Screening human genes for small alterations performing an enzymatic cleavage mismatched analysis (ECMA) protocol.

Many human diseases are caused by small alterations in the genes and in the majority of cases sophisticated protocols are required for their detection. In this study we estimated the efficacy of an enzymatic protocol, which using a new mismatch-specific DNA plant endonuclease from celery (CEL family) recognizes and cleaves mismatched alleles between mutant and normal PCR products. The protocol was standardized on a variety of known mutations, in 11 patients with cystic fibrosis (CF), Fabry's disease (FD), steroid 21-hydroxylase deficiency (21-HD) and Duchenne/Becker muscular dystrophy (DMD/BMD). The method does not require special equipment, labeling or standardization for every PCR product, since conditions of heteroduplex formation and enzyme digestion are universal for all products. The results showed that the method is rapid, effective, safe, reliable, and very simple, as the mutations are visualized on agarose or nusieve/agarose gels. The protocol was furthermore evaluated in three DMD patients with the detection of three alterations which after sequencing, were characterized as disease causative mutations. The proposed assay, which was applied for the first time in a variety of monogenic disorders, indicates that point mutation identification is feasible in any conventional molecular lab even for cases, where other techniques have failed.

Stress-induced DNA duplex destabilization (SIDD) in the E. coli genome: SIDD sites are closely associated with promoters.

We present the first analysis of stress-induced DNA duplex destabilization (SIDD) in a complete chromosome, the Escherichia coli K12 genome. We used a newly developed method to calculate the locations and extents of stress-induced destabilization to single-base resolution at superhelix density sigma = -0.06. We find that SIDD sites in this genome show a statistically highly significant tendency to avoid coding regions. And among intergenic regions, those that either contain documented promoters or occur between divergently transcribing coding regions, and hence may be inferred to contain promoters, are associated with strong SIDD sites in a statistically highly significant manner. Intergenic regions located between convergently transcribing genes, which are inferred not to contain promoters, are not significantly enriched for destabilized sites. Statistical analysis shows that a strongly destabilized intergenic region has an 80% chance of containing a promoter, whereas an intergenic region that does not contain a strong SIDD site has only a 24% chance. We describe how these observations may illuminate specific mechanisms of regulation, and assist in the computational identification of promoter locations in prokaryotes.

[Methods of computer modeling and conformational mobility of DNA duplexes]. / Metody komp'iuternogo modelirovaniia i konformatsionnaia podvizhnost' DNK-dupleksov.

The review is focused on issues of transferability of the context-sensitive conformational characteristics of DNA estimated from crystallographic structural data on the DNA in aqueous solution. The state of the art in molecular dynamics of charged biopolymers in aqueous solution is covered. Elaboration of expedient force fields and algorithms of calculating long-range electrostatic interactions and solving combined equations of atomic motion have made it possible to generate stable nanosecond trajectories of thermal atomic motion of the biopolymer in aqueous solution in the presence of counterions and salt ions over reasonable time. Tools for analyzing the atomic statistical trajectories of DNA duplexes in aqueous solution to infer context-sensitive conformational dynamic characteristics are discussed together with advances in simulating the mechanisms of global axial bend in DNA duplexes. These techniques allow one to consecutively analyze relationships between the contextual composition of the duplex and the basic modes of essential motions, their amplitude and extent of fluctuation. Development of satisfactory methods for estimating the free energy of biopolymer conformations in solution permits qualitative assessment of the conformational thermodynamic stability of biopolymers and their complexes.

Construction and characterization of mismatch-containing circular DNA molecules competent for assessment of nick-directed human mismatch repair in vitro.

The ability of cell-free extracts to correct DNA mismatches has been demonstrated in both prokaryotes and eukaryotes. Such an assay requires a template containing both a mismatch and a strand discrimination signal, and the multi-step construction process can be technically difficult. We have developed a three-step procedure for preparing DNA heteroduplexes containing a site-specific nick. The mismatch composition, sequence context, distance to the strand signal, and the means for assessing repair in each strand are adjustable features built into a synthetic oligonucleotide. Controlled ligation events involving three of the four DNA strands incorporate the oligonucleotide into a circular template and generate the repair-directing nick. Mismatch correction in either strand of a prototype G.T mismatch was achieved by placing a nick 10-40 bp away from the targeted base. This proximity of nick and mismatch represents a setting where repair has not been well characterized, but the presence of a nick was shown to be essential, as was the MSH2/MSH6 heterodimer, although low levels of repair occurred in extract defective in each protein. All repair events were inhibited by a peptide that interacts with proliferating cell nuclear antigen and inhibits both mismatch repair and long-patch replication.

