Software for machine-independent quantitative interpretation of SSCP in capillary array electrophoresis (QUISCA)

PCR single-stranded conformational polymorphism (SSCP) analysis is a simple and rapid electrophoretic technique for the sensitive detection of sequence variants of PCR products. Here we describe a cross-platform program package, quantitative interpretation of SSCP in capillary array (QUISCA), which allows semi-automated quantitative detection of sequence variants separated by multicolor fluorescence-based SSCP electrophoresis using various capillary array apparatus. The program, together with the QUISCAview as a graphical user interface, takes trace data in ASCII format and processes them with three modules: signal denoising/baseline subtraction, color-matrix construction/application, and calibration of peak positions between multiple capillary runs using internal standard peaks. QUISCA is compatible with data from various widely used capillary array sequencers and is suitable not only for finding or typing SNPs in individual DNAs but also for the accurate estimation of the allele frequencies of many SNPs using a pooled DNA strategy. QUISCA can also serve as a versatile core program for various fragment analyses.     

Single-strand conformational polymorphism markers associated with a major QTL for fusarium head blight resistance in wheat

A major quantitative trait locus (QTL) associated with resistance to Fusarium head blight (FHB) was identified on chromosome 3BS between simple sequence repeat (SSR) markers Xgwm389 and Xgwm493 in wheat 'Ning 7840', a derivative from 'Sumai 3'. However, the marker density of SSR in the QTL region was much lower than that required for marker-assisted selection (MAS) and map-based cloning. The objective of this study was to exploit new markers to increase marker density in this QTL region by using single-strand conformational polymorphism (SSCP) markers developed from wheat expressed sequence tags (ESTs) on 3BS bin 8-0.78-1.0. Sixty-nine out of 85 SSCP primer pairs amplified PCR (polymerase chain reaction) products from the genomic DNA of 'Chinese Spring'. Thirty-four primer pairs amplified PCR products that could form clear ssDNA (single strand DNA) bands through denaturation treatment. Ten SSCP markers had polymorphisms between 'Ning 7840' and 'Clark'. Five of the ten polymorphic SSCP markers were located on chromosome 3B by nulli-tetrasomic analysis. Three SSCP markers (Xsscp6, Xsscp20, and Xsscp21) were mapped into the region between Xgwm493 and Xgwm533, and possessed higher coefficient of determination (R2) than Xgwm493 and Xgwm533. The SSCP markers, Xsscp6, Xsscp20, and Xsscp21, can be used for map-based cloning of the QTL and for marker-assisted selection in FHB resistance breeding.     

Minisatellite isoalleles can be distinguished by single-stranded conformational polymorphism analysis in agarose gels.

Minisatellite isoallelism, i.e. the occurrence of minisatellite alleles with different internal sequence composition but indistinguishable length, is a common limitation of minisatellite allele length analysis. Internal sequence variation can be used to distinguish such isoalleles, provided that detailed sequence knowledge of its basis is available. We now show that minisatellite isoalleles can also be simply resolved by single-stranded conformational polymorphisms (SSCP) arising during agarose gel electrophoresis. SSCP on agarose gels can be used to distinguish minisatellite isoalleles either after PCR amplification, or by standard Southern blot analysis of genomic DNA.     

Four new nucleotide sequence polymorphisms in the LDL receptor gene detected by SSCP analysis

Seven nucleotide sequence polymorphisms were detected within exons of the low-density lipoprotein (LDL) receptor gene using single-strand conformation polymorphism (SSCP) analysis followed by direct sequence analysis on amplified DNA. Four nucleotide changes at nucleotide positions 1617, 1725, 2232, and 2635 were new nucleotide sequence polymorphisms not previously described. The remaining three nucleotide changes were identical with restriction fragment length polymorphisms and a previously reported nucleotide sequence polymorphism. These nucleotide sequence polymorphisms, detectable by SSCP analysis, are useful genetic markers for linkage analysis of the LDL receptor gene and familial hypercholesterolemia.     

