Two new mutations detected by single-strand conformation polymorphism analysis in cystic fibrosis from Russia

Single-strand conformation polymorphism (SSCP) analysis followed by direct sequencing of exons containing ATP-binding domains of the cystic fibrosis transmembrane conductance regulator (CFTR) gene was performed on 80 Russian DNA samples. Two new alterations — S1196X (exon 19) and W1282R (exon 20) — and two novel polymorphisms — 1525-61 (intron 9) and 1716+12 T-C (intron 10) — were identified. Mutation S1196X changes a TCA codon to TGA and destroys an EcoRI site. Alteration W1282R results from a T-to-C change at position 3976. It was found in one Russian patient and creates an AciI site; however, it is unclear whether this is a disease-causing mutation or a polymorphism. Polymorphism 1525-61 results from an A-to-G change. Alteration 1716+12 T-C was found in a Moldovian patient and creates a new MaeII site. It is not known whether this alteration affects the splicing of the mRNA. The previously described A4002G polymorphism was encountered in approximately 9% of Russian CF chromosomes. In addition, we have found the previously described 3732delA mutation in 7 CF chromosomes, making it the second (after F508) most frequent mutation in the Russian population.     

Automated DNA mutation analysis by single-strand conformation polymorphism using capillary electrophoresis with laser-induced fluorescence detection

Automation is essential for rapid genetic-based mutation analysis in clinical laboratory to screen a large number of DNA samples. We propose in this report an automatic process using Beckman Coulter P/ACE™ capillary electrophoresis (CE) with laser-induced fluorescence (LIF) system to detect a single-point mutation in the codon 12 of human K-ras gene. Polymerase chain reaction (PCR) using a fluorescently labeled reverse primer and a plain forward primer to specifically amplify a selected 50 bp DNA fragment in human K-ras gene. The amplified DNA is placed on the sample tray of the CE system with a pre-programmed step for single-strand conformation polymorphism (SSCP) analysis. Sample injection and denaturation processes are performed online along with separation and real-time data analysis. The concept of automation for rapid DNA mutation analysis using CE-LIF system for SSCP is presented.     

Toward a fluorescent single-strand conformation polymorphism technique that detects all mutations: F-DOVAM-S

Although DOVAM-S (detection of virtually all mutations-SSCP) in effect detects all mutations and is less costly than direct sequencing, the technique currently requires the use of radioactivity. F-DOVAM-S (fluorescent DOVAM-S) was developed to replace the isotopic label with fluorescence and to increase throughput via dye color multiplexing. As proof of principle, two multitemperature slab gel electrophoresis conditions were evaluated through the blinded analysis of mutations in the factor IX (FIX) genes of 88 hemophilia B (HB) patients and 7 wild-type controls. Using only two conditions, it was determined that F-DOVAM-S had a detection sensitivity of 97%. It is anticipated that when three or four optimized conditions are employed, F-DOVAM-S will detect all mutations. Three patient samples were multiplexed per well using three different fluorescent dyes (6FAM, VIC, and NED), demonstrating that it is possible to analyze up to 44 kb of diploid, color-coded amplification product per gel lane. This value corresponds to a throughput of approximately 4 Mb of DNA analyzed per 96-well gel, which is approximately triple that of conventional radiolabeled DOVAM-S. Throughput is further enhanced by the rapidity at which the fluorescent signal can be captured and the resultant multicolor chromatograms analyzed. Given these data, F-DOVAM-S has the potential to be a particularly powerful technology for clinical diagnosis because it allows the mutation analysis of multiple patients to be performed within 24h.     

Prediction of DNA single-strand conformation polymorphism: analysis by capillary electrophoresis and computerized DNA modeling

We have analyzed previously three representative p53 single-point mutations by capillary-electrophoresis single-strand conformation polymorphism (CE-SSCP). In the current study, we compared our CE-SSCP results with the potential secondary structures predicted by an RNA/DNA-folding algorithm with DNA energy rules, used in conjunction with a computer analysis workbench called STRUCTURELAB. Each of these mutations produces measurable shifts in CE migration times relative to wild type. Using computerized folding analysis, each of the mutations was found to have a conformational difference relative to wild type, which accounts for the observed differences in CE migration. Additional properties exhibited in the CE electropherograms were also explained using the computerized analysis. These include the appearance of secondary peaks and the temperature dependence of the electrophoretic patterns. The results yield insight into the mechanism of SSCP and how the conditions of this measurement, especially temperature, may be optimized to improve the sensitivity of the SSCP method. The results may also impact other diagnostic methods, which would benefit by a better understanding of DNA single-strand conformation polymorphisms to optimize conditions for enzymatic cleavage and DNA hybridization reactions.     

