Single-strand Conformation Polymorphism Patterns Research Articles

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.

Detection of human papillomavirus type 16 DNA in consecutive genital samples does not always represent persistent infection as determined by molecular variant analysis.

Persistent human papillomavirus (HPV) infection of the uterine cervix is a risk factor for progression to high-grade squamous intraepithelial lesions. Detection in consecutive genital samples of HPV-16 DNA, a frequently encountered HPV type, may represent persistent infection or reinfection. We undertook a study using PCR-single-strand conformation polymorphism (SSCP) analysis and sequencing of PCR products (PCR-sequencing) to determine if consecutive HPV-16-positive samples contained the same HPV-16 variant. Fifty women (36 human immunodeficiency virus [HIV] seropositive, 14 HIV seronegative) had at least two consecutive genital specimens obtained at 6-month intervals that contained HPV-16 DNA as determined by a consensus L1 PCR assay. A total of 144 samples were amplified with two primer pairs for SSCP analysis of the entire long control region. Fifteen different SSCP patterns were identified in our population, while 22 variants were identified by PCR-sequencing. The most frequent SSCP pattern was found in 75 (53%) samples from 27 (54%) women. The SSCP patterns obtained from consecutive specimens were identical for 46 (92%) of 50 women, suggesting persistent infection. Four women exhibited in consecutive specimens different HPV-16 SSCP patterns that were all confirmed by PCR-sequencing. The additional information on the nature of persistent infection provided by molecular variant analysis was useful for 6% of women, since three of the four women who did not have identical consecutive specimens would have been misclassified as having persistent HPV-16 infection on the basis of HPV typing.

Detection of arbuscular mycorrhizal fungi (Glomales) in roots by nested PCR and SSCP (Single Stranded Conformation Polymorphism)

PCR amplification of a region of the large subunit ribosomal DNA sequence with Glomus specific primers was used to detect arbuscular mycorrhizal fungi in root tissue of four plant species. The primers were specific to Glomus mosseae, Glomus caledonium, Glomus geosporum, Glomus coronatum, Glomus fragilistratum and Glomus constrictum, and did not recognise sequences from Glomus claroideum. Sequence differences between isolates were detected by Single Stranded Conformation Polymorphisms (SSCPs) in polyacrylamide gels under non-denaturing conditions. Isolates of G. mosseae, G. caledonium and G. coronatum could be separated by their SSCP patterns, while three isolates of G. geosporumshowed no variation. Specific SSCP patterns from isolates of G. mosseae and G. caledonium allowed detection of both fungi in the same root segment. Sequence differences leading to variations in SSCP patterns were confirmed by direct sequencing.

Mapping candidate genes in Eucalyptus with emphasis on lignification genes.

We used the single-strand conformation polymorphism (SSCP) technique to map eight genes on Eucalyptus urophylla and Eucalyptus grandis linkage maps. These included four genes involved in the common phenylpropanoid pathway (caffeic acid 3-0-methyltransferase, caffeoyl CoA 3-O-methyltransferase, 4-coumarate CoA ligase and phenylalanine ammonia-lyase), two genes involved in the `lignin specific' pathway (cinnamoyl CoA reductase and cinnamyl alcohol dehydrogenase), and two symbiosis regulated genes (EgHypar and EgTubA1). A novel source of variation which affects the SSCP pattern, i.e. the presence or absence of electrophoresis buffer upon loading the samples into the polyacrylamide gel, was found. The placement of these genes on the Eucalyptus maps was carried out using an interspecific hybrid mapping population. This will further facilitate the identification or exclusion of `positional' candidate genes for characterizing quantitative trait loci (QTL) for wood quality and vegetative propagation related traits.

