays and sieving matrix might limit the use of DCE in such studies. Thus, we have tested 72 different in-house formulated sieving matrices' ability to suppress EOF and separate PCR-amplified alleles with the DCE variant, cycling temperature CE (CTCE). The data herein demonstrate that alleles can be baseline-separated by use of PVP and poly(N,N-dimethyl acrylamide) polymers at various percentages and pH. Allele separation by CTCE is matrix-independent and consequently applicable to any capillary instrument used for DNA separation. Formulation of sieving matrix for CTCE was done by dissolving appropriate amount of polymer powder into the running buffers. Allele separation was observed at different pH (7.5-8.5), concentrations and molecular size of the polymer, without compromising the separation and reproducibility. Finally, the cost reduction of homemade matrices is more than 1000-fold as compared to commercial sieving matrices.     

Nonconventional synthesis and characterization of ultrahigh-molar-mass polyacrylamides

In spite of the significant progresses in the field of replaceable sieving matrices for separating DNA in capillary electrophoresis (CE), an intense research activity is still going on to improve the separation of large size DNA sequencing fragments. There are evidences, both from experimental and theoretical sides that the resolution of these fragments, at the single base, requires the use of sieving matrices comprised of long chain linear polymers. In the separation of DNA fragments by CE are of upmost importance: (i) the complete solubility of the polymer, (ii) the linearity of the chain, (iii) the achievement of ultrahigh viscosity in dilute solutions. The aim of this work is the synthesis of ultrahigh-molecular-weight polymers which possess the three requirements mentioned above by employing a nonconventional method. We demonstrate that the sieving performance of polyacrylamide is directly correlated to its intrinsic viscosity.     

Genotyping and haplotyping of the dopamine D4 receptor gene by capillary electrophoresis

In this paper we report on simultaneous genotyping of adjacent polymorphisms (referred to as haplotyping) by combining double-tube allele-specific polymerase chain reaction, restriction fragment length polymorphism and capillary gel electrophoresis analysis of the resulting fragments. Direct molecular haplotyping is of particular importance in the case of double heterozygote samples, since in these instances the haplotype structure cannot be constructed based on genotype data. Our approach provided a powerful tool for coincidental genotype analysis of the 48 base pair (bp) variable number of tandem repeats of the third exon and haplotype investigation of the -616CG and -521CT single nucleotide polymorphisms of the dopamine D4 receptor (DRD4) gene. The linear polyacrylamide sieving matrix was optimized for the size range of the double-stranded DNA fragments of interest varying from 35 to 763 bp. We demonstrated that capillary gel electrophoresis in combination with laser induced fluorescence detection offers a sensitive and accurate tool for automated haplotyping in clinical settings.     

Genotyping of two single nucleotide polymorphisms in 5,10-methylenetetrahydrofolate reductase by multiplex polymerase chain reaction and capillary electrophoresis

Two single nucleotide polymorphisms (SNPs) of 5,10-methylenetetrahydrofolate reductase (MTHFR) gene, A1298C and C677T, were widely considered to be related with various neoplasia disorders. We established a simple and effective capillary electrophoresis (CE) method for detection of two SNPs in MTHFR gene simultaneously. DNA samples were amplified by multiplex PCR with universal fluorescence-labeled primer and analyzed by single-strand conformation polymorphism (SSCP)-CE method. The CE method was performed using 1.5% hydroxyethyl cellulose in 1× TBE buffer containing 1 M urea. The PCR products after SSCP procedure were electrokinetically injected at −10 kV, 30 s. Separation voltage was −6 kV and the temperature was set at 20 °C. The optimal SSCP-CE method was applied to detect two polymorphisms in MTHFR gene of acute lymphoblastic leukemia (ALL) and attention-deficit/hyperactivity disorder (ADHD) patients. Genotyping results were evaluated in terms of relationships between outcomes for ADHD patients after ALL chemotherapy and ALL disease. The SSCP-CE method and multiplex PCR with universal fluorescence primer were used as the fast technique for screening two SNPs in MTHFR gene, A1298C and C677T. The genotyping data were coincident with DNA sequencing. This SSCP-CE method was found feasible for detecting mutation of MTHFR gene in populations.     

