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.     

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.     

Dideoxy fingerprinting of low-level nucleotide variation in mitochondrial DNA of the human blood fluke, Schistosoma japonicum.

Dideoxy fingerprinting (ddF) is an efficient method for the detection of sequence changes in polymerase chain reaction (PCR)-amplified DNA fragments. It is a hybrid between single-strand conformation polymorphism (SSCP) and dideoxy sequencing, employing only one dideoxynucleotide in the reaction. We report the application of ddF for the display of low-level nucleotide variation in the mitochondrial (mt) NADH dehydrogenase subunit 1 (ND1) (404 bp) in the human blood fluke, Schistosoma japonicum. Variant samples differing by 1-6 nucleotides could be readily differentiated from one another by their characteristic and reproducible ddF profiles. The findings indicate the potential of this method to screen for point mutation in any parasite genes.     

Rapid identification of the ABO genotypes by their single-stand conformation polymorphism

The ABO locus on chromosome 9 contains many more alleles than are currently used routinely in forensic science. The use of single-strand conformation polymorphism (SSCP) can separate sequence polymorphisms that differ by only one base. The SSCP process used allows for both single- and double-stranded polymerase chain reaction (PCR) products to be visualized. The six ABO genotypes can be differentiated by the allele-specific B and O SSCP patterns. The double-stranded DNA produced 'hybrid' bands due to heterozygous samples and allowed sequence diversity to be detected between alleles of heterozygotes. These 'hybrid' bands are valid markers to confirm genotypes of specimens.     

Single-strand conformation polymorphism analysis with high throughput modifications, and its use in mutation detection in familial hypercholesterolemia. The IFCC Scientific Division: Committee on Molecular Biology Techniques.

The identification of the specific mutation causing an inherited disease in a patient is the framework for the development of a rationale for therapy and of DNA-based tests for screening relatives. We present here a review of the single-strand conformational polymorphism (SSCP) method, which allows DNA fragments that have been amplified with specific primers and PCR to be scanned rapidly for any sequence variation. The general principles of the method are described, as are the major factors that must be considered in developing an optimal SSCP strategy, namely length of the PCR fragment and the temperature of the gel run. Options for sample denaturing gel characteristics and detection of DNA fragments are discussed. In addition, several modifications are presented that have been developed for high-throughput mutational analysis. The application of these techniques to screen for mutations in the LDL receptor gene in patients with familial hypercholesterolemia are described.     

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.     

Genotyping Taenia tapeworms by single-strand conformation polymorphism of mitochondrial DNA.

To overcome limitations in identifying tapeworms of the genus Taenia by traditional approaches, we have established a single-strand conformation polymorphism (SSCP) method utilizing two different regions of mitochondrial (mt) DNA as targets. The NADH dehydrogenase 1 and the cytochrome c oxidase subunit I genes were amplified from genomic DNA by polymerase chain reaction (PCR), denatured and subjected to electrophoresis in mutation detection enhancement gels. SSCP analysis achieved delineation among eight different species of Taenia from different hosts based on characteristic profiles and enabled the detection of intraspecific variability in profiles for some taxa. This SSCP-based typing method has important implications for taxonomy, diagnosis and for studying the genetic structure of Taenia populations.     

结直肠癌组织中K-ras基因突变的毛细管电泳检测

通过对PCR扩增的76例结直肠癌组织及癌旁正常组织DNA基因组共152个样本纯化变性后,采用毛细管电泳-激光诱导荧光检测(CE-LIF)结合单链构象多态性(SSCP)分析方法检测了人结直肠癌组织及癌旁正常组织中K-ras基因第12/13位密码子突变。所检测的76例结直肠癌患者中有30例患者存在基因突变,并对异常片段进行测序验证,测序证实以碱基G→A点突变为主。结果表明所建立的CE-LIF技术结合SSCP分析检测K-ras基因突变的方法高效、快速、灵敏、准确,适合于临床上大样本结直肠癌中K-ras基因突变分析,对选择抗结直肠癌药物有一定的指导作用。     