Characterization of Citrus Tristeza Virus Isolates by Single-Strand Conformation Polymorphism Analysis of DNA Complementary to Their RNA Population

Citrus tristeza virus (CTV) isolates, like other RNA viruses, contain a population of genomic variants, the composition of which may affect their biological characteristics. Analysis of single-strand conformation polymorphism (SSCP) is a simple technique that allows detection of minor variations in the nucleotide sequence of DNA fragments without sequencing. To assess if SSCP could be used to characterize the population of sequence variants present in CTV isolates, dsRNA from two isolates, differing by their SSCP profile of the gene p20, was combined at various ratios and the mixtures used as templates to synthesize cDNA by RT-PCR. The SSCP profile obtained from each dsRNA mix was a composite of the individual profiles of the two isolates, and the intensities of the cDNA bands reflected the relative proportion of each isolate in the dsRNA mix. The SSCP pattern of each component could be observed in the composite pattern when its dsRNA was in a ratio of at least 10%. Graft or slash-inoculation of CTV isolate T317 from citron to sweet orange, or from citrus to lime and then to sweet orange, caused changes in its SSCP profile, indicating host selection of the predominant sequence variants in the population. In some cases, this change was reversed when the isolate was back inoculated into the first host species, whereas in others the change was permanent. Variations in the viral population after change of host may help to explain biological diversity observed among CTV isolates.

Absence of mutations in the kinase domain of the Met gene and frequent expression of Met and HGF/SF protein in primary gastric carcinomas.

Hepatocyte growth factor/scatter factor (HGF/SF) and its receptor, Met, are known to play important roles in tumor cell migration, invasion, and metastasis. We analyzed the expression of these genes and the mutations in the kinase domain of the Met gene in 43 gastric carcinomas. Of the 43 cases, the Met and HGF/SF protein were expressed in 29 (67%) and 22 (51%), respectively. All of the cases with HGF/SF immunopositivity also expressed Met. Of 22 cases with HGF/SF immunopositivity, 13 (59%) expressed HGF/SF in tumor cells as well as fibroblasts. We detected no aberrant single-strand conformational polymorphism patterns, suggesting that there are no genetic alterations in the kinase domain of the Met gene. These results indicate that HGF/SF-mediated autocrine and/or paracrine stimulation and overexpression rather than structural alteration of its receptor may contribute to the development and progression of gastric carcinoma, and that expression of Met and HGF/SF may confer a growth advantage to neoplastic cells.

P53 mutations as a marker of malignancy in bladder washing samples from patients with bladder cancer.

The diagnosis and follow-up of patients with bladder cancer rely on invasive procedures (cystoscopy with biopsy). Polymerase chain reaction (PCR)-based technologies may allow the sensitive detection of cancer-related genetic mutations in exfoliated tumour material, potentially allowing the development of less invasive techniques. This pilot study investigated the feasibility of detecting mutations in exons 5–8 of the p53 gene using single-stranded conformational polymorphism (SSCP) analysis in bladder-washing specimens from patients with bladder cancer. Bladder-washing samples (31) were collected from patients (27) with bladder cancer. An abnormal additional SSCP band was detected in five samples from five different patients suggesting the presence of a p53 mutation. In all five cases the same abnormal SSCP pattern was demonstrated in samples of the corresponding bladder tumour. In one case bladder washings were available from the same patient on two separate occasions with one washing demonstrating a mutation and the other not. In two further cases a mutation was demonstrated in the bladder tumour but not in the corresponding washing. It is concluded that it is possible to detect and characterize p53 mutations in bladder-washing samples from patients with bladder cancer. Improved sensitivity in detecting mutations in a sample containing a mixture of normal and malignant cells may lead to the clinical applicability of molecular methods of disease monitoring. © 2000 Cancer Research Campaign

Rapid test for identification of a human papillomavirus 16 E6 L83V variant.

A variant of human papillomavirus (HPV) 16 has been shown recently to be more prevalent in invasive cervical carcinoma than in preinvasive lesions. This HPV 16 variant possesses a common mutation (T to G) in nucleotide 350 (codon 83) of the E6 gene, resulting in an amino acid shift, L83V, in the E6 protein. This mutation was believed to signify preinvasive cervical lesions with a high probability of progression to invasive carcinoma. The purpose of the present investigation is to describe a rapid method for the detection of this variant HPV 16, E6 (L83V). Paraffin blocks of 18 gynecologic biopsy specimens were collected, all displaying the morphology of cervical intraepithelial neoplasia (CIN I-III) and a positive HPV 16 test. Sections from these blocks were used for DNA extraction. A DNA sequence of the E6 gene containing 176 bases (including codon 83) was amplified by polymerase chain reaction (PCR) and analyzed by non-radioactive single-strand conformational polymorphism (SSCP) analysis. A divergent SSCP pattern was observed in 7 of the HPV 16 positive biopsy specimens. A DNA sequence analysis of the PCR products revealed the conversion of Leu to Val in codon 83 of the E6 gene, correlating to the divergent band pattern. This PCR-SSCP method can be used to test for HPV 16 in women who are at serious risk of developing invasive cervical carcinoma.