Detection of the major mutation M467T causing cystinuria by single-strand conformation polymorphism analysis using capillary electrophoresis

We present a fast detection of M467T, the major mutation causing cystinuria, by capillary electrophoresis version of single-strand conformation polymorphism (SSCP). The DNA fragment (317 bp) carrying the point mutation was amplified by polymerase chain reaction (PCR) on the exon 8 of the SLC3A1 gene, which encodes for the transmembrane glycoprotein rBAT, a part of the active cystine and dibasic amino acids transporter. The complementary strands of the fragment were labeled by fluorescein and TAMRA, respectively. Thus, the electromigration of both strands was recorded independently as a laser-induced fluorescence (LIF) signal, what enabled an effective optimization of separation conditions. The injected sample was denatured by immersing the inlet of the separation capillary into a vial with 0.1 M solution of NaOH prior to analysis. Under optimum conditions, the SSCP analysis in poly(vinyl alcohol) (PVA)-coated silica-fused capillary of an effective length of 15 cm, filled with 4% linear polyacrylamide (LPA) solution, was accomplished in approximately 6 min. The experimentally observed mobility shifts of single-stranded DNA (ssDNA) fragments were compared to the appearance of their calculated two-dimensional conformations using Version 3.0 of MFOLD software. The number of nucleotides involved in the duplex regions of theoretical structures correlates well with their real migration order in the sieving medium.     

Imperfect units of an extended microsatellite structure involving single nucleotide changes

Short tandem repeats (STRs) and single nucleotide polymorphisms (SNPs) are widely used as markers in human genome studies. We have characterized a highly polymorphic STR locus (D20S85) with (AAAG)n repeats, by a combination of direct DNA sequencing and single-strand conformation polymorphism (SSCP) analysis. Eight STR alleles were first identified on denaturing gels, and SSCP gels were then used to demonstrate the existence of previously indistinguishable multiple alleles at the locus on the basis of variable allelic flanking sequences. This was confirmed by direct sequencing of the alleles. Four transitions, two G to A and two A to G in the 5'-flanking region of the locus at positions 14, 22, 24, and 26 effectively subdivided the STR alleles into two groups, with frequencies of 0.431 and 0.569, respectively. The mutational processes that generated the polymorphisms involved both simple changes in the number of AAAG repeats and single nucleotide mutations in the region flanking the repeat. The findings have potential application in the avoidance of false linkage and association. A composite locus of this nature, with separate STR and SNP evolutionary histories and resulting from different mutational processes, also could have wide application in studies of selection, drift, migration and inbreeding.     

Low viscosity DNA sieving matrices for capillary electrophoresis

The rapid development of DNA capillary electrophoresis (CE) technology has increased the demand of new low viscosity sieving matrices with high separation capacity. The high throughput, resolution and automatic operation of CE systems have stimulated the application of the technique to different kinds of DNA analysis, including DNA sequencing, separation of restriction fragments, PCR products and synthetic oligonucleotides. In addition specific methods for PCR-based mutation assays for the study of known and unknown point mutations have been developed for use in CE. The key component for a large scale application of CE to DNA analysis is the availability of appropriate sieving matrices. This article gives an overview of the linear polymers used as DNA separation matrices with particular emphasis on the polymers that combine high sieving capacity, low viscosity and chemical resistance.     

Multitemperature single-strand conformation polymorphism.

Changes of gel temperature during single-strand conformation polymorphism (SSCP) electrophoresis increase the sensitivity of mutation detection in polymerase chain reaction (PCR) products and significantly reduce the overall time and costs of analysis. Based on these findings, a new method for single nucleotide polymorphism (SNP) and point mutation detection--multitemperature single-strand conformation polymorphism (MSSCP) was devised. In order to control the gel temperature with 0.1 degrees C accuracy during electrophoresis, new equipment was developed. We demonstrated that increasing the gel temperature by 8 degrees C or decreasing it by 10 degrees C from 23 degrees C led to the disappearance of all electrophoretic differences between five alleles of exon 8 of the human p53 gene during the SSCP analysis. The interesting result was the detection of two additional SNPs (out of seven analyzed) in exon 7 of the human PAH gene during a one hour MSSCP electrophoresis. This result is better than that obtained by three classical SSCP analyses of the same samples at different but constant gel temperatures. We advocate the MSSCP technology as a fast, reliable, and cost-effective tool for the screening and preselection stage of genomics surveys, especially when a high variability of the analyzed DNA fragment is expected.     

Single-strand conformation polymorphism analysis to detect the p53 mutation in colon tumor samples by capillary electrophoresis

The polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) technique is developed for the detection of point mutations in DNA samples, and is very useful in the research of tumors. The traditional SSCP was carried out with slab gel electrophoresis (SGE), but this is time-consuming and labor-intensive, particularly for clinical diagnoses. We have developed a capillary electrophoresis (CE) method for SSCP detection with a linear polyacrylamide gel solution as the sieving matrix. Twenty colon tumor samples were detected with SSCP-CE and the point mutation in exon 7 of the p53 gene was found in six of the samples. Based on the sequencing results, the accuracy of SSCP-CE was better than that of SSCP-SGE. We hope this rapid and convenient method could be applied in the clinical diagnosis of tumors soon.     

