Cathepsin D: screening for new polymorphisms using single-strand conformation polymorphism analysis.

Cathepsin D (CTSD) is a lysosomal protease involved in the pathogenesis of several diseases such as breast cancer and possibly Alzheimer's disease (AD). Previous findings revealed a significant association between the T allele of the 224 C/T (A58V) polymorphism in exon 2 of the CTSD gene and late onset AD. The exonic regions of the CTSD gene were screened for further polymorphic variations using polymerase chain reaction and single-strand conformation polymorphism analysis. In addition to the known 224 C/T polymorphism and two silent mutations in exons 3 and 4 we detected two new polymorphisms in introns 5 and 8. Combination of these sequence variations results in three different haplotypes; one of these haplotypes is due to the new polymorphism in intron 5. We detected no further missense mutations except for the known 224 C/T polymorphism in exon 2. Thus, if sequence variations within the CTSD gene influence the risk for various diseases, the pathogenic mechanism is likely to be linked to the amino acid substitution in the profragment of CTSD.     

Genotyping of human cytomegalovirus using non-radioactive single-strand conformation polymorphism (SSCP) analysis

Genetic variation in the glycoprotein B (gB) gene may play a role in human cytomegaloviruses (HCMVs) pathogenesis. Using restriction analysis of the gB gene product (PCR-RFLP), amplified by the nested polymerase chain reaction, the HCMV strains can be compared and classified into at least four HCMV groups. PCR single-strand conformation polymorphism (PCR-SSCP) is one of the techniques used to identify a mutant sequence or a polymorphism in a known gene. SSCP analysis has the advantage over RFLP analysis on detection of DNA polymorphisms and point mutations at a variety of positions in DNA fragments. However, the original SSCP protocols using the incorporation of radioactive label and polyacrylamide gel electrophoresis for detection are labour intensive and time-consuming. A simplified SSCP protocol is described to identify HCMV strains and the gB genotype, allowing the detection of sequence variations not residing in the endonuclease recognition sites.     

A rapid bioassay screen for quantifying nucleopolyhedroviruses (Baculoviridae) in the environment.

A quantitative bioassay method for the detection of Helicoverpa armigera (Lepidoptera: Noctuidae) singly encapsulated nucleopolyhedroviruses (HaSNPVs) in soil is described. Calibration curves used for estimating soil virus titres in environmental samples were generated by incorporating a sterilised soil into a semi-synthetic insect diet then inoculating known concentrations of an HaSNPV into the soil-diet mixture. Calibration curves were constructed for soil diets containing varying proportions of soil: 0, 1, 5, 10 and 25% soil (w/v). Their accuracy was assessed in a series of blind tests in which the actual soil virus concentration fell within the estimated mean 95% confidence region for each of three samples. The five soil-diet incorporation rates were compared in terms of larval survivorship and growth rate. There was no significant difference in larval survivorship after 10 days (i.e. for the duration of the bioassay period). The stage structure of bioassay larvae at 10 days and pupal weight at 20 days was significantly different for individuals reared on 25% soil-diet in terms of both a slower growth rate and a lower mean pupal weight compared to individuals reared on 10, 5 and 1% soil diets. This did not, however, appear to lead to greater variability in bioassay response at the high soil rate at 10 days. The level of sensitivity of virus detection achieved using this method was extremely good with the LC10 value for mid-first instar H. armigera larvae reared on the 25% soil-diet estimated at 26 polyhedral inclusion bodies (PIBs) per gram of soil. The suitability of using this approach for quantifying Helicoverpa NPVs in Australian soils was assessed by comparing percent bioassay infection across a range of five isolates known to be present in Australia. The effect of soil pH and soil management (cultivated versus non-cultivated) on percent bioassay infection was also examined. In both cases, no significant differences were observed. Finally, percent idopathic mortality, percent NPV infection and estimates of Helicoverpa SNPV concentration in a selection of samples from the Australian environment are presented.     

A novel HLA-DR13 allele (DRB11314) identified by single-strand conformation polymorphism analysis and confirmed by direct sequencing

A novel variant of the HLA-DR13 group is described. The new allele was found in a DR 10, 13 heterozygous patient of Turkish origin, two HLA genotypically identical children of the patient typed as DR11, 13, and one child typed as DR13, 13. DR13 subtyping of the patient was initially performed by SSCP analysis of PCR-amplified DNA, using the 11th IHWS primers DRBAMP-3 and DRBAMP-B. Due to an unusual SSCP banding pattern, the PCR product was subjected to solid-phase sequencing. The sequence of the new allele, DRB1^*1314, is different from that of DRB 1^*1307 by a single nucleotide substitution in codon 47, with T replacing consensus A. This results in a single amino acid change of tryptophan to phenylalanine in the first domain of the DRβ chain.     