Comparison of heteroduplex analysis, direct sequencing, and enzyme mismatch cleavage for detecting mutations in a large gene, FBN1.

Analysis of large genes for mutations of clinical relevance is complicated by intragenic heterogeneity, sensitivity, and cost of the methods available, and in the case of many conditions, specificity of the genetic alterations detected. We examined the FBN1 gene for mutations in people who had Marfan syndrome using three methods: single-chain polymorphism analysis (SSCP) with heteroduplex (HA) analysis, enzyme-mediated cleavage (EMC) of heteroduplexes, and direct sequencing. We also used these methods to search for mutations in the P53 gene in patients with hepatocellular carcinoma. The results showed that EMC was most efficient for detecting mutations. However, the cost favored SSCP with heteroduplex analysis, provided conditions did not need to be optimized to detect a mutation. Until more cost-effective and sensitive methods are developed to detect unknown mutations in large genes, diagnosis of many genetic disorders will depend on the willingness of an investigator who is studying a particular disorder to perform clinical molecular testing and have the laboratory accredited.

Denaturing high-performance liquid chromatography detects reliably BRCA1 and BRCA2 mutations.

Denaturing high-performance liquid chromatography (DHPLC) is a recently developed method of comparative sequencing based upon heteroduplex detection. To assess the reliability of this method, 180 different mutations (54 deletions, 12 insertions, and 117 single base substitutions) in BRCA1 and BRCA2 were tested. Second, 25 index individuals with complete DHPLC analysis of BRCA1 were reanalyzed by dye-terminator sequencing. Third, 41 index individuals were analyzed concomitantly by both DGGE and DHPLC. Of the 180 different BRCA1 and BRCA2 mutations, 179 showed heterozygous DHPLC elution profiles. Dye-terminator sequencing of the entire BRCA1 gene, including 5592 bp of coding sequence and 5206 bp of flanking noncoding sequence, in 25 index individuals did not reveal additional variants missed by DHPLC. The concomitant analysis of 41 index cases showed that 4 probably disease-associated mutations were identified by DHPLC while only 3 of those 4 sites were detected by denaturing gradient gel electrophoresis. We conclude that DHPLC is a sensitive and cost-effective method for the screening of BRCA1 and BRCA2.

Critical factors in the performance and cost of two-dimensional gene scanning: RB1 as a model.

Two-dimensional (2-D) gene scanning (TDGS) is a method for mutation detection based on the electrophoretic separation of PCR-amplified DNA fragments according to size and base pair sequence. The use of denaturing gradient gel electrophoresis (DGGE) as the second separation step provides virtually 100% sensitivity, while the 2-D format allows the inspection of multiple gene fragments simultaneously. Analysis of many exons in parallel is greatly facilitated by extensive PCR multiplexing based on preamplification by long-distance PCR. Recently, TDGS has been applied to detect mutations in the retinoblastoma tumor suppressor gene RB1. Using RB1 as a model, we have now analyzed each step of the protocol, presenting overall improvements and a detailed cost analysis, where the total cost of the assay is found to be about $40 (US). An overall picture of TDGS cost-performance, as compared to direct sequencing, is provided as a function of the number of target fragments.

Insights into the dynamic nature of DNA duplex structure via analysis of nuclear Overhauser effect intensities.