The number of genes having different alleles between rice cultivars estimated by SNP analysis

Identification of single nucleotide polymorphisms (SNPs) in a large number of genes will enable estimation of the number of genes having different alleles in a population. In the present study, SNPs between 21 rice cultivars including 17 Japanese cultivars, one upland rice, and three indica cultivars were analyzed by PCR–RF–SSCP. PCR–RF–SSCP analysis was found to be a more efficient method for detecting SNPs than mismatch–cleavage analysis, though both PCR–RF–SSCP and mismatch–cleavage are useful for screening SNPs. The number of DNA fragments showing polymorphism between Japanese cultivars was 134 in the 1,036 genes analyzed. In 137 genes, 638 DNA polymorphisms were identified. Out of 52 genes having polymorphisms in the exons, one had a frame-shift mutation, three had polymorphism causing amino acid insertions or deletions, and 16 genes had missense polymorphisms. The number of genes having frame-shift mutations and missense polymorphisms between the 17 Japanese cultivars was estimated to be 41 and 677 on average, respectively, and those between japonica and indica to be 425 and 6,977, respectively. Chromosomal regions of cultivars selected in rice breeding processes were identified by SNP analysis of genes. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The single strand conformational analysis of cattle and human single nucleotide polymorphisms may be biased towards specific sequence motifs that minimize local secondary structure of single strand DNA

Single strand conformational polymorphisms (SSCP) resulting from point mutations were found to be associated preferentially with two DNA sequence motifs. These motifs are (1) three or more of the same base but in which the polymorphism is not due to length variation and (2) a region of polypurine or polypyrimidine bases. These motifs were identified after SSCP alleles from cattle were sequenced. The sequence difference and flanking sequence for each single nucleotide polymorphism are shown. The motifs were also found in SSCP from humans chosen at random from the literature, in which the alleles had been sequenced. There is a low level of complementarity of adjacent bases in these motifs and they should represent regions of low secondary structure in the single stranded DNA. Regions of high secondary structure, such as palindromes, were found in the same sample to have allelic variation that was not detected by SSC analysis. These results give a rule of thumb for selecting the particular part of a DNA fragment to be selected for testing for polymorphisms, but this rule clashes with rules used to design primers to amplify sequences using the PCR, namely, minimise hydrogen bonding within and between primers and reduce self-complementarity.     

Efficient mutation detection in mismatch repair genes using a combination of single-strand conformational polymorphism and heteroduplex analysis at a controlled temperature

Single-strand conformational polymorphism analysis (SSCP) and heteroduplex analysis (HD) were tested as methods for mutation screening with respect to experimental variation, sensitivity, and specificity. Thirty-nine fluorescently labeled PCR products covering the two mismatch repair genes, hMLH1 and hMSH2, were tested in 15 patients for pattern changes, using SSCP and HD at two temperatures, in a total of 2340 runs. SSCP was most efficient in detecting base changes, whereas HD was the method of choice when detecting deletions. SSCP and HD at 20 degrees C were most effective (sensitivity 97%, specificity 49%), and SSCP and HD at 10 degrees C gave no additional information, except in one case where an exon had two base changes. Several mutations only showed a small pattern change in one of the two strands, most explicit at 20 degrees C. No correlation between the type of base change and the size or direction of the pattern changes could be found.     

Label-free protein quantification using LC-coupled ion trap or FT mass spectrometry: Reproducibility, linearity, and application with complex proteomes