A new intragenic polymorphism detected by the single-strand conformation polymorphism (SSCP) assay in the dystrophin gene.

We have employed the single strand conformation polymorphism (SSCP) technique to examine a group of patients with Duchenne or Becker muscular dystrophy who do not contain deletions detectable by multiplex PCR or Southern/cDNA, in an attempt to identify uncommon mutations within the dystrophin gene. In SSCP analysis, a mutated sequence can be detected as a change of mobility in a nondenaturing polyacrylamide gel. During the course of this investigation, we detected and characterized a new polymorphism at the 3' end of intron 16. The G-to-T base change creates a TaqI restriction site which allows for rapid typing of the polymorphism by restriction digestion and electrophoresis of PCR amplified products. Its localization inside the 5' region of the dystrophin gene and its high heterozygosity makes it a useful and easy tool for rapid carrier and prenatal diagnosis.     

A fluorescence-based polymerase chain reaction-linked single-strand conformation polymorphism (F-PCR-SSCP) assay for the identification of Fasciola spp

The present study aimed to establish a fluorescence-based polymerase chain reaction-linked single-strand conformation polymorphism (F-PCR-SSCP) assay for the identification of Fasciola spp. Based on the sequences of the second internal transcribed spacer (ITS-2) of the nuclear ribosomal DNA, we designed a set of genus-specific primers for the amplification of Fasciola ITS-2, with an estimated size of 140 bp. These primers were labelled by fluorescence dyes, and the PCR products were analyzed by capillary electrophoresis under non-denaturing conditions (F-PCR-SSCP). Capillary electrophoresis analysis of the fluorescence-labelled DNA fragments displayed three different peak profiles that allowed the accurate identification of Fasciola species: one single peak specific for either Fasciola hepatica or Fasciola gigantica and a doublet peak corresponding to the “intermediate” Fasciola. Validation of our novel method was performed using Fasciola specimens from different host animals from China, Spain, Nigeria, and Egypt. This F-PCR-SSCP assay provides a rapid, simple, and robust tool for the identification and differentiation between Fasciola spp.     

Optimization of the single-strand conformation polymorphism (SSCP) technique for detection of point mutations

The efficiency of detection of single base substitutions by single-stranded conformation polymorphism (SSCP) analysis was tested on 86 randomly distributed point mutations in a 193-bp-long DNA fragment of the mouse β-globin gene. Multiple parameters were varied, including electrophoresis temperature, buffer concentration, gel concentration, acrylamide-to-bis-acrylamide ratio, and/or addition of different compounds to the gel matrix. Gels with a higher concentration of acrylamide and lower crosslinking gave optimal separation, and all 86 mutations can be clearly distinguished from the wild type on a 5% or 7.5% (2.6% C) acrylamide gel at 4°C. Most of the mutations are also resolvable from wild type on gels with 5% urea or formamide, or 10% dimethylsulfoxide or sucrose. The relative position of the purine and pyrimidine-rich single strands were followed by an asymmetric PCR–SSCP technique. We found that most of the informativity comes from the purine rich strand, which appears to be much more sensitive to changes in the gel. The position or type of mutation showed no correlation with its ability to be detected. However, the neighboring base sequence around the mutation appears to have an effect on mobility. For example, A→G substitutions in GC-rich regions significantly increase the mobility shift of the purine-rich strand, while most G→A changes decrease it. We conclude that SSCP is a very efficient method for the detection of point mutations, if the parameters that effect the separation are optimized for a particular DNA fragment. © 1993 Wiley-Liss, Inc.     

Improving the sensitivity of single-strand conformation polymorphism (SSCP) to study the variability of PLMVd

Single-strand conformation polymorphism (SSCP) was used to characterize viroids. Eight cDNA clones, which showed identical profiles in preliminary existing SSCP analysis but had different sequences, were chosen to develop a sensitive SSCP technique for identifying the variability of Peach latent mosaic viroid (PLMVd). Polyacrylamide Gel Electrophoresis (PAGE) conditions were optimized to improve the sensitivity of the existing SSCP, and a modified SSCP protocol was developed. The results indicated that the modified SSCP protocol provided an overall sensitivity in identifying the variability of these clones, and showed higher resolution than the existing one and its improved versions. As shown by sequence analyses of cDNA clones of PLMVd and the modified SSCP profiles, there is no close correlation between the number of base changes and variation of the modified SSCP band patterns. The potential use of the modified SSCP analysis is discussed as a tool for viroids characterization.     