Sequence-dependent structures of DNA duplexes in solution can be reliably determined using NMR data if care is taken to determine restraint bounds accurately. This entails use of complete relaxation matrix methods to analyze nuclear Overhauser effect (NOE) spectroscopic cross-peak intensities, yielding accurate distance restraints. NMR studies of various DNA duplexes have suggested that there may be some limited internal motions. First, it is typically not possible to reconcile all vicinal proton coupling constants in deoxyribose rings with a single conformer. In addition, with the increased accuracy of interproton distance measurements afforded by the complete relaxation matrix algorithm MARDIGRAS, we find inconsistencies in certain distances which can most readily be ascribed to limited conformational flexibility, since conformational averaging is nonlinear. As base-sugar interproton distances depend on both sugar pucker and glycosidic torsion angle chi, motion involving these structural variables should be reflected by experimental data. Possible motional models have been considered to account for all of the data for three DNA duplexes. Analysis of intraresidue base-sugar interproton NOE bounds patterns suggests a motional model with individual sugars in equilibrium between S (2'-endo) and N (3'-endo) conformations, with S being preferred. As sugar repuckering is correlated with changes in glycosidic torsion angle chi, different sugar conformers imply different values for chi, but this is insufficient to account for all data. A two-state jump between anti and syn glycosidic conformers was considered, but it was incapable of accounting for all data. However, a model with restricted diffusion (rocking) about the glycosidic bond in addition to sugar repuckering was capable of accommodating all experimental data. This motional model is in qualitative agreement with experimental 13C relaxation-derived order parameter values in a DNA duplex.

Nuclear magnetic resonance structure of d(GCATATGATAG). d(CTATCATATGC): a consensus sequence for promoters recognized by sigma K RNA polymerase.

The three-dimensional structure of d(GCATATGATAG).d(CTATCATATGC), from the promoter region of a gene regulating sporulation in Bacillus subtilis mother cells, was determined utilizing two-dimensional nuclear Overhauser effect (2D NOE) and double-quantum-filtered COSY (2QF-COSY) spectra. To minimize the effect of methods used to obtain restraints and refine structure, several variables were studied. Interproton distance bounds were calculated very conservatively by running the complete relaxation matrix program MARDIGRAS hundreds of times using 2D NOE spectra for exchangeable and for nonexchangeable protons at different mixing times, assuming different overall correlation times and different starting structures. The 435 distance restraints were used with two different structural refinement methods: restrained molecular dynamics (rMD) and restrained Monte Carlo calculations (rMC). Refinement using different procedures and starting structures resulted in essentially the same structure (<0.8 A rmsd), indicating that the structure is defined by experimental restraints and not the refinement method or variables used. R factors indicate the structures fit the experimental NOE data very well. Some helical parameters, notably large negative X displacement, are characteristic of A-DNA, but others are characteristic of B-DNA. As with TG.CA steps in other duplex DNA sequences studied in our laboratory, the two TG.CA steps have a positive roll, with T6-G7 exhibiting the largest, and consequently a bent helix axis. The converged structure represents a time-averaged structure. However, multiple conformations, especially in deoxyriboses, were evident from vicinal coupling constants obtained from quantitative simulations of 2QF-COSY cross-peaks and from persistent inconsistencies in experimental distances due to nonlinear conformational averaging.

Detection of mutations in multi-exon genes: comparison of conformation sensitive gel electrophoresis and sequencing strategies with respect to cost and time for finding mutations.

This report compares the relative advantages and disadvantages of two alternative strategies with respect to cost and time for finding mutations in the COL2A1 gene in patients with degenerative diseases of the joint. The coding region of the COL2A1 gene, 30 kb in length and containing 52 exons, can be analyzed using 26 genomic PCR products. The results indicate that the most efficient and cost effective way to screen large genes is a prescreen of the coding sequences followed by sequencing of a limited number of variant-PCR products.

Assessment of T-cell receptor beta-chain diversity by heteroduplex analysis.

The aim of this work was to search for a simple and alternative approach to the currently used methodologies for the analysis of T-cell receptor repertoire diversity. To this end we studied whether the heteroduplex analysis could be adapted to study the clonality of the T-cell receptor beta chain (TCRBV). We therefore analyzed, by sequencing, the molecular characteristics of the V-D-J junctions of numerous TCRBV chains from a variety of patients and from normal individuals, and compared the results with those obtained with the heteroduplex analysis. The latter procedure involves the amplification of the target TCRBV chains and the denaturation and renaturation of the amplified product to permit the random association of the distinct DNA strands encoding the different junctional regions. Whereas amplified material from polyclonal lymphoid cells migrates on a polyacrylamide gel as a "smear" of bands composed of different-sized polyclonal PCR fragments, the mismatched chains derived from oligoclonal populations migrate as discrete "heteroduplexes" and can be separated from the matched "homoduplex" obtained from homogeneous clonal cells. Our results provide evidence demonstrating that heteroduplex analysis can successfully be applied to the analysis of T-cell clonality in a variety of samples and can be complementary or substitute for the standard approach of TCR cloning and multiple sequencing of junctional regions. Thus, the procedure should facilitate the implementation of the analysis of TCR in diagnostic routine and should find applications in numerous physiologic and pathologic conditions.

