Comparison of different methods to produce single-strand DNA for identification of canned tuna by single-strand conformation polymorphism analysis

By using single-strand conformation polymorphism (SSCP) analysis of three amplicons of the cytochrome b gene obtained by the polymerase chain reaction (PCR) it was possible to differentiate between various species of tunas and bonitos processed as canned fish. Four different techniques were used to produce single-strand DNA (ssDNA): (i) Denaturation of double-strand DNA (dsDNA) by formamide and alkali, (ii) two-step asymmetrical PCR, (iii) one-step asymmetrical PCR, and (iv) exonuclease digestion of the phosphorylated strand of dsDNA. The technique rendering optimal results depended on the type of amplicon (i.e. the sequence).     

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 p53 gene mutations in human gliomas by polymerase chain reaction-based single-strand conformation polymorphism and DNA sequencing

This report documents the effects of malaria epidemic and how it was controlled in one highland district of Kenya. The effects of the epidemic are presented in terms of mortality, morbidity and school absenteeism; information is from routine and verbal reports. Treatment with chloroquine, amodiaquine and sulphonamide pyrimethamine combinations, limited vector control, and health education were used to control the epidemic. Hospital mortality per month increased by 8.6 times during the epidemic while morbidity went up by 3.7 times. Of the 103 deaths attributed to malaria, 64 (62.1%) occurred in hospital and 39 (37.9%) at home. Most of the home deaths (92.3%), occurred in areas that border the malaria endemic Lake Victoria Basin. The rate of pupil absenteeism ranged from 17.6% to 54.4% in primary schools. The policy implications of the report are discussed.     

Nonradioactive single-strand conformation polymorphism analysis for detection of fluoroquinolone resistance in mycobacteria

The synovium from a patient with mixed connective tissue disease and destructive ankle monoarthritis was studied in detail to determine its immunohistological characteristics. Fibrinoid necrotic tissue on the surface of the synovium, multi-layered lining cells, increased numbers of capillaries, interstitial edema, infiltration of macrophages, relatively small numbers of lympho-plasma cells and polymorphonuclear leukocytes, scattered bone fragments, and multinucleated giant cells were observed. Many cells in the lining and sublining area were positive for CD68 and MAC387. Lower layers of increased lining cells which had a spindle shape were positively stained with anti-HLA-DR antibody. The small arteries in the deeper part of the synovium revealed obstruction or highly stenotic change. These findings suggest that obstructive circulatory disturbance due to endothelial injury might influence the progression of arthritis.     

Multiplex-PCR-based single-strand conformation polymorphism protocol for simultaneous analysis of up to five fragments of the low-density-lipoprotein receptor gene

Single-strand conformation polymorphism has become a screening method for the detection of mutations in different genes. For analysis of the promotor region and the coding sequence of the low-density-lipoprotein receptor gene by standard protocols, 21 radiolabeled PCRs and electrophoreses have to be performed. To accelerate this procedure, we developed a nonradioactive multiplex approach of the single-strand conformation polymorphism analysis. Multiplex PCRs were established, each resulting in the amplification of 4 or 5 fragments of this gene. The heat-denatured, single-stranded multiplex-PCR products were electrophoresed, blotted on a nylon membrane and visualized using a chemiluminescence detection system. The simultaneously amplified fragments were clearly resolved by their different mobility on the gel. Comparing the pattern of bands of each separately amplified PCR product and the multiplex-PCR products allowed identification of each band as one exon, part of an exon or the promotor region of the gene. To determine the sensitivity of this method, the low-density-lipoprotein receptor gene of 11 patients with 11 different mutations was analyzed. All mutations could be identified in the multiplex reactions. We conclude that a multiplex-PCR-based, single-strand conformation polymorphism protocol is much faster but equally sensitive compared to standard protocols.     

Rapid detection of K-ras gene mutations in canine lung cancer using single-strand conformational polymorphism analysis

A total of 126 spontaneous lung tumors from pet dogs were examined for K-ras mutations within exon 1 and exon 2 using a non-radioisotope single-strand conformational polymorphism analysis (SSCP) detection method on PCR products. Mutations were confirmed by direct DNA sequencing. Tumors were classified as adenomas (9), bronchioloalveolar carcinomas (59), adenocarcinomas (30), adenosquamous carcinomas (16), squamous cell carcinomas (3) and anaplastic carcinomas (9). Nineteen mutations were detected in the malignant tumors: 18 occurred in exon 1 codon 12 and one in exon 2 codon 61. No mutations were present in the adenomas. The most common mutation was a G-->A transition (11/19) in the second position of codon 12. Based on this study, K-ras mutations occur in canine non-small cell lung carcinomas. The frequency and type of mutation more closely matches tumors from human non-smokers with K-ras mutations than smokers. With the application of screening techniques such as SSCP, large numbers of dog tumors can be examined to provide a large animal model for comparative studies of carcinogenesis.     