Sequence variability in p27 gene of Citrus Tristeza Virus (CTV) revealed by SSCP analysis

Citrus tristeza closterovirus (CTV), is a phloem-limited virus transmitted by aphids in a semipersistent manner. The genome of CTV is composed of a ssRNA with two capsid proteins: CP, covering about 95% of the particle length, and a diverged coat protein (dCP), present only in one end of the particle, forming a rattlesnake structure. dCP is the product of p27 gene for which it is also postulated a function in the transmissibility by aphid vectors. Hybridization analysis showed a p27 gene region, which exhibits different patterns with two probes derived from two biological distinct CTV isolates. In an attempt to screen whether that gene region differs in mild and severe strains, six CTV isolates belonging to different biogroups were compared for variations in their p27 gene by analysis of singlestrand conformation polymorphism (SSCP). The p27 gene was reverse transcribed and amplified by PCR and thirty clones of each isolate were obtained. From each clone, two fragments of the gene were amplified by PCR: fragment (a), 459 bp long, and fragment (b), 281 bp long. Sequence variations in both gene fragments were studied by SSCP analysis. A variety of SSCP patterns was obtained from each isolate, being isolates belonging to the groups II-IV and III those with the higher and lower number of them. Moreover, SSCP analysis provided a rapid procedure to screen the genetic heterogeneity of the viral isolates reducing considerably the amount of nucleic acid sequenciation necessary to gain that knowledge.

Determination of failure of treatment of plasmodium falciparum infection by using polymerase chain reaction single-strand conformational polymorphism fingerprinting.

The inability to distinguish failures of treatment of Plasmodium falciparum infection from new infections is an important impediment to the evaluation of antimalarial drugs. On the basis of a pilot study utilizing polymerase chain reaction (PCR) single-strand conformational polymorphism (SSCP) analysis to genotype P. falciparum isolates, we sought to confirm that PCR SSCP analysis could reliably distinguish infections for which treatment failed from unrelated infections with a sample size adequate to estimate the accuracy of this technique. PCR SSCP analysis of the MSP-1, MSP-2, and GLURP genes was performed on 72 paired isolates recovered from 36 individuals for whom treatment failed in Thailand. In every case (100% [95% confidence interval (CI), 90%-100%]), the PCR SSCP pattern of the recrudescent isolates matched that of the primary isolate. We determined whether PCR SSCP analysis could separate unrelated infections by comparing each recrudescent isolate with each of the unrelated primary isolates. Of 1,260 comparisons, 1,258 (99.8% [95% CI, 99.4%-100%]) were unique. The results indicate that PCR SSCP analysis can be used to differentiate infections for which treatment failed from reinfections.

Fluorescence-based single-strand conformation polymorphism analysis of the low density lipoprotein receptor gene by capillary electrophoresis

We describe here a new method to screen for unknown mutations in the low density lipoprotein (LDL) receptor gene by the use of capillary electrophoresis in single-strand conformation polymorphism (SSCP) analysis. To analyze the promoter and all 18 exons, 20 different amplification reactions were necessary. For each polymerase chain reaction (PCR), the forward and reverse primers were 5′ fluorescent-labelled with FAM and HEX, respectively. To test the accuracy of the newly developed method, 61 genetic variants distributed in 16 exons were analyzed. Under identical electrophoresis conditions (13 kV, 30°C, 30 min), 59 mutations were detected by a distinct abnormal SSCP pattern. The two remaining mutations showed only slight abnormalities, which could be amplified by increasing the electrophoresis temperature. The high accuracy, the degree of automation and the speed of analysis make fluorescence-based SSCP analysis with capillary electrophoresis ideal for rapid mutation screening and the technique is well-suited for clinical applications.