Direct genomic multiplex PCR for BRCA1 and application to mutation detection by single-strand conformation and heteroduplex analysis

Most mutation detection methods are based on analysis of PCR amplified segments and the application of multiplex PCR is one central approach to improving screening efficiency. Genes like the breast-ovarian cancer susceptibility gene BRCA1 pose a difficult challenge to efficient mutation screening because of large coding regions, numerous exons, and complex mutational spectra. The application to BRCA1 of a general approach to effective multiplex PCR is described here. Fifteen triplex PCRs and a single PCR reaction condition were used for amplification of all BRCA1 coding regions and the BRCA1-specific segments from the duplicated promoter region. SSCP/HDX gel analysis of the multiplex products detected mobility distinctions for 34/34 sets of allelic BRCA1 fragments. A novel polymorphism was found, CTTCT(4)CT(10)CT(12) >CT(4)CT(11), a compound deletion in a region beginning at the +33 position of IVS7 and resulting in a net deletion of 15 bp. This change was shown to be one of the common polymorphisms that define the two major haplotypes of the BRCA1-RNU2 region in a large proportion of the world population. A triplex PCR for SSCP detection of this deletion and two other distantly located common polymorphisms may be used to screen haplotype content and facilitate comparison of samples with similar haplotypes in subsequent mutation screening. The approach for robust multiplex amplification is generally applicable and allows rapid development of efficient testing for a wide variety of mutations in any gene(s) encompassing a large coding region or numerous exons and including as many as 50 different genomic PCR products.     

MS-qFRET: A quantum dot-based method for analysis of DNA methylation

DNA methylation contributes to carcinogenesis by silencing key tumor suppressor genes. Here we report an ultrasensitive and reliable nanotechnology assay, MS-qFRET, for detection and quantification of DNA methylation. Bisulfite-modified DNA is subjected to PCR amplification with primers that would differentiate between methylated and unmethylated DNA. Quantum dots are then used to capture PCR amplicons and determine the methylation status via fluorescence resonance energy transfer (FRET). Key features of MS-qFRET include its low intrinsic background noise, high resolution, and high sensitivity. This approach detects as little as 15 pg of methylated DNA in the presence of a 10,000-fold excess of unmethylated alleles, enables reduced use of PCR (as low as eight cycles), and allows for multiplexed analyses. The high sensitivity of MS-qFRET enables one-step detection of methylation at PYCARD, CDKN2B, and CDKN2A genes in patient sputum samples that contain low concentrations of methylated DNA, which normally would require a nested PCR approach. The direct application of MS-qFRET on clinical samples offers great promise for its translational use in early cancer diagnosis, prognostic assessment of tumor behavior, as well as monitoring response to therapeutic agents.Aberrant DNA hypermethylation is observed at classic tumor-suppressor genes, which are known to be genetically mutated and cause inherited forms of cancer (Jones and Baylin 2002). Tumor cells display a larger number of genes inactivated by promoter hypermethylation than by genetic mutations (Schuebel et al. 2007). Furthermore, these abnormal epigenetic changes appear to be an early event that precedes detection of genetic mutations (Esteller et al. 1999; Feinberg and Tycko 2004; Yamada et al. 2005). Thus, detection of promoter hypermethylation is a valuable tool for early diagnosis of cancer, monitoring tumor behavior, as well as measuring response of tumors to targeted therapy (Esteller et al. 2000; Gore et al. 2006b; Brock et al. 2008).The number of tools available to assess DNA methylation demonstrates the extensive interest that has been invested in understanding the role of epigenetics in carcinogenesis (Laird 2003). One of the more common techniques used for the detection of methylation is methylation-specific PCR (MSP) (Herman et al. 1996). The technique relies on sodium bisulfite treatment of DNA, which converts unmethylated cytosines to uracils while leaving methylated cytosines unaffected. The modified sequences are then amplified with specific primers, and the amplified products are identified using gel electrophoresis. However, this standard MSP approach offers only qualitative analysis and cannot discern relative amounts of methylation. Although real-time PCR-based MSP methods (Lo et al. 1999; Eads et al. 2000) enable quantitative analysis, they may lack the sensitivity for direct screening of challenging samples, such as sputum, where the DNA from tumor cells is minimal, thereby requiring a nested PCR approach (Brandes et al. 2005; Belinsky et al. 2006; Machida et al. 2006; Kim et al. 2007).Methylation-specific quantum dot fluorescence resonance energy transfer (MS-qFRET) combines the high specificity of MSP and the high sensitivity and simplicity of the quantum dot FRET (QD-FRET) technology (Zhang et al. 2005). MS-qFRET facilitates a straightforward approach for both a qualitative and quantitative detection of methylated DNA, as well as allowing detection of low-abundance methylated DNA. The sensitivity of the MS-qFRET is first examined here, followed by a demonstration of its ability to quantify methylation, both in cell lines, as well as in myelodysplastic syndrome (MDS) patient samples. The advantages of MS-qFRET are also highlighted by its capability of multiplexing reactions and its potential application for high-throughput screening. Finally, the sensitivity of this technique is validated in patient sputum samples that contain very low concentrations of DNA.     