A critical step in protein biomarker discovery is the ability to contrast proteomes, a process referred generally as quantitative proteomics. While stable-isotope labeling (e.g., ICAT, 18O- or 15N-labeling, or AQUA) remains the core technology used in mass spectrometry-based proteomic quantification, increasing efforts have been directed to the label-free approach that relies on direct comparison of peptide peak areas between LC-MS runs. This latter approach is attractive to investigators for its simplicity as well as cost effectiveness. In the present study, the reproducibility and linearity of using a label-free approach to highly complex proteomes were evaluated. Various amounts of proteins from different proteomes were subjected to repeated LC-MS analyses using an ion trap or Fourier transform mass spectrometer. Highly reproducible data were obtained between replicated runs, as evidenced by nearly ideal Pearson's correlation coefficients (for ion's peak areas or retention time) and average peak area ratios. In general, more than 50% and nearly 90% of the peptide ion ratios deviated less than 10% and 20%, respectively, from the average in duplicate runs. In addition, the multiplicity ratios of the amounts of proteins used correlated nicely with the observed averaged ratios of peak areas calculated from detected peptides. Furthermore, the removal of abundant proteins from the samples led to an improvement in reproducibility and linearity. A computer program has been written to automate the processing of data sets from experiments with groups of multiple samples for statistical analysis. Algorithms for outlier-resistant mean estimation and for adjusting statistical significance threshold in multiplicity of testing were incorporated to minimize the rate of false positives. The program was applied to quantify changes in proteomes of parental and p53-deficient HCT-116 human cells and found to yield reproducible results. Overall, this study demonstrates an alternative approach that allows global quantification of differentially expressed proteins in complex proteomes. The utility of this method to biomarker discovery is likely to synergize with future improvements in the detecting sensitivity of mass spectrometers.     

Autosomal dominant retinal dystrophy (Rdy) in Abyssinian cats: exclusion of PDE6G and ROM1 and likely exclusion of Rhodopsin as candidate genes

Retinal dystrophy (Rdy) is an autosomal dominant photoreceptor dysplasia of Abyssinian cats and a model for autosomal dominant retinitis pigmentosa (ADRP) in man. We have pursued a candidate gene approach in the search for the causal mutation in Rdy. The genes RHO (encoding rhodopsin), ROM1 (encoding the structural retinal outer-membrane protein-1) and PDE6G (encoding the gamma subunit of the visual transduction protein cyclic guanosine monophosphate-phosphodiesterase) were polymerase chain reaction-amplified from normal feline genomic DNA. Leader, coding and 3' untranslated regions of each gene, and parts of introns were sequenced. Single-stranded conformation polymorphism (SSCP) analysis of Rdy-affected and normal cats was used to identify intragenic polymorphisms within ROM1 and PDE6G. DNA sequencing of all three genes in Rdy-affected cats was used to confirm results from SSCP. For both ROM1 and PDE6G polymorphisms identified by SSCP and sequencing showed disconcordance between the polymorphism and the disease phenotype within an Rdy disease pedigree. SSCP analysis of RHO performed across the 5' untranslated region, the entire coding sequence and the intron/exon boundaries in Rdy-affected and control cats failed to identify any intragenic polymorphisms that could be used for linkage analysis. DNA sequencing of these regions showed no differences between Rdy-affected and control cats. Mutations in ROM1 or in PDE6G are not causative of feline Rdy. The absence of potentially pathogenic polymorphisms in sequenced portions of the RHO gene makes it unlikely that a mutation in this gene is the cause of Rdy.     

A PCR-based diagnostic tool for distinguishing grape skin color mutants

Berry skin color mutants are phenotypically different from their original cultivars, but they show identical molecular profile if analysed by using microsatellite markers. This work gives an easy, inexpensive and quick diagnostic tool to discriminate these somatic variants. We distinguished some grape (Vitis vinifera L.) skin color mutants from white to red or pink and from black to grey, pink or white and we investigated their molecular bases by single-strand conformational polymorphism (SSCP), single base primer extension and coding sequence analysis of anthocyanin biosynthetic enzyme genes and by polymerase chain reaction (PCR) analysis of VvmybA1 regulatory gene. Analyses of structural genes did not reveal polymorphisms between wild type and mutant cultivars but only among different varieties, whereas the study of VvmybA1 regulatory gene has given important outcomes for color mutants characterisation. The discrimination between white wild type and its derived colored mutant and between black wild type and white mutant has been obtained through a simple test of amplification for presence/absence. The discrimination between black wild type and less colored mutant has occurred through a quantitative result on agarose gel confirmed by real-time PCR analysis: the amount of functional allele in less colored somatic variants genome was about one-fourth of the correspondent quantity in original black cultivars genome.     