Mutations in von Willebrand Factor Multimerization Domains Are not a Common Cause of Classical Type 1 von Willebrand Disease

Type 1 von Willebrand disease (vWD) is an autosomal dominant bleeding disorder of variable penetrance. It is characterised by a mild to moderate bleeding tendency and a quantitative deficiency of von Willebrand factor (vWF) with the full range of vWF multimers. Few mutations have been described which account for the mode of inheritance in dominant vWD type 1. We screened the vWF multimerization domains (regions D1-D3 of the vWF gene) of 12 unrelated patients with dominant vWD type 1 to investigate the hypothesis that multimerization of vWF sub-units may be inhibited or reduced by a "dominant negative" mechanism. Platelet-derived RNA was reverse transcribed and the resulting vWF cDNA amplified by the polymerase chain reaction (PCR) in a series of overlapping fragments. These were subjected to a combination of single-strand conformation polymorphism (SSCP) and heteroduplex analysis. This approach identified mobility shifts on acrylamide gels that represented 12 distinct SSCP and/or heteroduplex patterns in our patient group. DNA sequencing of the region encompassing each mobility shift showed these variants to represent previously described polymorphisms within the vWF coding sequence. Examination in all 12 patients for the previously described G3389T and T3445C mutations proved negative. The molecular pathology of classical type 1 vWD remains enigmatic, mutations having been identified in only a small minority of patients. A common mechanism underlying this disease state has still to be elucidated.

Glycogen storage disease type Ia: four novel mutations (175delGG, R170X, G266V and V338F) identified. Mutations in brief no. 220. Online.

Deficient activity of glucose-6-phosphatase (G6Pase) causes glycogen storage disease type Ia (GSD Ia). We analysed the G6Pase gene of 16 GSD Ia patients using single strand conformation polymorphism (SSCP) analysis prior to automated sequencing of exon(s) revealing an aberrant SSCP pattern. In all GSD Ia patients we were able to identify mutations on both alleles of the G6Pase gene, indicating that this method is a reliable procedure to identify mutations. Four novel mutations (175delGG, R170X, G266V and V338F) were identified.

Genomic Variation of Human Papillomavirus Type 16 and Risk for High-Grade Cervical Intraepithelial Neoplasia

Background Epidemiologic studies have demonstrated strong and consistent associations between the detection of human papillomavirus (HPV) type 16 DNA and the risk of cervical intraepithelial neoplasia (CIN) and cervical cancer. However, HPV16 is also the most common type of HPV in the normal population, and only a minority of women with HPV16 infection develop cervical cancer. Studies of genomic heterogeneity in HPV16 have demonstrated the presence of multiple variant forms in all human populations examined to date. It is conceivable that the natural variants of HPV16 in a given population may not have the same biologic behavior. Purpose This study was designed to determine the association between natural variants of HPV16 and the risk of biopsy-confirmed CIN 2 or 3, the most important precancerous lesions of the uterine cervix. Methods Prospective studies were conducted among 1) women attending a university and 2) women presenting to a sexually transmitted disease clinic. Subjects were eligible for inclusion in this investigation if the initial cytologic findings did not reveal CIN 2-3 and HPV16 DNA was detected by means of a polymerase chain reaction (PCR)-based method in one or more cervical or vulvovaginal samples. Eligible subjects were followed every 4 months with cervical Pap smears and colposcopic examinations. Women were referred for biopsy if cytology or colposcopy suggested CIN 2-3. Two groups of HPV16 variants, prototype-like and nonprototype-like, were determined by means of single-strand conformation polymorphism (SSCP) analysis of PCR products from the noncoding region of the viral genome. Representative SSCP patterns from HPV16 variants were further characterized by direct DNA sequencing of the PCR products. Relative risks (RRs) and 95% confidence intervals (CIs) were calculated by Cox regression analysis. Results Prototype-like variants accounted for 79% of the HPV16 detected in university students and 86% of the virus detected in patients presenting to the sexually transmitted disease clinic. CIN 2-3 was confirmed by biopsy in nine of 57 HPV16-positive women attending the university and in 10 of 66 HPV16-positive women presenting to the sexually transmitted disease clinic. Among university students, those with HPV16 nonprototype-like variants were 6.5 (95% CI = 1.6-27.2) times more likely to develop CIN 2-3 than those with prototype-like variants. A similar association was observed among women presenting to the sexually transmitted disease clinic (RR = 4.5; 95% CI = 0.9-23.8). Conclusions This study suggests that the risk of developing CIN 2-3 is not the same with all variants of HPV16 and that nonprototype-like variants confer a greater risk compared with prototype-like variants. The important genomic differences underlying this increased risk of CIN 2-3 remain to be determined.