Optimization of a simple and rapid single-strand conformation analysis for detection of mutations in the PROS1 gene: identification of seven novel mutations and three novel, apparently neutral, variants

Anticoagulant protein S (PS) deficiency is a known risk factor for thrombophilia. The structure and high allelic heterogeneity of the PS gene (PROS1), together with the presence of a 97% homologous pseudogene, complicates PROS1 analysis. We have optimized a simple, fast, and non-isotopic Single-Strand Conformation Analysis (SSCA or SSCP) method for PROS1 mutation detection. This is accomplished through the analysis of the single-stranded and heteroduplex DNA fragments corresponding to 15 PCR segments that include part of the 5'-upstream region and the 15 PROS1 exons with their intron boundaries. To standardize the method, 13 known PROS1 mutations or allele variants in 10 different fragments were analyzed under different electrophoretic conditions. The results indicated that, using a combination of two different electrophoretic settings, all the allele variants could be detected as a single-strand band shift and/or by the presence of a heteroduplex. This method was used to analyze the PROS1 gene in 31 propositi with different types of PS deficiency and thrombosis. Ten different cosegregating mutations, seven of which are novel (143C->G, L-27H, G96X, M599T, P626L, 1418delA, and 1877delT), were identified in the five families suffering from type I or quantitative PS deficiency and in four of the nine families with coexistence of type I and type III phenotypes. No clearly co-segregating PROS1 mutations were identified in any of the 17 type III propositi analyzed, although eight of them were heterozygotes for the uncommon P460 allele of the S/P460 variant. Furthermore, five apparently neutral allelic variants, three of which are novel (-296C->T, 182G->C and T57S), were identified in a normal control, two type I/III and two type III PS-deficient pedigrees.     

Sensitivity of single-strand conformation polymorphism and heteroduplex method for mutation detection in the cystic fibrosis gene

The gene responsible for cystic fibrosis (CF) contains 27 codingexons and more than 300 Independent mutations have been identified.An efficient and optimized strategy is required to identifyadditional mutations and/or to screen patient samples for thepresence of known mutations. We have tested several differentconditions for performing single-stranded conformation polymorphism(SSCP) analysis in order to determine the efficiency of themethod and to Identify the optimum conditions for mutation detection.Each exon and corresponding exon boundaries were amplified.A panel of 134 known CF mutations were used to test the efficiencyof detection of mutations. The SSCP conditions were varied byaltering the percentage and cross-linking of the acrylamide,employing MDE (an acrylamide substitute), and by adding sucroseand glycerol. The presence of heteroduplexes could be detectedon most gels and in some cases contributed to the ability todistinguish certain mutations. Each analysis condition detected75–98% of the mutations, and all of the mutations couldbe detected by at least one condition. Therefore, an optimized(SSCP) analysis can be used to efficintly screen for mutationsIn a large gene.     

Polymerase chain reaction--single-strand conformation polymorphism analysis of polymorphism in DPA1 and DPB1 genes: a simple, economical, and rapid method for histocompatibility testing.

A new technical trial was carried out to detect polymorphism in HLA-DP genes, based on the diversity in electrophoretic mobility of single-stranded DNA (single-strand conformation polymorphism, SSCP). Genomic DNAs from 31 cell lines homozygous for 2 and 14 different DPA1 and DPB1 alleles, respectively, and from peripheral blood cells of a normal individual homozygous for another DPB1 allele were subjected to polymerase chain reaction (PCR) to amplify the polymorphic exon 2 of DPA1 or DPB1 genes. The PCR samples were denatured by heating in the presence of formamide to obtain single-stranded DNA, electrophoresed in a neutral polyacrylamide gel, and visualized by silver staining. Allelic differences were detected by the distinctive electrophoretic pattern of each single strand, depending on the sequence-specific conformation. Fifteen DPB1 alleles showed 11 distinct electrophoretic patterns, leaving four allelic combinations not distinguished. These four allelic combinations could be further distinguished by using another couple of primers in PCR, with which a part of the exon was amplified, and by subsequent SSCP analysis. The use of four pairs of primers in PCR allowed for discrimination of all the 15 DPB1 alleles tested. Two allelic differences in exon 2 of DPA1 gene could be clearly demonstrated. In addition, putative new alleles of DPA1 and DPB1 genes were detected by SSCP analyses. The PCR-SSCP analysis is simple and rapid, requires neither radioactive materials nor restriction enzymes, and is expected to be a useful tool for investigating the fine HLA-matching required for clinical transplantation of organs.     