A Study of Polymorphisms in Three Loci Known to Influence Defensive Behavior Using PCR-SSCP and Direct Sequencing in the Iranian Honey Bee Population

Polymerase Chain Reaction (PCR) and subsequent separation of PCR products on polyacrylamide gels to find Single Stranded Conformation Polymorphisms (SSCP) was used for three loci, known from studies in North America to affect defensive behavior (Quantitative Trait Loci (QTL) sting1–3), on samples of Iranian honey bee (Apis mellifera L.). In the present study these loci were amplified with specific primers for sting1–3. The analysis of these loci using SSCP, created different patterns among samples. The polymorphisms observed in this study of the Iranian population of honey bees are a first step toward the eventual implementation of a genetic marker-based selection program to reduce defensive behavior. Now, because of this study, the markers are available to conduct more research so that the association of these polymorphic loci and aggressive traits in the Iranian honey bee population can be determined.     

Precise estimation of allele frequencies of single-nucleotide polymorphisms by a quantitative SSCP analysis of pooled DNA

We show that single-nucleotide polymorphisms (SNPs) of moderate to high heterozygosity (minor allele frequencies >10%) can be efficiently detected, and their allele frequencies accurately estimated, by pooling the DNA samples and applying a capillary-based SSCP analysis. In this method, alleles are separated into peaks, and their frequencies can be reliably and accurately quantified from their peak heights (SD <1.8%). We found that as many as 40% of publicly available SNPs that were analyzed by this method have widely differing allele frequency distributions among groups of different ethnicity (parents of Centre d'Etude Polymorphisme Humaine families vs. Japanese individuals). These results demonstrate the effectiveness of the present pooling method in the reevaluation of candidate SNPs that have been collected by examination of limited numbers of individuals. The method should also serve as a robust quantitative technique for studies in which a precise estimate of SNP allele frequencies is essential-for example, in linkage disequilibrium analysis.     

Single-strand conformational polymorphism markers associated with a major QTL for fusarium head blight resistance in wheat

A major quantitative trait locus (QTL) associated with resistance to Fusarium head blight (FHB) was identified on chromosome 3BS between simple sequence repeat (SSR) markers Xgwm389 and Xgwm493 in wheat “Ning 7840”, a derivative from “Sumai 3”. However, the marker density of SSR in the QTL region was much lower than that required for marker-assisted selection (MAS) and map-based cloning. The objective of this study was to exploit new markers to increase marker density in this QTL region by using single-strand conformational polymorphism (SSCP) markers developed from wheat-expressed sequence tags (ESTs) on 3BS bin 8-0.78-1.0. Sixty-nine out of 85 SSCP primer pairs amplified PCR (polymerase chain reaction) products from the genomic DNA of “Chinese Spring”. Thirty-four primer pairs amplified PCR products that could form clear ssDNA (single strand DNA) bands through denaturation treatment. Ten SSCP markers had polymorphisms between Ning 7840 and “Clark”. Five of the ten polymorphic SSCP markers were located on chromosome 3B by nullitetrasomic analysis. Three SSCP markers (Xsscp6, Xsscp20, and Xsscp21) were mapped into the region between Xgwm493 and Xgwm533 and possessed a higher coefficient of determination (R²) than Xgwm493 and Xgwm533. The SSCP markers, Xsscp6, Xsscp20, and Xsscp21, can be used for map-based cloning of the QTL and for marker-assisted selection in FHB resistance breeding.     

Detection of exon polymorphisms in the human lactoferrin gene.