Identification of the commonest cystic fibrosis transmembrane regulator gene DeltaF508 mutation: evaluation of PCR--single-strand conformational polymorphism and polyacrylamide gel electrophoresis

In the present study we investigated whether single-strand conformational polymorphism (SSCP) and polyacrylamide gel electrophoresis (PAGE) could be used for the identification of the CFTR DeltaF508 gene mutation, which is commonest in the Greek population. Using DNA from patients carrying this mutation, the appropriate 98 bp region of the CFTR gene was amplified by PCR and the reaction products were analysed by non-radioactive SSCP-electrophoresis using silver staining for band visualization and non-denaturating PAGE to confirm the results. SSCP electrophoretic analysis has been optimized for several parameters in order to achieve the best resolution. Single-strand DNA fragments gave a reproducible pattern of bands, characteristic for the particular mutation. Comparison of the obtain patterns with control samples allowed the detection of the DeltaF508 mutation in the patients studied by SSCP assay and these results were confirmed by the independent method of PAGE. Although SSCP and PAGE can be used for detection of this mutation, PAGE resulted in more distinct patterns than SSCP. It is, therefore, proposed that PAGE can be reliably used for the detection and identification of such a mutation in patients provided that suitable controls are available. The applicability of PAGE to identification of the mutation in carriers, particularly useful for population screening, is also discussed.     

多重聚合酶链反应-毛细管电泳-激光诱导荧光法检测三种食源性致病菌

建立了食品中常见致病菌大肠杆菌O157:H7的uidA基因、沙门菌的invA基因和志贺菌的ipaH基因的多重聚合酶链反应（PCR）产物的毛细管电泳快速检测方法。根据这3种致病菌的特异性基因序列设计多重PCR引物,优化PCR扩增反应体系,采用7.0 g/L 甲基纤维素为筛分介质,毛细管电泳-激光诱导荧光检测法同时检测了3种常见致病菌的PCR扩增产物。在优化的多重PCR反应和毛细管筛分电泳条件下,该方法可以同时检测沙门菌、志贺菌和大肠杆菌O157:H7基因的多重PCR扩增产物,22 min内即可完成3种常见致病菌的毛细管电泳检测。迁移时间的相对标准偏差为1.47%~2.07%。与凝胶电泳法比较,该法简便快速,灵敏度高,可用于多种致病菌脱氧核糖核酸的检测,为食品安全提供了一种可靠的快速检测方法。     

A simple and rapid method for HLA-DQA1 genotyping by polymerase chain reaction-single strand conformation polymorphism and restriction enzyme cleavage analysis.

A simple and rapid method for identification of alleles at the human leucocyte antigen (HLA)-DQA1 locus is described. The polymorphic second exon of the HLA-DQA1 locus was amplified by the polymerase chain reaction (PCR) method. The amplified DNA was analyzed by single-strand conformation polymorphism (SSCP) and restriction enzyme cleavage assay. Using this method, the eight known DQA1 alleles could be distinguished from each other. This paper suggests that the method can be used for quick genotyping of DQA1 alleles, but detecting point mutations at various positions in a fragment as well as new HLA-DQA1 genotypes should also be possible.     

Electrophoretic detection of population variation within Contracaecum ogmorhini (Nematoda: Ascaridoidea: Anisakidae)