Fluorescence-based SSCP Analysis with Automatic Allele Detection Demonstrated for the Factor V Leiden Mutation

Resistance of coagulation factor V to activated protein C is caused by a point mutation in which a G→A substitution at nucleotide 1691 of the factor V gene leads to the replacement of Arg506 by Gln (1) . Because of the high prevalence of the factor V Leiden mutation, it would be reasonable to perform a rapid and simple DNA test. In recent publications, several strategies of DNA testing were described. These tests included restriction enzyme digestion, the introduction of new cutting sites, allele-specific PCR, oligonucleotide ligation assay, and single-strand conformation polymorphism (SSCP) analysis (1)(2)(3)(4)(5) . The high sensitivity in mutation detection and the simple application have led to wide use of the SSCP method. Currently in most laboratories, SSCP analysis is performed in polyacrylamide gels in combination with silver staining. In this report, we adapted SSCP analysis to capillary electrophoresis. This was the first step toward automation because preparation of a gel was no longer necessary. The denatured DNA samples were placed in a sample tray, and after each electrophoretic run, new gel was injected from the system into the capillary. The SSCP patterns …

Ras-related TC21 is activated by mutation in a breast cancer cell line, but infrequently in breast carcinomas in vivo.

Activating ras mutations are found in many types of human tumour. Mutations in Harvey (H-), Kirsten (K-) and neuronal (N-) ras are, however, rarely found in breast carcinomas. TC21 is a ras family member that shares close homology to H-, K- and N-ras, and activating mutations have been found in ovarian carcinoma and leiomyosarcoma cell lines. We have examined panels of cDNAs from breast, ovarian and cervical cell lines, and primary and metastatic breast tumours for mutations in TC21 using a single-strand conformational polymorphism (SSCP)-based assay. One breast cancer cell line, CAL51, exhibited an altered SSCP pattern, compared with normal tissue, which was due to an A-T base change in codon 72, causing a predicted Gln-Leu activating mutation. Of nine primary and 15 metastatic breast tumour cDNAs analysed, none exhibited an altered pattern by SSCP. The apparently wild-type pattern by SSCP analysis was confirmed by sequence analysis of some of the cDNAs assayed. Thus, we conclude that mutations in TC21 are uncommon in breast carcinomas.

Optimized PCR labeling in mutational and microsatellite analysis

To optimize the labeling and visualization of PCR products we tested different variables, including deoxynucleotide concentration and ratio, dilution of labeled product, number of PCR cycles, and use of one-step or nested labeling protocols. Labeling was achieved using a fixed amount of labeled dATP, whose relative specific activity was varied by adding increasing amounts of cold dATP. Optimal PCR-labeling intensity was reached at dATP concentrations between 0.9 and 7.0 micromol/L, with a peak at 1.8 micromol/L. This concentration corresponded to an optimal ratio between the increase in specific activity and the decrease in DNA yield. Nucleotide imbalances >1:2 were not advantageous. Mutational analysis by single-strand conformational polymorphism (SSCP) was used to validate PCR-labeling protocols. The limiting nucleotide concentrations did not affect SSCP. Clear SSCP patterns were obtained using DNA templates of different sizes derived from several genes. SSCP patterns obtained using one-step or nested PCR-labeling protocols were equivalent and were visualized after overnight exposure, using [alpha35S]dATP as the label. Dilutions of labeled products ranging between 1:10 and 1:2.5 influenced SSCP patterns, and the lowest dilution tested produced better-defined and more-intense signals. Optimized SSCP conditions allowed the detection of novel and previously characterized nucleotide variants. Clear microsatellite typing was also obtained using optimized protocols and [alpha35S]dATP as the label.

