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.     

Single-strand conformation polymorphism analysis of candidate genes for reliable identification of alleles by capillary array electrophoresis

We investigated the reliability of capillary array electrophoresis-single strand conformation polymorphism (CAE-SSCP) to determine if it can be used to identify novel alleles of candidate genes in a germplasm collection. Both strands of three different size fragments (160, 245 and 437 bp) that differed by one or more nucleotides in sequence were analyzed at four different temperatures (18 degrees C, 25 degrees C, 30 degrees C, and 35 degrees C). Mixtures of amplified fragments of either the intron interrupting the C-terminal WRKY domain of the Tc10 locus or the NBS domain of the TcRGH1 locus of Theobroma cacao were electroinjected into all 16 capillaries of an ABI 3100 Genetic Analyzer and analyzed three times at each temperature. Multiplexing of samples of different size range is possible, as intermediate and large fragments were analyzed simultaneously in these experiments. A statistical analysis of the means of the fragment mobilities demonstrated that single-stranded conformers of the fragments could be reliably identified by their mobility at all temperatures and size classes. The order of elution of fragments was not consistent over strands or temperatures for the intermediate and large fragments. If samples are only run once at a single temperature, small fragments could be identified from a single strand at a single temperature. A combination of data from both strands of a single run was needed to identify correctly all four of the intermediate fragments and no combination of data from strands or temperatures would allow the correct identification of two large fragments that differed by only a single single-nucleotide polymorphism (SNP) from a single run. Thus, to adequately assess alleles at a candidate gene locus using SSCP on a capillary array, fragments should be < or =250 bp, samples should be analyzed at two different temperatures between 18 degrees C and 30 degrees C to reduce the variability introduced by the capillaries, data should be combined from both strands and both temperatures, and undenatured double-stranded (ds)DNA molecular weight standards, such as ROX 2500, should be included as internal standards.     

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.     

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.     

Subtyping of orosomucoid 1 (ORM1) by isoelectric focusing in agarose and polyacrylamide gels

Genetic polymorphism of orosomucoid (ORM) has been demonstrated in several populations and comprises two structural gene loci, ORM1 and ORM2. In Caucasians three common ORM1 alleles have been shown, while the ORM2 locus is almost monomorphic. ORM1 phenotyping by isoelectric focusing in agarose or polyacrylamide gel combined with either print immunofixation or enzyme-linked immunoblotting is described, and population and family data from Denmark and Southern Germany are given. It is proposed to use a different alpha-numerical nomenclature for the phenotypes of the ORM1 and ORM2 systems.     

An integrated method for mutation detection using on-chip sample preparation, single-stranded conformation polymorphism, and heteroduplex analysis

This work integrates rapid techniques for mutation detection by producing single-stranded DNA and (renatured) double-stranded DNA on-chip, labeling these with fluorescent DNA stains and then performing two complementary methods of mutation detection-single stranded conformation polymorphism (SSCP) analysis and heteroduplex analysis (HA). This involves the denaturation of double-stranded polymerase chain reaction (PCR) product into single-stranded DNA, the mutation analysis of the single-stranded DNA by SSCP and the rehybridized double-stranded DNA by HA. These steps were performed entirely on-chip within several minutes of operation. The combination of these two mutation detection methods on-chip provides a highly sensitive method of mutation detection for either genotyping or screening. Many mutation analysis methods rely upon fluorescently labeled samples from a PCR with fluorescently labeled primers. By labeling on-chip we not only attain improved signal strength, but the method is considerably more versatile. Although we used PCR products in this work, this method could be used to analyze DNA from any source. We believe that this combination of several procedures on a single chip represents a significant step in the development of higher levels of integration upon microfluidic devices.     

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.     

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.     

High-resolution electrophoretic procedures for the identification of five Eimeria species from chickens, and detection of population variation

To overcome limitations of conventional approaches for the identification of Eimeria species of chickens, we have established high resolution electrophoretic procedures using genetic markers in ribosomal DNA. The first and second internal transcribed spacer (ITS-1 and ITS-2) regions of ribosomal DNA were amplified by polymerase chain reaction (PCR) from genomic DNA samples representing five species of Eimeria (E. acervulina, E. brunetti, E. maxima, E. necatrix and E. tenella), denatured and then subjected to denaturing polyacrylamide gel electrophoresis (D-PAGE) or single-strand conformation polymorphism (SSCP) analysis. Differences in D-PAGE profiles for both the ITS-1 and ITS-2 fragments (combined with an apparent lack of variation within individual species) enabled the unequivocal identification of the five species, and SSCP allowed the detection of population variation between some isolates representing E. acervulina, which remained undetected by D-PAGE. The establishment of these approaches has important implications for controlling the purity of laboratory lines of Eimeria, for diagnosis and for studying the epidemiology of coccidiosis.     