Transverse formamide gradients as a simple and easy way to optimise DNA single-strand conformation polymorphism analysis.

Although widely used, the detection of DNA mutations by the single-strand conformation polymorphism (SSCP) method is often hampered by the need to examine a large set of electrophoretic conditions in order to select the one suited to the DNA sequence under study. We show here that the use of transverse chemical gradient gels allows for a quick and easy optimisation of SSCP analysis, as exemplified on two mutations in exon 2 of the alpha-1-antitrypsin gene.     

Analysis of sequence variation in Gnathostoma spinigerum mitochondrial DNA by single-strand conformation polymorphism analysis and DNA sequence

Morphological variations were observed in the advance third stage larvae of Gnathostoma spinigerum collected from swamp eel (Fluta alba), the second intermediate host. Larvae with typical and three atypical types were chosen for partial cytochrome c oxidase subunit I (COI) gene sequence analysis. A 450 bp polymerase chain reaction product of the COI gene was amplified from mitochondrial DNA. The variations were analyzed by single-strand conformation polymorphism and DNA sequencing. The nucleotide variations of the COI gene in the four types of larvae indicated the presence of an intra-specific variation of mitochondrial DNA in the G. spinigerum population.     

Single-strand conformation polymorphism for detection of mutations and base substitutions in phenylketonuria.

In the past few years, more than 20 different mutations have been reported in hyperphenylalaninemias. In southwestern Europe and Mediterranean countries, however, the mutant genotypes reported account for only a fraction (27%) of all mutant alleles at the phenylalanine hydroxylase (PAH) locus, and most of the mutations causing the disease remain unknown. In order to develop a strategy for rapid detection of mutation-containing exons, we applied the single-strand conformation-polymorphism (SSCP) technique to exons 3, 5, 7, and 12 of the PAH gene. We observed five abnormal patterns of migration in mutant PAH genes, and we consistently found base substitutions in the corresponding exons, with no false-positive results. By this procedure, two novel putative mutations were detected in the seventh exon of the PAH gene, (A259V and Y277D) and we were able to demonstrate that the delta I94, R158Q, R408W, and E280K mutations were easily detectable by the SSCP technique. This procedure is therefore of particular interest for rapid detection of mutation-containing exons and for determination of further genotype-phenotype correlations in hyperphenylalaninemias.     

Rapid differentiation of phenotypically similar yeast species by single-strand conformation polymorphism analysis of ribosomal DNA

Single-strand conformation polymorphism (SSCP) analysis of ribosomal DNA (rDNA) was investigated for rapid differentiation of phenotypically similar yeast species. Sensitive tests indicated that some yeast strains with one, most strains with two, and all strains with three or more nucleotide differences in the internal transcribed spacer 1 (ITS1) or ITS2 region could be distinguished by PCR SSCP analysis. The discriminative power of SSCP in yeast species differentiation was demonstrated by comparative studies of representative groups of yeast species from ascomycetes and basidiomycetes, including Saccharomyces species, medically important Candida species, and phylloplane basidiomycetous yeast species. Though the species within each group selected are closely related and have relatively similar rDNA sequences, they were clearly differentiated by PCR-SSCP analysis of the ITS1 region, given the amplified fragments were less than 350 bp in sizes. By using SSCP analysis for rapid screening of yeast strains with different rDNA sequences, species diversity existing in a large collection of yeast strains from natural sources was effectively and thoroughly investigated with substantially reduced time and cost in subsequent DNA sequencing.     

The detection of mitochondrial DNA mutations using single stranded conformation polymorphism (SSCP) analysis and heteroduplex analysis

A number of mitochondrial (mt) point mutations have been associated with inherited disorders. These pathogenic mutations are usually heteroplasmic. Here we describe the identification of three heteroplasmic mtDNA point mutations using the techniques of single stranded conformation polymorphism (SSCP) and heteroduplex analysis.     

Monitoring of a pyrite-oxidising bacterial population using DNA single-strand conformation polymorphism and microscopic techniques

The single-strand conformation polymorphism (SSCP) technique was used to study the evolution of a bacterial consortium during the batch oxidation of a cobaltiferous pyrite in two types of bio-reactor: a bubble column and a classical stirred tank. Sequencing 16S rDNA revealed the presence of three organisms affiliated to Leptospirillum ferrooxidans, Acidithiobacillus thiooxidans and Sulfobacillus thermosulfidooxidans, respectively. Attempts were made to determine the proportions of bacteria attached to solid particles or freely suspended in the medium using a combination of PCR-SSCP and a microscopic technique. Ac. thiooxidans-related bacteria were dominant in the liquid during the early phase of the batch, but were later supplanted by L. ferrooxidans-related bacteria. L. ferrooxidans-related organisms were always in the majority on the solids. The growth of S. thermosulfidooxidans-related bacteria seemed to be favoured by the bubble-column reactor.     