Assessment of mitochondrial gene in proliferative vitreoretinal tissues from patients with familial diabetes mellitus.

Fibrovascular tissues obtained at therapeutic vitrectomy from 22 patients with proliferative diabetic retinopathy were studied for a mutation of tRNALEU(UUR) at nucleotide position (np) 3243 of mitochondrial DNA (mtDNA). We found a mutation of mtDNA in the vitreous sample of one patient, a 44-year-old woman who had maternally inherited, familial, non-insulin-dependent diabetes mellitus of 25-year duration with recurrent vitreous hemorrhages due to proliferative retinopathy. Heteroduplex analysis also detected the mutant-type mtDNA in the vitreous sample of this patient. The abnormality was, however, not observed in the peripheral blood sample from either this patient orr her family members with diabetes mellitus. The other 21 patients were negative for the mutation in vitreous specimens as well as in peripheral blood. Although a firm conclusion cannot be drawn from a single case report, our observations suggest that this case was an example of tissue-specific expression of mtDNA mutation. Further studies of a larger number of patients with familial diabetes mellitus seem justified.

Experimental evaluation of the ribonuclease protection assay method for the assessment of genetic heterogeneity in populations of RNA viruses.

The ribonuclease (RNase) protection assay (RPA) was evaluated as a method to estimate genetic distances among sequence variants of RNA viruses. The patterns of fragments generated, under different RPA conditions, by three sets of RNA sequence variants of known nucleotide sequence, were analyzed. Both the effectiveness of cleavage (i.e. the probability of cleavage in a certain heteroduplex) and its degree (i.e. in all the molecules in the assay or in a part of them) varied largely according to the nature of the mismatch. Probability and degree of cleavage were also dependent on distant sequence context effects. No correlation could be established between context and cleavage, so that the pattern of fragments in RPA cannot be unequivocally predicted from sequence information. Accordingly, nucleotide sequence differences between two sequence variants cannot be directly derived from RPA data. For all three sequence sets linear relationships were found between the number of non-shared fragments in the RPAs of two variants and their nucleotide sequence differences. Nevertheless, both linearity and the linear regression parameters varied largely according to the sequence set and according to RPA conditions, in a non-predictable way. Thus, under experimental conditions, RPA may not be as appropriate a method to estimate genetic distances between RNA sequences as simulation under an ideal model suggested. Possible ways to diminish the gap between the ideal model and the experimental procedure are proposed.

An alternative approach to the assessment of gamma delta T-cell clonality in celiac disease intestinal lesions through cDNA heteroduplex analysis of T-cell receptor VJ junctions.

We have investigated the clonality of the gamma delta T lymphocytes infiltrating the intestinal mucosa of CD patients and control subjects by means of a simple and powerful method based on the heteroduplex analysis of the TCR VJ junctions. Each V-specific TCR chain, amplified either from fresh biopsy material or intestinal T-cell-line cDNA, is denatured and renatured to allow the random reshuffling of the various strands carrying different junctional sequences, coamplified in the same reaction. The mismatched chains (heteroduplexes) are separated from the matched ones (homoduplexes) through polyacrylamide gel electrophoresis, and whenever one or more T-cell clones are emerging over the polyclonal background, discrete bands are visible by ethidium-bromide staining. Through this method, we have estimated the diversity of the V delta 1-3 chains and a newly described V gene (V delta 8) whose homologue in mice is abundantly expressed in gamma delta iLs. We demonstrate that the well-documented expansion of V gamma 1+ gamma delta lymphocytes in the jejunum of CD patients is polyclonal. Overall, the heteroduplex analysis on fresh intestinal and peripheral blood lymphocytes from both healthy and affected subjects shows a polyclonal pattern of all the V delta+ subsets. In contrast, most intestinal T-cell lines produce oligoclonal patterns, suggesting a dramatic in vitro selection effect. The cell expansion in culture is generally not required for the TCR heteroduplex analysis, which can therefore be applied to rapidly monitor the T-cell response in a variety of physiologic and autoimmune reactions, substituting the standard approach of TCR cloning and multiple VJ sequencing.