Human herpesvirus type 6 variants identified by single-strand conformation polymorphism analysis

Six human herpesvirus 6 (HHV-6) variants were analyzed for heterogeneity using the polymerase chain reaction (PCR) and single-strand conformation polymorphism (SSCP). Two independent DNA regions were selected: a fragment of the gene U11 (position 18966–21578) coding for a basic phosphoprotein, the major antigenic structural protein pp100; and a fragment from an open reading frame (ORF) area of the gene U67, previously referred to as 13R (position 102458–103519), coding for a product of unknown function. The two PCR systems based on the above DNA sequences yielded products of 187 bp and 223 bp, respectively. DNA obtained from three laboratory reference strains (U1102, R104 and St.W.) and from HHV-6 infected peripheral white blood cells of bone marrow transplant patients and blood donors was used to test the applicability of two different SSCP analysis systems for the identification of HHV-6 variants using amplicons derived by PCR from the two genomic regions described above (U11 [pp100], U67 [13R-ORF]). The generation of characteristic SSCP patterns enables the rapid differentiation of HHV-6 A and B strains for the classification of variants derived from clinical samples, reducing the need for expensive and time-consuming direct sequencing analyses.

Identification of the Oka strain of the live attenuated varicella vaccine from other clinical isolates by molecular epidemiologic analysis.

A method was developed to distinguish the Oka vaccine strain of varicella-zoster virus (VZV) from other clinical isolates. The molecular characteristics of 52 clinical isolates from varicella or zoster patients with no history of VZV vaccination and the Oka strain, including vaccine and parental viruses, were analyzed by PstI cleavage of the PstI site-less (PSL) region. This was followed by single-strand conformational polymorphism (SSCP) after polymerase chain reaction amplification of repeating region 2 (R2). Most of the clinical isolates tested, especially recent isolates, had a PstI site in the PSL region, but the Oka strain did not. The SSCP patterns of R2 in Oka strain virus differed from those of other viruses. These results suggest that analysis of the PstI site followed by SSCP of R2 will be useful for identifying the Oka vaccine virus in isolates.

Single-strand conformation polymorphism (SSCP)-based mutation scanning approaches to fingerprint sequence variation in ribosomal DNA of ascaridoid nematodes.

In this study, we assessed single-strand conformation polymorphism (SSCP)-based approaches for their capacity to fingerprint sequence variation in ribosomal DNA (rDNA) of ascaridoid nematodes of veterinary and/or human health significance. The second internal transcribed spacer region (ITS-2) of rDNA was utilised as the target region because it is known to provide species-specific markers for this group of parasites. ITS-2 was amplified by PCR from genomic DNA derived from individual parasites and subjected to analysis. Direct SSCP analysis of amplicons from seven taxa (Toxocara vitulorum, Toxocara cati, Toxocara canis, Toxascaris leonina, Baylisascaris procyonis, Ascaris suum and Parascaris equorum) showed that the single-strand (ss) ITS-2 patterns produced allowed their unequivocal identification to species. While no variation in SSCP patterns was detected in the ITS-2 within four species for which multiple samples were available, the method allowed the direct display of four distinct sequence types of ITS-2 among individual worms of T. cati. Comparison of SSCP/sequencing with the methods of dideoxy fingerprinting (ddF) and restriction endonuclease fingerprinting (REF) revealed that also ddF allowed the definition of the four sequence types, whereas REF displayed three of four. The findings indicate the usefulness of the SSCP-based approaches for the identification of ascaridoid nematodes to species, the direct display of sequence variation in rDNA and the detection of population variation. The ability to fingerprint microheterogeneity in ITS-2 rDNA using such approaches also has implications for studying fundamental aspects relating to mutational change in rDNA.