Comparison of sieving matrices for on-the-fly fluorescence lifetime detection of dye-labeled DNA fragments

Commercially available, replaceable sieving matrices and their solvent modulated forms were evaluated for use in on-the-fly fluorescence lifetime detection of dye-labeled DNA fragments in capillary electrophoresis. The fragments were labeled with dyes that can be excited by the 488 nm line of an argon ion laser and have lifetimes in the range of 0.8 ns to 3.8 ns. The sieving matrices and buffer systems included poly(vinylpyrrolidone) (PVP), poly(ethyleneoxide) (PEO), hydroxyethylcellulose (HEC), Tris-borate-EDTA (TBE) and Tris-TAPS-EDTA buffers modified with DMSO and formamide. Selection of the optimal sieving matrix is based on the separation efficiency and the enhancement of lifetime resolution of DNA fragments. Best results for both electrophoretic resolution and lifetime detection were obtained using a poly(ethyleneoxide)/TBE gel buffer in the presence of 10% formamide.     

毛细管电泳无胶筛分介质分离DNA的机理

快速、高效而灵敏的分离技术对于DNA的分析是至关重要的。使用无胶筛分介质的毛细管电泳是最重要的DNA分离技术之一,通常使用无交联的高分子溶液作为无胶筛分介质。本文在介绍高分子溶液理论的基础上,综述了DNA在毛细管电泳无胶筛分介质(缠结溶液和稀溶液)中的分离机理,主要包括Ogston筛分模型、各种修正的爬行模型、瞬态缠结偶合机理及其改进机理等。     

A Novel Sieving Medium for Separation of DNA Fragments- Poly(acrylamide-dimethylacrylamide)

IntroductionCapillary electrophoresis (CE) in polymer solution has become an attractive alternative to slab gel electrophoresis for DNA analysis1, 2. This technique has been successfully applied for mutation detection, genotyping, DNA sequencing and gene expression3-7. In order to perform DNA analysis, the inner surface of the capillary must be modified, usually by covalent bonding of hydrophilic polymers. However, the covalent coating will increase the cost and often causes problems rela…     

Comparison of sieving matrices for on-the-fly fluorescence lifetime detection of dye-labeled DNA fragments

Commercially available, replaceable sieving matrices and their solvent modulated forms were evaluated for use in on-the-fly fluorescence lifetime detection of dye-labeled DNA fragments in capillary electrophoresis. The fragments were labeled with dyes that can be excited by the 488 nm line of an argon ion laser and have lifetimes in the range of 0.8 ns to 3.8 ns. The sieving matrices and buffer systems included poly(vinylpyrrolidone) (PVP), poly(ethyleneoxide) (PEO), hydroxyethylcellulose (HEC), Tris-borate-EDTA (TBE) and Tris-TAPS-EDTA buffers modified with DMSO and formamide. Selection of the optimal sieving matrix is based on the separation efficiency and the enhancement of lifetime resolution of DNA fragments. Best results for both electrophoretic resolution and lifetime detection were obtained using a poly(ethyleneoxide)/TBE gel buffer in the presence of 10% formamide. Received: 25 August 2000 / Revised: 7 November 2000 / Accepted: 14 November 2000     

Rapid identification of pathogenic bacteria by capillary electrophoretic analysis of rRNA genes

Molecular diagnosis is playing an increasingly important role in the rapid detection and identification of pathogenic organisms in clinical samples. The genetic variation of ribosomal genes in bacteria offers an alternative to culturing for the detection and identification of these organisms. Here 16S rRNA and 16S-23S rRNA spacer region genes were chosen as the amplified targets for single-strand conformation polymorphism (SSCP) and restriction fragment length polymorphism (RFLP) capillary electrophoresis analysis and bacterial identification. The multiple fluorescence based SSCP method for the 16S rRNA gene and the RFLP method for the 16S-23S rRNA spacer region gene were developed and applied to the identification of pathogenic bacteria in clinical samples, in which home-made short-chained linear polyacrylamide (LPA) was used as a sieving matrix; a higher sieving capability and shorter analysis time were achieved than with a commercial sieving matrix because of the simplified template preparation procedure. A set of 270 pathogenic bacteria representing 34 species in 14 genera were analyzed, and a total of 34 unique SSCP patterns representing 34 different pathogenic bacterial species were determined. Based on the use of machine code to represent peak patterns developed in this paper, the identification of bacterial species becomes much easier.     