This study examined genetic variation among specimens of Contracaecum ogmorhini from different otariid hosts and geographical origins using a polymerase chain reaction (PCR)-based mutation detection approach. The first (ITS-1) and second (ITS-2) internal transcribed spacers (ITS) of ribosomal DNA (rDNA) were amplified individually by PCR, scanned for sequence variation by single-strand conformation polymorphism (SSCP), and samples displaying variable SSCP profiles were subjected to cycle sequencing. While C. ogmorhini individuals from Arctocephalus pusillus pusillus (CoAPP) from South Africa and those from Arctocephalus pusillus doriferus (CoAPD) from Australia had very similar SSCP profiles for both ITS-1 and ITS-2, individuals of C. ogmorhini from Zalophus californianus (CoZC) from Pacific Canada could be unequivocally distinguished based on their profiles. In accordance with SSCP results, both CoAPP and CoAPD had identical ITS consensus sequences, whereas CoZC differed in sequence from both CoAPP and CoAPD populations by 0.2% (one base in the ITS-1) and 0.7% (two bases in the ITS-2). Based on the nucleotide difference in the ITS-2 sequence, a PCR-linked restriction fragment length polymorphism (RFLP) could be employed to distinguish individuals representing CoZC from those of both CoAPP and CoAPD. The findings suggest that C. ogmorhini may represent a complex of at least two species.     

PCR/SSCP detects reliably and efficiently DNA sequence variations in large scale screening projects

A simple and fast method with high reliability is necessary for the identification of mutations, polymorphisms and sequence variants (MPSV) within many genes and many samples, e.g. for clarifying the genetic background of individuals with multifactorial diseases. Here we review our experience with the polymerase chain reaction/single-strand conformation polymorphism (PCR/SSCP) analysis to identify MPSV in a number of genes thought to be involved in the pathogenesis of multifactorial neurological disorders, including autoimmune diseases like multiple sclerosis (MS) and neurodegenerative disorders like Parkinson s disease (PD). The method is based on the property of the DNA that the electrophoretic mobility of single stranded nucleic acids depends not only on their size but also on their sequence. The target sequences were amplified, digested into fragments ranging from 50-240 base pairs (bp), heat-denatured and analysed on native polyacrylamide (PAA) gels of different composition. The analysis of a great number of different PCR products demonstrates that the detection rate of MPSV depends on the fragment lengths, the temperature during electrophoresis and the composition of the gel. In general, the detection of MPSV is neither influenced by their location within the DNA fragment nor by the type of substitution, i.e., transitions or transversions. The standard PCR/SSCP system described here provides high reliability and detection rates. It allows the efficient analysis of a large number of DNA samples and many different genes.     

Validation of three rapid screening methods for detection of verotoxin-producing Escherichia coli in foods: interlaboratory study

An interlaboratory study was conducted for the validation of 3 methods for the detection of all verotoxin-producing Escherichia coli (VTEC) in foods. The methods were a multi-analyte 1-step lateral flow immunoassay (LFIA) for detection of E. coli O157 and verotoxin (VT); an enzyme-linked immunosorbent assay targeted against VT1, VT2, and VT2c (VT-ELISA); and a polymerase chain reaction (PCR) method for detection of VT genes (VT-PCR). Aliquots (25 g or 25 mL) of 4 food types (raw minced [ground] beef, unpasteurized milk, unpasteurized apple juice [cider], and salami) were individually inoculated with low numbers (<9 to 375 cells/25 g) of 6 test strains of E. coli (serogroups O26, O103, O111, O145, and O157) with differing VT-producing capabilities. Five replicates for each test strain and 5 uninoculated samples were prepared for each food type. Fourteen participating laboratories analyzed samples using the LFIA, 9 analyzed the samples by ELISA, and 9 by PCR. The LFIA for O157 and VT had a specificity (correct identification of negative samples) of 92 and 94%, respectively, and a sensitivity (correct identification of positive samples) of 94 and 55%, respectively. The VT-ELISA and VT-PCR had a specificity of 98 and 99%, respectively, and a sensitivity of 89 and 72%, respectively.     