Rapid and precise genotyping of porcine microsatellites

Microsatellites are useful markers for genetic mapping and linkage analysis because they are highly polymorphic, abundant in genomes and relatively easily scored with polymerase chain reaction (PCR). A rapid genotyping system for microsatellites was developed, which included multiplex PCRs, multiple use of Hydrolink gels, automated fluorescent detection of fragments on an A.L.F. DNA sequencer, automatic assignment of alleles to each locus and verification of genotypes with a self-developed computer program "Fragtest". Eight multiplex PCRs have been developed to genotype 29 microsatellites for genetic and quantitative trait loci (QTL) mapping on pig chromosomes 6, 7, 12 and 13. Three to six microsatellites could be amplified in one multiplex PCR. Each multiplex reaction required only different concentrations of each pair of primers and a low concentration of dNTP (100 microM). A dNTP concentration of 100 microM proved to be optimal for the coamplification of microsatellites under the concentration of 1.5 mM MgCl2. Using four internal size standards added in each sample, the 5% Hydrolink gel could subsequently be used up to five times (total running time of 500 min) on the A.L.F. automated sequencer without significant loss of resolution and precision of fragment length analysis. Automatic assignment of alleles on each locus using "Fragtest" significantly increased the efficiency and precision of the genotyping. This system is thus a rapid, cheap, and highly discriminating genotyping system.     

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 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.     

Automated DNA fragment collection by capillary array gel electrophoresis in search of differentially expressed genes

An automatic DNA fragment collector using capillary array gel electrophoresis has been developed. A sheath flow technique is used for not only detection but also collection of DNA fragments. In a sheath flow cell, the DNA fragments separated by 16 capillaries flow independently into corresponding sampling capillaries. The fraction collector consists of 16 sampling trays and each sampling tray is set beneath each end of the sampling capillaries to collect the flow-through DNA fragments. Certain DNA fragments are automatically sorted by controlling the movement of the sampling trays according to the signals from the system. The collector experimentally separated two mixtures of polymerase chain reaction (PCR) products: one prepared by using eight different sizes (base lengths from 161 to 562) of DNAs; and the other prepared by a differential display (DD) method with cDNA fragments. Collected DNA fragments are amplified by PCR and measured by electrophoresis. DNA fragments with base length differences of one (base lengths 363 and 364) were successfully separated. A separated DNA fragment from the DD sample was also successfully sequenced. In addition, differentially expressed DNA fragments were automatically sorted by comparative analysis, in which two similar cDNA fragment groups, labeled by two different fluorophores, respectively, were analyzed in the same gel-filled capillary. These results show that the automatic DNA fragment collector is useful for gene hunting in research fields such as drug discovery and DNA diagnostics.     

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.     

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.     

Genetic polymorphism of 13 non‐CODIS STR loci in three national populations from China

The aim of this study was to investigate a 13 non‐CODIS STR loci database using three national populations from China. A new multiplex PCR system that simultaneously amplified 13 loci in the same PCR reaction was developed. This multiplex system included the 13 STR markers (D3S2402, D3S2452, D3S1766, D3S4554, D3S2388, D3S3051, D3S3053, D4S2364, D4S2404, AC001348A, AC001348B, D17S975, and D17S1294), which were successfully analyzed by using 441 DNA samples from three national populations in China (154 Mongol, 177 Kazakh, and 110 Uigur). Allele frequencies and mutation rates of the 13 non‐CODIS STR loci were investigated. A total of 4–10 alleles at each locus were observed and altogether 84, 88, and 87 alleles for the all selected loci were found in the Mongol, Kazakh, and Uigur, respectively. Eight mutations were detected from the 13 selected loci in 9880 meioses in kinship cases. These results indicate that this multiplex system may provide significant polymorphic information for kinship testing and relationship investigations.     

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.     

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.     

A single-strand conformation polymorphism method for the large-scale analysis of mutations/polymorphisms using capillary array electrophoresis

We present a high-throughput single-strand conformation polymorphism (SSCP) method, performed on a commercially available capillary array DNA sequencer. We tested various sieving matrices and electrophoretic conditions, using 51 DNA fragments which included 45 fragments carrying only one single nucleotide polymorphism (SNP), 4 fragments having two SNPs and 2 fragments with insertion or deletion. Resolution of alleles was improved by increasing concentrations of both sieving matrices and buffers, and all examined polymorphisms of DNA fragments were detected, most of them (45 fragments) as clearly split allele peaks in heterozygotes. Allele frequencies of SNPs can be estimated accurately by determining the relative amounts of alleles in pooled DNA. In this method, the turn-around time for the analysis of 96 samples is less than 3 h. These results demonstrate that capillary array-based SSCP is an efficient and accurate technique for the large-scale quantitative analysis of mutations/polymorphisms.     

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.