Validation of fluorescence-labeled artificial nonhuman sequences for single-strand conformation polymorphism mutation detection in familial hypercholesterolemia

We developed a two-in-one, polymerase chain reaction (PCR)-based method with a specific amplification step and a universal amplification step in one tube to screen for the presence of DNA variations. The method relies on fluorescence-labeled artificial nonhuman sequences for mutation detection. To document utility, we applied this method as a high-throughput capillary single-strand conformation polymorphism screening system to identify 30 mutations in the low-density lipoprotein receptor gene. The sensitivity of mutant allele detection compared to wild-type allele detection was 93%. We conclude that the "two-in-one PCR" is sensitive, simple, and cost effective.     

A single base pair polymorphism in the WT1 gene detected by single-strand conformation polymorphism analysis

The Wilms' tumor predisposition gene, WT1, was analysed exon-by-exon in a variety of tumours using the single-strand conformation polymorphism (SSCP) technique. A consistent variation in the usual band pattern for exon 7 was detected in this survey. On sequencing, a silent mutation was noted in codon 313 resulting in an AG transition in an arginine codon. The AG transition destroys an AflIII restriction enzyme recognition site, which provides a rapid means of identifying heterozygotes at this locus. Analysis of the segregation of this polymorphism in families demonstrated a co-dominant inheritance pattern. In an analysis of 21 randomly selected individuals 25% were heterozygous at this locus, which makes this polymorphism useful in a variety of genetic analyses.     

Photogrammetry: a useful tool for three‐dimensional morphometric analysis of small mammals

Recently, the improvement of methods for shape analysis has revolutionized the field of morphometrics. While three‐dimensional (3D) imaging technology is increasingly available, many studies of 3D structures still use two‐dimensional (2D) data, even when this may result in the loss of important information. This is particularly conspicuous in the study of small mammals, as devices precise enough for 3D digitization of small objects are the most expensive. Thus, the development of low‐cost methods aimed to recover 3D shape from small mammals would be of wide interest. Photogrammetry allows for obtaining 3D data with a lower cost than other 3D techniques, but it has not been previously applied to the study of small mammals. Accordingly, here we test the suitability of photogrammetric techniques to obtain 3D landmarks on mouse skulls as a model for small mammals. Shape and size of 3D models obtained with photogrammetric techniques were consistent among replicates, even when different sets of photographs were used. The linear measurements obtained from 3D models produced here were highly correlated with measurements obtained with callipers on actual crania, and differences among both sets of measures were smaller than those among individuals in most of the tested measures. These results show for the first time that photogrammetry is a precise technique for 3D shape analysis of small mammals. Photogrammetry also proved to be accurate for obtaining linear measurements between 3D landmarks; however, further studies are needed to demonstrate that this technique is also accurate to recreate 3D shapes.     

'Cold SSCP': a simple, rapid and non-radioactive method for optimized single-strand conformation polymorphism analyses.

A rapid (< 2.5 hrs) method for single-strand conformation polymorphism (SSCP) analysis of PCR products that allows the use of ethidium bromide staining is described. PCR products ranging in size from 117 to 256 bp were evaluated for point mutations and polymorphisms by 'cold SSCP' in commercially available pre-cast polyacrylamide mini-gels. Several electrophoretic parameters (running temperature, buffers, denaturants, DNA concentration, and gel polyacrylamide concentration) were found to influence the degree of strand separation and appeared to be PCR fragment specific. Use of the 'cold' SSCP technique and the mini-gel format allowed us to readily optimize the electrophoretic conditions for each PCR fragment. This greatly increased our ability to detect polymorphisms compared to conventional, radioisotope-labeled 'hot' SSCP, typically run under two standard temperature conditions. Excellent results have been obtained in resolving mutant PCR fragments from human p53 exons 5 through 8, human HLA-DQA, human K-ras exons 1 and 2, and rat K-ras exon 3. Polymorphisms could be detected when mutant DNA comprised as little as 3% of the total gene copies in a PCR mixture. Compared to standard 'hot' SSCP, this novel non-isotopic method has additional advantages of dramatically increased speed, precise temperature control, reproducibility, and easily and inexpensively obtainable reagents and equipment. This new method also lacks the safety and hazardous waste management concerns associated with radioactive methods.