We previously demonstrated that lactoferrin gene polymorphisms occur in cancer cells of patients with leukemia and breast cancer. In this study, we established a non-radioactive polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis, one of the most sensitive and simplest methods to detect polymorphisms and mutations of the human lactoferrin gene. We optimized the PCR conditions for nine different DNA templates and 16 pairs of exon primers for SSCP analysis. The DNA templates used in the analyses were prepared from a cosmid clone (CT6-1) that contains the human lactoferrin gene, human placental tissue, leukocytes from 10 normal volunteers, leukemic cells of two patients, and previously established three breast and two leukemic cell lines. With the appropriate exon-primer sets, PCR products from exon I to exon 16 of the lactoferrin gene were generated from the DNA templates and analyzed by SSCP. Compared with the homogenous cloned DNA, lactoferrin gene polymorphisms were detected within exons 2, 5, 7, 9, 13, 14, and 15 of the normal placental and leukocyte DNA. In addition, abnormal migration patterns of the lactoferrin gene in cancer cells were detected in exons 4, 5, 13, 14, and 15. The PCR-SSCP band migration patterns can be attributed either to gene polymorphism in normal cells or to DNA mutations in cancer cells and the employed method cannot distinguish between them. Nonetheless, the present analysis suggests that genetic polymorphisms of the lactoferrin gene exist in selected exons and additional mutations of the lactoferrin gene do occur in the cancer cells.     

Sequence alterations can mask each other's presence during screening with SSCP or heteroduplex analysis: BRCA genes as examples

For mutation detection, various screening techniques are widely used because DNA sequencing, the gold-standard method, is still considered to be expensive and laborious for high-throughput screening. Single-strand conformation polymorphism (SSCP) analysis, heteroduplex analysis (HA) and their variant techniques are popular and frequently used for this purpose. It is widely accepted that when searching for unknown sequence variations, any revealed distinct pattern should always be sequenced. We give examples here of the BRCA1 and BRCA2 genes where the SSCP/HA techniques can produce ambiguous predictions if used to detect known genetic variants compared to positive controls. Using direct DNA sequencing, we provide evidence that in such cases, mutations or polymorphisms can mask each other's presence. This phenomenon can often influence the results of any DNA testing because genetic variations such as single-nucleotide polymorphisms occur frequently in the human genome. We suggest that even in the case of known electrophoretic patterns of well-characterized genetic alterations, every sequence alteration should be confirmed by direct DNA sequencing, especially if genetic testing is carried out for diagnostic purposes.     

A simple method for non-radioactive PCR-SSCP using MDE gel solution and a midi gel format: application for the detection of variants in the GLUT1 and CTLA-4 genes

The need to identify disease-causing mutations and DNA polymorphisms has increased with the continuing identification of new candidate genes. PCR single-strand conformation polymorphism (PCR-SSCP) is one of the techniques most widely used to identify a mutant sequence or a polymorphism in a known gene. However, the original SSCP protocols using the incorporation of radioactive label and polyacrylamide gel electrophoresis on sequencing gels for detection were labour intensive and time-consuming. Here we describe a simple SSCP protocol using MDE gel solution and a midi gel format to detect SSCP variations in the glucose transporter gene GLUT1, that we have previously analysed with the standard radioactive SSCP protocol, and we have also tested this method on the previously described point mutation (A/G transition in exon 1) of the CTLA-4 (cytotoxic T lymphocyte associated-4) gene. All known variants were detected. Based on the results, this technique appears to be simple, with no use of radioactive labels and with easy handling of the gel. Furthermore, it needs little optimisation, is relatively rapid and highly sensitive. We propose this method for the first screening for candidate gene variants.     