Allelic loss on chromosome 9 in bladder cancer tissues and urine samples detected by blunt-end single-strand DNA conformation polymorphism

BACKGROUND: A high incidence of backache with radiating pains to the lower extremities, termed transient radicular irritation (TRI), has been reported following the use of 5% hyperbaric lidocaine. This has been attributed to a neurotoxic reaction. METHODS: A retrospective audit has been carried out in our hospital on the postoperative anaesthetic records of all patients from the 1st of January 1993 to the 1st of September 1996, who received spinal anaesthesia with either hyperbaric lidocaine or hyperbaric bupivacaine for day-care surgery. RESULTS: Backache was reported in 1.9% of patients (6/322) receiving hyperbaric lidocaine and in 2.4% of patients (1/41) receiving hyperbaric bupivacaine. This was not associated with any sensory, motor or sphincter disturbances. One patient complained of backache with bilateral pain referred to the gluteal area (TRI), which was assessed as acute facet joint syndrome. Unilateral accidental block of the femoral nerve was observed in 3 patients, sensory disturbances in the L2/3 dermatome was reported in a further 3 patients at 24 h, following wound infiltration with local anaesthetic during hernia repair. CONCLUSIONS: The low incidence of backache at 24 h and the absence of associated symptoms of neurogenic pain, sensory and motor disturbances, does not support the hypothesis that TRI is a neurotoxic reaction, subsequent to the use of hyperbaric lidocaine.     

Detection of rifampin resistance by single-strand conformation polymorphism analysis of cerebrospinal fluid of patients with tuberculosis of the central nervous system

Mutations in a 69-bp region of the rpoB gene of Mycobacterium tuberculosis are associated with rifampin resistance (Rif[r]). These have been detected with mycobacterial DNA extracted from bacterial suspensions or respiratory specimens that were acid-fast smear positive. We experimented with a strategy for the rapid detection of Rif(r) in cerebrospinal fluid (CSF) samples. The strategy involves the amplification of the 69-bp region of rpoB by means of PCR and the identification of nucleotide mutations by single-strand conformation polymorphism (SSCP) analysis of the amplification products. Sixty-five CSF specimens collected from 29 patients (19 patients were coinfected with human immunodeficiency virus) with culture or autopsy-confirmed (22 patients) or highly probable (7 patients) tuberculosis of the central nervous system (CNS-TB) were processed. Amplified products suitable for evaluation by SSCP analysis were obtained from 37 CSF specimens from 25 subjects (86.2%). PCR-SSCP of CSF correctly identified the rifampin susceptibility phenotype of isolates from all 17 patients for whom the results of susceptibility tests carried out with strains cultured from CSF or respiratory samples were available. Moreover, this assay revealed the rifampin susceptibility genotype of isolates from the eight patients (three patients with culture-confirmed CNS-TB and five patients in whom CNS-TB was highly probable) for whom no susceptibility test results were available; the PCR-SSCP data obtained for these patients were concordant with the outcome after a standard antituberculosis treatment. The evolution of a mutation in the rpoB gene was documented in a patient during the course of treatment. PCR-SSCP analysis of CSF seems to be an efficacious method of predicting Rif(r) and would reduce the time required for susceptibility testing from approximately 4 to 8 weeks to a few days.     

Detection of missense mutations by single-strand conformational polymorphism (SSCP) analysis in five dysfunctional variants of coagulation factor VII

Five unrelated subjects with dysfunctional coagulation factor VII (FVII) were studied in order to identify missense mutations affecting function. Exons 2 to 8 and the intron-exon junctions of their FVII genes were amplified from peripheral white blood cell DNA by PCR and screened by SSCP analysis. DNA fragments showing aberrant mobility were sequenced. The following mutations were identified: in case 1 (FVII:C < 1%, FVII:Ag 18%) a heterozygous A to G transition at nucleotide 8915 in exon 6 results in the amino acid substitution Lys-137 to Glu near the C-terminus of the FVIIa light chain; in case 2 (FVII:C 7%, FVII:Ag 47%) a heterozygous A to G transition at nucleotide 7834 in exon 5 results in the substitution of Gln-100 by Arg in the second EGF-like domain; in case 3 (FVII:C 20%, FVII:Ag 76%) a homozygous G to A transition at nucleotide position 6055 in exon 4 was detected resulting in substitution of Arg-79 by Gln in the first EGF-like domain; in case 5 (FVII:C 10%, FVII:Ag 52%) a heterozygous C to T transition at nucleotide position 6054 in exon 4 also results in the substitution of Arg79, but in this case it is replaced by Trp; case 4 (FVII:C < 1%, FVII:Ag 100%) was homozygous for a previously reported mutation (G to A) at nucleotide position 10715 in exon 8, substituting Gln for Arg at position 304 in the protease domain. Cases 1, 2 and 5 evidently have additional undetected mutations.     