毛细管电泳-限制性片段长度多态性分析检测胃癌H-ras基因点突变

建立一种毛细管电泳快速高效检测限制性内切酶酶切产物的方法, 使其更好地用于基因诊断. 以甲基纤维素(Methyl cellulose, MC)为筛分介质, 用pUC19 DNA/Msp I (Hpa II) Marker标准DNA片段为实验对象, 通过考察筛分介质的浓度、pH值、毛细管的温度和运行电压优化出分离小于600 bp的双链DNA片段的最适条件, 并将此方法应用于临床59例胃癌患者肿瘤组织H-ras基因12位密码子点突变情况的检测. MC是一种良好的筛分介质, 运用其进行毛细管电泳对于遗传性疾病的诊断将更加快速、准确、简便、灵敏.     

Detection of two variant Vero toxin genes in Escherichia coli by capillary electrophoresis

Three methods [capillary electrophoresis (CE)-allele-specific PCR, CE-single-strand conformation polymorphism (SSCP) and CE-cleavase fragment length polymorphism (CFLP)] were developed in order to effect rapid and specific analysis of the vero toxin (VT)1 and VT2 genes of O157. The allele-specific polymerase chain reaction (PCR) method, which utilized specific duplex PCR with specific primers for VT1 and VT2, showed that VT1 and VT2 consisted of 174 and 128 bp, respectively. Subsequent CE analysis was carried out. Separation time was 4 min. SSCP, which utilized one primer set which reacted with both VT1 and VT2 in the PCR method, was followed by CE analysis of secondary structure of single-strand DNA. Two genes could be analyzed in approximately 18 min. CFLP, like SSCP, is a method for detecting mutation-induced changes in secondary structure of single-stranded DNA. The endonuclease cleavase I recognizes and cleaves the 5' side of hairpin loops in self-annealed single-strand DNA of PCR product 169 bp obtained from VT1 and VT2. The produced DNA fragments are analyzed by CE and the electrophelogram reveals a sequence-specific CFLP. Separation time was 6 min. These techniques are suitable for the detection and the identification of O157.     

Optimum sample medium for single-nucleotide polymorphism and mutation detection by capillary electrophoresis

Capillary electrophoresis (CE) is a versatile analytical platform widely used for nucleic acids analysis. Its applications in research and diagnostics include scanning and screening for mutations and polymorphisms by such reliable methods as single-strand conformation polymorphism (SSCP), heteroduplex analysis (HA), and combined SSCP/HA. This study, aimed at the further development of these methods, is focused on detailed sample-media characteristics. Factors affecting single-strand conformer stability and DNA intake efficiency were analyzed. The sample media optimal for efficient mutation or SNP detection were determined, and complex SSCP-CE patterns arising from unpurified PCR products were explained. It turns out, that the nondenaturing aqueous media assure both efficient DNA intake, and single-strand conformers stability required for the SSCP and combined SSCP/HA. The results of this study are applicable to all these areas of biomedical research, in which capillary electrophoresis is used for the characterization of nucleic acids.     

Sieving properties of end group‐halogenated Pluronic polymer matrix in DNA separation under nondenaturing CE analysis

CE‐SSCP analysis is a well‐established DNA separation method that is based on variations in mobility caused by sequence‐induced differences in the conformation of single‐stranded DNA. The resolution of CE‐SSCP analysis was improved by using a Pluronic polymer matrix, and it has been successfully applied in various genetic analyses. Because the Pluronic polymer forms a micellar cubic structure in the capillary, it provides a stable internal structure for high‐resolution CE‐SSCP analysis. We hypothesized that formation of micellar cubic structure is influenced by the end hydroxyl group of the Pluronic polymer, which affords structural stability through hydrogen bonding. To test this hypothesis, the hydroxyl group was halogenated to eliminate the hydrogen bonding without disturbing the polarity of polymer matrix. CE‐SSCP resolution of two DNA fragments with a single base difference was significantly worse in the halogenated polymer matrices due to band broadening. The viscoelastic properties of control (which has hydroxyl group), chlorinated, and brominated F108 solution upon heating were also investigated by rheological experiments, and we found that gelation was significantly associated with resolution. In this series of experiments, the effect of the hydroxyl group in Pluronic polymer matrix on separation resolution of CE‐SSCP analysis was demonstrated.