A simple and economical DRB1 typing procedure combining group-specific amplification, DNA heteroduplex and enzyme restriction analysis.

A new procedure for HLA-DRB1 typing is proposed. The method combines six group-specific amplifications with heteroduplex analysis and, in some cases, enzyme restriction analysis. This technique, which is as discriminative as oligotyping, is simple, economical and does not require probes. These characteristics make this approach a valid alternative to other HLA genomic typing procedures, especially in those cases such as donor-recipient pairs matching where a small number of samples has to be managed at once.

[Structure of the hydration envelope of the B-form of polydeoxyribonucleotides poly(dA-dC).poly(dG-dT) and poly(dA-dG).poly(dTs-dT) from the data of Monte-Carlo simulation]. / Struktura gidratnoi obolochki polidezoksiribonukleotidov v B-forme poli(dA-dTs).poli(dG-dT) i poli(dA-dG).(poli(dTs-dT) soglasno rezul'tatam simuliatsii metodom Monte-Karlo.

The results of a Monte Carlo simulation of the hydration shell of two polynucleotides poly (dA-dC).poly(dG-dT) and poly(dA-dG).poly(dC-dT) are reported. This study is a part of a series of Monte Carlo computations of the hydration of regular polydeoxyribonucleotides with dinucleotide repeat aimed at looking for dependences of hydration shell structure on base sequence. The coordinates of the main local maximal of water density near the polymers and the topology of the most probable one- and two-membered water bridges are published. For most of the sequences a common primary hydration of base edges of successive base pairs is characteristic. The AT-homopolymeric sequence represents an exception with autonomous primary hydration of a base pair in both grooves, which correlates with the sequence-dependent flexibility and the occurrence of bends of DNA.

Genomic analysis I: inheritance units and genetic selection in the rapid discovery of locus linked DNA markers.

We propose, and test using a Monte-Carlo analysis (a computer-based numerical analysis using a random number generator), a novel and efficient method to obtain sets of DNA markers linked to any inherited genetic locus. The method consists of a targeted search that is based on the common inheritance among members of an outbred pedigree, of discrete chromosome lengths, which we call inheritance units, to obtain DNA markers linked to the locus. In cases where two individuals inherit the same trait through two different lines of descent from a common ancestor, the set of inheritance units in each of the two genomes includes an inheritance unit that is identical in both individuals for a substantial distance on both sides of the DNA sequence which confers the trait. The power of the technique derives from the genetic selection that reduces the size and number of the inheritance units as the generational distance between the two individuals being compared increases.

Assessment of the base sequence homology between the two subtypes of equine herpesvirus 1.

The magnitude of the genetic relatedness of the two antigenic subtypes of equine herpesvirus 1 (EHV-1) was determined by DNA-DNA reassociation kinetics. Denatured, labeled viral DNA from one EHV-1 subtype was allowed to reassociate in the presence or absence of the unlabeled heterologous viral DNA. The initial rate of reassociation of either labeled viral DNA was increased by the presence of the heterologous viral DNA to an extent indicating 10 to 20% homology between the two EHV-1 genomes. Similar estimates of the amount of homology between the genomes of the two EHV-1 subtypes were obtained by determining the maximum fraction of labeled viral DNA that could be made resistant to S1 nuclease by hybridization with a large molar excess of the unlabeled, heterologous viral DNA. Analysis of the thermal stability of the subtype 1-subtype 2 heteroduplex DNA indicated approximately 30% base pair mismatching within the hybrid DNA molecules. Cross-hybridization of 32P-labeled virion DNA to nitrocellulose blots of restriction endonuclease cleavage fragments of each EHV-1 subtype DNA indicated that the observed homology between the two viruses was nonuniformly distributed with the viral genome. No homology could be detected between the DNA of either EHV-1 subtype and that of a strain of equine cytomegalovirus (EHV-2). The data suggest that the two biotypes of EHV-1 have arisen by divergent evolution from a common progenitor herpesvirus.