Development of a Sensitive Deaminated Single-Strand Conformation Polymorphism (DSSCP)

The single-strand conformation polymorphism (SSCP), accompanied by sequencing, is a useful methods for identifying mutations in a DNA fragment. In this study, we have developed a modified SSCP with the aid of sodium bisulfite treatment. The corresponding PCR products for exon 3 of Hb gene were sequenced and samples with homozygote and heterozygote single nucleotide substitutions were identified. The PCR products were treated with sodium bisulfite, which deaminates all the cytosine residues. The reaction mixture was then analyzed on non-denaturing polyacrylamide gels. The modified method, which is called deaminated SSCP (DSSCP), was applied successfully in analysis of mutations in the beta-globin gene at positions relevant to codon 6. DSSCP is a very effective and reproducible method providing clear results that are easy to interpret without the involvement of radioactivity.     

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.     

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.     

Use of capillary electrophoresis for high throughput screening in biomedical applications. A minireview

Diagnosis of inherited diseases or cancer predispositions frequently involves determination of specific mutations or polymorphisms. The number of characterized monogenetic and polygenetic diseases is significantly rising every year. As a result, an increasing number of patient samples with a rising complexity of genetic diseases require molecular diagnostics. In order to apply genetic analyses to large groups of patients or population screening, automation of a sensitive and precise method is highly desirable. Capillary electrophoresis (CE) facilitates the development of methods which can rapidly process large number of patient samples in an automated fashion. In contrast, conventional techniques including Southern blotting, sequencing or standard gel electrophoresis are time consuming, cost ineffective and require substantial amounts of each specimen. Robustness, ease of operation, good reproducibility and low cost are the main advantages of CE. Currently, most protocols adapted to automated CE represent (i) analyses of DNA fragment length or DNA restriction patterns (RFLP), (ii) analyses of single-strand conformation polymorphism (SSCP) and (iii) microsatellite analyses. Recently, automated detection of variations in the FRAXA (CGG)n region (fragile X syndrome), LDL receptor gene, p53 gene, MTHFR (methylenetetrahydrofolate reductase) gene, HFE gene and others has been established on CE systems. These applications clearly demonstrate the suitability of CE for high throughput screening in medical applications.     

Effect of polymer matrix and glycerol on rapid single-strand conformation polymorphism analysis by capillary and microchip electrophoresis for detection of mutations in K-ras gene

We present the rapid single-strand conformation polymorphism (SSCP) analysis by capillary and microchip electrophoresis to detect the mutations in K-ras gene. Parameters that might affect the analysis of mutation in K-ras gene, such as the polymer and the additive in the sieving matrix, have been studied systematically. Under the optimal conditions, the analysis of seven mutants of K-ras gene could be finished within 10 min by capillary electrophoresis (CE). Furthermore, with the wild-type gene as the inner standard, the analysis accuracy of mutations could be improved. In addition, by studying the properties of polymer solutions, the matrix suitable for microchip electrophoresis was found, and the detection of mutations in K-ras gene could be further shortened to 1 min.     

Genotyping Cryptosporidium parvum by single-strand conformation polymorphism analysis of ribosomal and heat shock gene regions

Polymerase chain reaction (PCR)-coupled single-strand conformation polymorphism (SSCP) approaches utilizing nuclear DNA regions of the small subunit (SSU) of ribosomal RNA and heat shock protein 70 gene (HSP70) were established for genotyping Cryptosporidium parvum. The regions were amplified (individually or in a multiplex reaction) by PCR from DNA extracted from oocysts from ruminant or human hosts, then denatured and subjected to electrophoresis in a mutation detection enhancement (nondenaturing) gel matrix. Single-strand profiles produced in SSCP allowed the unequivocal identification/differentiation of the two common (human, 1 and cattle, 2) genotypes of C. parvum and the direct display of sequence variability within some samples, reflecting population variation. As these are considered among the most closely related genotypes (based on SSU and HSP70 sequence data), these findings and other preliminary results for C. felis (from cat) C. serpentis (from snake) and C. baileyi (from bird) indicate that the SSCP approaches established could be employed to identify any of the currently recognised genotypes and species of Cryptosporidium.