Typing of Pneumocystis carinii f. sp. hominis by single-strand conformation polymorphism of four genomic regions

To better investigate Pneumocystis carinii f. sp. hominis epidemiology, we have developed a molecular typing method. Because of the limited genetic variability of the P. carinii hominis genome, a multitarget approach was used. Four variable regions of the genome were amplified by PCR, polymorphism in each region was assessed by the single-strand conformation polymorphism (SSCP) technique, and the results for the four regions of each patient were combined. Bronchoalveolar lavage specimens collected from 11 patients were examined. Four patients were probably infected by a single strain, since their specimens yielded simple SSCP patterns (two bands corresponding to one allele). The combinations of these patterns were unique, suggesting that the strains which infected these patients were different. For the other seven patients, complex patterns were found (three or four bands corresponding to two alleles). The presence of more than one allele of a region in a patient is likely to be due to coinfection. Polymorphism was also assessed by sequencing, which revealed variations at nucleotide positions previously reported to vary. About half of the observed alleles had already been reported by laboratories in different countries. Multitarget typing of P. carinii hominis by PCR-SSCP should allow investigation of strain diversity and thus be useful for future epidemiological studies.     

A molecular model of the serine protease domain of activated protein C: application to the study of missense mutations causing protein C deficiency

A molecular model of the serine protease domain of protein C was constructed by standard comparative methods. Individual missense mutations were inserted into the model and plausible explanations for their interference with protein C structure/function were derived through consideration of location, steric effects and protein stability. A hydrophilic cluster of many Arg and Lys residues, found adjacent to the active site cleft, is proposed to be involved in thrombomodulin and/or protein S interactions. Analysis of comparative binding studies also suggested the presence of an extended substrate binding pocket in the model.     

Identification of p53 mutations in endometrial adenocarcinoma by polymerase chain reaction-single-strand conformation polymorphism

OBJECTIVE: We determined whether mutations in p53 exons 5-6-7-8, as detected in the polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) test, might be correlated with stage or grade in endometrial adenocarcinoma. METHODS: We amplified sequences containing exons 5, 6, 7, or 8 in DNA from tumors and controls. Mutation within the amplified sequences was indicated by changes in electrophoretic mobility (band shifts) in the SSCP test. The results were analyzed statistically and compared with the results of other, similar studies. RESULTS: We identified 15 band shifts among 47 endometrial tumors studied (band shifts in two different exons in two cases) and none among 42 controls. Band shifts in exons 5 and 8 were associated uniformly with grade 2 or grade 3 histology. In other studies p53 mutations were correlated with advanced-stage malignancy. CONCLUSION: Further evaluation of particular p53 mutations and their relation to disease course in endometrial adenocarcinoma seems warranted. The PCR-SSCP test seems well-suited to this purpose.     

Inferring the fitness effects of DNA mutations from polymorphism and divergence data: statistical power to detect directional selection under stationarity and free recombination

The fitness effects of classes of DNA mutations can be inferred from patterns of nucleotide variation. A number of studies have attributed differences in levels of polymorphism and divergence between silent and replacement mutations to the action of natural selection. Here, I investigate the statistical power to detect directional selection through contrasts of DNA variation among functional categories of mutations. A variety of statistical approaches are applied to DNA data simulated under Sawyer and Hartl's Poisson random field model. Under assumptions of free recombination and stationarity, comparisons that include both the frequency distributions of mutations segregating within populations and the numbers of mutations fixed between populations have substantial power to detect even very weak selection. Frequency distribution and divergence tests are applied to silent and replacement mutations among five alleles of each of eight Drosophila simulans genes. Putatively "preferred" silent mutations segregate at higher frequencies and are more often fixed between species than "unpreferred" silent changes, suggesting fitness differences among synonymous codons. Amino acid changes tend to be either rare polymorphisms or fixed differences, consistent with a combination of deleterious and adaptive protein evolution. In these data, a substantial fraction of both silent and replacement DNA mutations appear to affect fitness.     

Neonatal purpura fulminans due to homozygous protein C deficiency

All aspects of surgical decision making,--why, what, when, where, how and to whom--are being subjected to an increasingly accurate analysis. The principles of this analysis should form an essential part of the theoretical curriculum of all surgeons, young and old. Only by these means can the production of health benefits be maximized and the definitive goal of all surgical care be attained: the maximization of produced health benefits. This article provides a short outline of decision analysis in surgery.     

Rapid detection of sequence polymorphisms in the human mitochondrial DNA control region by polymerase chain reaction and single-strand conformation analysis in mutation detection enhancement gels

The article describes a rapid approach for the detection of sequence polymorphisms in the mitochondrial (mt)DNA control region that involves enzymatic amplification of each entire mtDNA control region (HV1 and HV2) and the subsequent analysis of the PCR products by single-strand conformation analysis (SSCA) in mutation detection enhancement (MDE) gels, followed by silver stain detection. HV1 and HV2 SSC reference ladders were developed to standardize the classification of the different mtDNA types. Twenty-five mtDNA types were observed among the 45 Spanish individuals analyzed: 11 types were observed in the HV1 region as compared with 10 types in the HV2 region. This mutation scanning strategy could be a promising method of potential use not only in forensic genetics but also in population and evolutionary studies.