Quantitative real-time RT-PCR using hybridization probes and imported standard curves for cytokine gene expression analysis

Quantitative real-time or kinetic RT-PCR is increasingly used for the quantification of specific mRNA targets, especially in clinical applications. To quantify the mRNA of cytokines and their receptors, which play important roles in the pathogenesis of autoimmune diseases such as multiple sclerosis, we have developed quantitative two-step RT-PCR assays for IL-4, IL-4R, IFN-gamma, IFN-beta, and the housekeeping gene porphobilinogen deaminase (PBGD). The LightCycler system was used to quantify the copy numbers with the sequence-specific hybridization probe detection format. The quantification was carried out on the basis of standard curves generated with external homologous plasmids for each different parameter in relation to the gene expression of PBGD. Therefore, this procedure represents a relative quantification method with external standards, as the standard curves were used to obtain an absolute value for the copy numbers of the targets and the reference (PBGD). The new software version 3.5 of the LightCycler system allows the construction of a single parameter-dependent plasmid standard curve for the quantification of unknown samples from different runs. Here we demonstrate how to achieve precise and reproducible quantification, even when using measurements from different PCR runs.     

Screening strategies for a highly polymorphic gene: DHPLC analysis of the Fanconi anemia group A gene

Introduction: Patients with Fanconi anemia (Fanc) are at risk of developing leukemia. Mutations of the group A gene (FancA) are most common. A multitude of polymorphisms and mutations within the 43 exons of the gene are described. To examine the role of heterozygosity as a risk factor for malignancies, a partially automatized screening method to identify aberrations was needed. We report on our experience with DHPLC (WAVE (Transgenomic)). Methods: PCR amplification of all 43 exons from one individual was performed on one microtiter plate on a gradient thermocycler. DHPLC analysis conditions were established via melting curves, prediction software, and test runs with aberrant samples. PCR products were analyzed twice: native, and after adding a WT-PCR product. Retention patterns were compared with previously identified polymorphic PCR products or mutants. Results and discussion: We have defined the mutation screening conditions for all 43 exons of FancA using DHPLC. So far, 40 different sequence variations have been detected in more than 100 individuals. The native analysis identifies heterozygous individuals, and the second run detects homozygous aberrations. Retention patterns are specific for the underlying sequence aberration, thus reducing sequencing demand and costs. DHPLC is a valuable tool for reproducible recognition of known sequence aberrations and screening for unknown mutations in the highly polymorphic FancA gene.     

Detection of polymorphisms at exons 3 (Tyr113-->His) and 4 (His139-->Arg) of the microsomal epoxide hydrolase gene using fluorescence PCR method combined with melting curves analysis

An association between exon 3 polymorphisms of the gene encoding microsomal epoxide hydrolase (mEH) and susceptibility to the development of chronic obstructive pulmonary disease (COPD) has been described. We have developed two methods for detecting polymorphisms at exons 3 (Tyr113-->His) and 4 (His139-->Arg) of the mEH gene based on different melting temperatures (T(m)) of fluorescent-labeled oligonucleotide hybridization probes using single-step assays that combine fluorescence PCR and melting curve analysis (LightCycler methodology). DNA was extracted from blood in 79 COPD patients and 146 healthy controls. Results were compared with those obtained by restriction fragment length polymorphism (RFLP) analysis to detect Tyr113His variants and a single-strand conformation polymorphism (SSCP) assay for His139Arg detection. The T(m) of the exon 3 polymorphisms were 61.3 degrees C for Tyr113 (wild type) and 67.5 degrees C for His113 (mutant). The T(m) values of the exon 4 polymorphisms were 67.5 degrees C for His139 (wild type) and 59.2 degrees C for Arg139 (mutant). The within- and between-run melting peaks for the same allele differed by less than 0.5 degrees C for both the exon 3 and the exon 4 polymorphisms. Thus, melting analysis allowed easy and unambiguous assignment of genotyping by means of the respective melting curves. The proportion of individuals who were homozygous mutant for exon 3 was significantly higher in the COPD group than in the control group (p=0.004). LightCycler fluorescence genotyping of exon 4 polymorphisms correlated perfectly with SSCP results. RFLP assay classified 2 patients as homozygous mutant while LightCycler analysis genotyped them as heterozygous. DNA analysis by PCR and sequencing confirmed the LightCycler result. These high-speed (about 40 min for 32 samples), highly sensitive, and specific small-volume assays with low labor requirements hold great promise as tools for rapid detection of COPD susceptibility.