High-resolution mapping of heteroduplex DNA formed during UV-induced and spontaneous mitotic recombination events in yeast.

In yeast, DNA breaks are usually repaired by homologous recombination (HR). An early step for HR pathways is formation of a heteroduplex, in which a single-strand from the broken DNA molecule pairs with a strand derived from an intact DNA molecule. If the two strands of DNA are not identical, there will be mismatches within the heteroduplex DNA (hetDNA). In wild-type strains, these mismatches are repaired by the mismatch repair (MMR) system, producing a gene conversion event. In strains lacking MMR, the mismatches persist. Most previous studies involving hetDNA formed during mitotic recombination were restricted to one locus. Below, we present a global mapping of hetDNA formed in the MMR-defective mlh1 strain. We find that many recombination events are associated with repair of double-stranded DNA gaps and/or involve Mlh1-independent mismatch repair. Many of our events are not explicable by the simplest form of the double-strand break repair model of recombination.

Electrophoretic mobility of duplex DNA cross-linked by mechlorethamine at a cytosine-cytosine mismatch pair.

The common nitrogen mustard, mechlorethamine, can form a covalent cross-link between the two bases of a cytosine-cytosine mismatch pair within a DNA duplex. The cross-linked species can be readily separated from DNA monoadducts and unreacted strands using denaturing polyacrylamide gel electrophoresis. Here, using DNA 19 mer duplexes that are mechlorethamine cross-linked at a C(4)-C(35), C(7)-C(32), C(10)-C(29), or C(13)-C(26) mismatch pair, we show that the denaturing polyacrylamide gel electrophoresis mobility of the cross-linked species is particularly sensitive to the proximity of the C-C cross-link to the duplex end. Species that are cross-linked at a C(4)-C(35) mismatch have greater mobilities than those cross-linked at C(7)-C(32) or C(13)-C(26), and the species with a central C(10)-C(29) cross-link have the lowest mobility. The mobility is also dependent on the proximity of the cross-link to a 5'-(32)P-phosphate or a 5'-fluorescein label. We interpret these results in terms of the conformational properties of the cross-linked species in the denaturing gel. The results are consistent with the retention of partial duplex character at the end proximal to the cross-link, with an influence on the mobility of the GC/AT ratio proximal to the cross-link and at the duplex end, and a small but discernible effect of the label.

Applying and testing the conveniently optimized enzyme mismatch cleavage method to clinical DNA diagnosis.

Establishing a simple and effective mutation screening method is one of the most compelling problems with applying genetic diagnosis to clinical use. Because there is no reliable and inexpensive screening system, amplifying by PCR and performing direct sequencing of every coding exon is the gold standard strategy even today. However, this approach is expensive and time consuming, especially when gene size or sample number is large. Previously, we developed CEL nuclease mediated heteroduplex incision with polyacrylamide gel electrophoresis and silver staining (CHIPS) as an ideal simple mutation screening system constructed with only conventional apparatuses and commercially available reagents. In this study, we evaluated the utility of CHIPS technology for genetic diagnosis in clinical practice by applying this system to screening for the COL2A1, WRN and RPS6KA3 mutations in newly diagnosed patients with Stickler syndrome (autosomal dominant inheritance), Werner syndrome (autosomal recessive inheritance) and Coffin-Lowry syndrome (X-linked inheritance), respectively. In all three genes, CHIPS detected all DNA variations including disease causative mutations within a day. Direct sequencing of all coding exons of these genes confirmed 100% sensitivity and specificity. We demonstrate high sensitivity, high cost performance and reliability of this simple system, with compatibility to all inheritance modes. Because of its low technology, CHIPS is ready to use and potentially disseminate to any laboratories in the world.

An assay of gene copy number and its application based on heteroduplex products.

The aim of this study was to set up a simple and efficient method for detecting gene copy number, based on heteroduplex products from single-tube PCR/DHPLC. Single-nucleotide polymorphisms (SNPs) on the α-globin gene and chromosome 21 were used as examples. And the formula for quantitative calculation of gene copy number was deduced-based on the peak heights of homoduplexes and heteroduplexes on the DHPLC pattern. 27 samples (14 normal DNA and 13 cases of trisomy-21) were assessed with this method, and 160 samples (48 normal DNA and 112 α-thalassemia samples) were assessed with this method combined with a duplex PCR/DHPLC. Results for 184 of 187 cases were concordant with the known genotypes; three cases of trisomy-21 could not be detected because the target SNPs were homozygous. In conclusion, quantitative assessment of heteroduplex products from single-tube PCR/DHPLC is simple and rapid, and can be used to detect α-thalassemia gene deletions (α(-3.7), α(-4.2)) and trisomy-21.

Superfamily I helicases as modular components of DNA-processing machines.

Helicases are a ubiquitous and abundant group of motor proteins that couple NTP binding and hydrolysis to processive unwinding of nucleic acids. By targeting this activity to a wide range of specific substrates, and by coupling it with other catalytic functionality, helicases fulfil diverse roles in virtually all aspects of nucleic acid metabolism. The present review takes a look back at our efforts to elucidate the molecular mechanisms of UvrD-like DNA helicases. Using these well-studied enzymes as examples, we also discuss how helicases are programmed by interactions with partner proteins to participate in specific cellular functions.

[The comparative determination mutations in mtDNA of irradiated mice using mismatch-specific endonuclease and TTGE-technique].