A rapid and simple method for sex identification by heteroduplex analysis, using denaturing high-performance liquid chromatography (DHPLC)

A novel method for sex identification, using a denaturing high-performance liquid chromatography (DHPLC) system, is described. Among many methods for identifying sex, the most popular and credible system has been the polymerase chain reaction (PCR) method, using nucleotide primer sets of the amelogenin gene, which is shared on both the X and Y chromosomes. With this conventional method, the judgment depends on detection of the size difference between the PCR products derived from the X and Y chromosomes. In this study, we adopted DHPLC to detect the difference by checking heterodu-plex formation between the products, which enabled us to shorten the PCR products to 45 bp and the separation time to within a period of 8 min per sample. This new system may have wide applications in many different fields, such as forensic medicine, prenatal diagnosis, inbreeding of animals, and anthropology. Received: October 31, 2000 / Accepted: February 5, 2001     

限制酶切指纹-单链构象多态性--一种有效的大片段中突变的检测方法

目的；建立一种简便、价廉、有效地在大片段中检测突变的限制酶切指纹-单链构象多态性（restriction endonuclease fingerprinting-single strand conformation polymorphis，REF-SSCP）方法。方法：以耳聋患者基因组DNA为模板，扩增Cx26基因片段，通过对扩增产物进行单链构象多态性、限制酶切指纹-单链构象多态性和DNA测序，比较REF-SSCP技术对大片段中突变的检出效率。结果：长度为724bp的扩增产物SSCP结果未显示差异，REF-SSCP显示3种不同带型，经测序发现该基因中的79G→A突变，突变情况与REF-SSCP带型完全吻合，检出率为100%。结论：所建立的REF-SSCP技术适用于大样本量地检测大片段DNA中的突变。     

Herpes simplex virus genotyping: multiple optional PCR-based RFLP systems and a non-isotopic single-strand conformation polymorphism method

Polymerase chain reaction (PCR)-based methods for herpes simplex virus (HSV) types I/II genotyping are described. These methods are based on the single-stranded conformation properties of DNA molecules obtained by PCR (PCR-SSCP) and restriction fragment analysis of PCR products (PCR-RFLP). With the aim to analyse these two genotyping techniques, genomic DNA from the standard viral strains KOS and G, and also 79 normal cervical samples were studied for HSV I/II. Sequence analysis showed 19 different RFLP possible systems for HSV typing. Five systems were used in this study and all led to the expected fragments obtained by sequence analysis. PCR-SSCP showed distinct patterns for both viral types and had 100% of concordance with PCR-RFLP results. The prevalence of herpes simplex virus in the normal cervical samples were 21.5% (17 out of 79), the most frequent viral type was HSV-2, with a prevalence of 71% (12 out of 17). Both techniques appeared suitable for HSV I/II genotyping and are easy to perform in most clinical laboratories.     

Correlation of MFOLD-predicted DNA secondary structures with separation patterns obtained by capillary electrophoresis single-strand conformation polymorphism (CE-SSCP) analysis

We have studied 57 different mutations within three beta-globin gene promoter fragments with sizes 52 bp, 77 bp, and 193 bp by fluorescent capillary electrophoresis CE-SSCP analysis. For each mutation and wild type, energetically most-favorable predicted secondary structures were calculated for sense and antisense strands using the MFOLD DNA-folding algorithm in order to investigate if any correlation exists between predicted DNA structures and actual CE migration time shifts. The overall CE-SSCP detection rate was 100% for all mutations in three studied DNA fragments. For shorter 52 bp and 77 bp DNA fragments we obtained a positive correlation between the migration time shifts and difference in free energy values of predicted secondary structures at all temperatures. For longer 193 bp beta-globin gene fragments with 46 mutations MFOLD predicted different secondary structures for 89% of mutated strands at 25 degrees C and 40 degrees C. However, the magnitude of the mobility shifts did not necessarily correlate with their secondary structures and free energy values except for the sense strand at 40 degrees C where this correlation was statistically significant (r = 0.312, p = 0.033). Results of this study provided more direct insight into the mechanism of CE-SSCP and showed that MFOLD prediction could be helpful in making decisions about the running temperatures and in prediction of CE-SSCP data patterns, especially for shorter (50-100 bp) DNA fragments.