We defined the mutations in mtDNA of X-irradiated mice brair using mismatch-specific endonuclease (CEL I-nuclease method) and by temporal temperature gradient gel electrophoresis (TTGE-technique). The comparison of the received by both methods, allows to conclude, that CEL I-nuclease method gives more qualitative results, than TTGE-technique. Moreover, CEL I-nuclease method is more sensitive, in contrast with TTGE-technique. The CEL I-nuclease method allows simultaneously to conduct the analysis of big amount of sample DNA, to get the reproducible results. It does not require complex equipment and economical. The analysis of mutations in mtDNA of brain of X-irradiated mice by CEL I-nuclease method has shown, that the amount of mutant copies mtDNA is essentially reduced (in 2-3 times) with 8 up to 28 days of the post-radiation period. However the amount mtDNA copies in brain tissue of the irradiated animals is remains during all post radiation time without change though lower, concerning given control group. The results permit the suggestion that mutant mtDNA copies are eliminated from the tissues of irradiated animals in the post-radiation period.

Nonradioactive heteroduplex tracking assay for the detection of minority-variant chloroquine-resistant Plasmodium falciparum in Madagascar.

BACKGROUND: Strains of Plasmodium falciparum genetically resistant to chloroquine (CQ) due to the presence of pfcrt 76T appear to have been recently introduced to the island of Madagascar. The prevalence of such resistant genotypes is reported to be low (< 3%) when evaluated by conventional PCR. However, these methods are insensitive to low levels of mutant parasites present in patients with polyclonal infections. Thus, the current estimates may be an under representation of the prevalence of the CQ-resistant P. falciparum isolates on the island. Previously, minority variant chloroquine resistant parasites were described in Malawian patients using an isotopic heteroduplex tracking assay (HTA), which can detect pfcrt 76T-bearing P. falciparum minority variants in individual patients that were undetectable by conventional PCR. However, as this assay required a radiolabeled probe, it could not be used in many resource-limited settings. METHODS: This study describes a digoxigenin (DIG)-labeled chemiluminescent heteroduplex tracking assay (DIG-HTA) to detect pfcrt 76T-bearing minority variant P. falciparum. This assay was compared to restriction fragment length polymorphism (RFLP) analysis and to the isotopic HTA for detection of genetically CQ-resistant parasites in clinical samples. RESULTS: Thirty one clinical P. falciparum isolates (15 primary isolates and 16 recurrent isolates) from 17 Malagasy children treated with CQ for uncomplicated malaria were genotyped for the pfcrt K76T mutation. Two (11.7%) of 17 patients harboured genetically CQ-resistant P. falciparum strains after therapy as detected by HTA. RFLP analysis failed to detect any pfcrt K76T-bearing isolates. CONCLUSION: These findings indicate that genetically CQ-resistant P. falciparum are more common than previously thought in Madagascar even though the fitness of the minority variant pfcrt 76T parasites remains unclear. In addition, HTAs for malaria drug resistance alleles are promising tools for the surveillance of anti-malarial resistance. The use of a non-radioactive label allows for the use of HTAs in malaria endemic countries.

HyBeacon probes for rapid DNA sequence detection and allele discrimination.

HyBeacon probes are single-stranded oligonucleotides with one or more internal base(s) labeled with a fluorescent dye. When a probe forms a duplex with its target sequence, the level of fluorescence emission increases considerably. HyBeacons have been developed as new tools for rapid sequence detection and discrimination and have been employed in a wide variety of applications including infectious diagnostics and analysis of human polymorphisms. Single-labeled (FVG1) and dual-labeled (FVG11) probes were designed to analyze the factor V Leiden (R506Q) polymorphism which causes an increased risk of deep vein thrombosis and pulmonary embolism. Detection and identification of factor V alleles is performed by melting curve analysis and determination of probe melting temperature (T(m)). HyBeacon hybridization to the glutamine allele (Q) causes the formation of mismatched DNA duplexes that are detected through decreases in T(m). HyBeacon probes are included in homogeneous PCR assays to genotype samples with respect to the factor V polymorphism within 20 min, using purified DNAs and unpurified saliva/blood samples. This paper describes the preparation of homogeneous PCR assays, LightCycler target amplification, and subsequent melting curve analysis. This chapter also describes the use of homologous oligonucleotides and melting curve analysis as a method for probe evaluation.

High-throughput simultaneous detection of point mutations and large-scale rearrangements by CE.

The detection of unknown mutations is important both in population genetics research and in diagnosis. At present, two different methods must be used to detect either point mutations or large-scale genetic rearrangements, which is costly and time-consuming. We describe here a new method for the simultaneous detection of these two types of mutations. It is based on electrophoretic heteroduplex analysis (HDA) using enhanced mismatch mutation analysis (EMMA) and semiquantitative multiplexed PCR conditions. The use of such conditions allows the simultaneous search of any kind of mutation in up to five different fragments per capillary, in a single or multi-CE system. The method was validated on patient samples with mutations in the breast predisposition gene BRCA1. It leads to highly reliable and high-throughput mutation detection at low cost, as compared with classical methods.

Minority-variant pfcrt K76T mutations and chloroquine resistance, Malawi.

Genotyping of the chloroquine-resistance biomarker pfcrt (Plasmodium falciparum chloroquine resistance transporter gene) suggests that, in the absence of chloroquine pressure, Plasmodium falciparum parasites in Malawi have reverted to chloroquine sensitivity. However, malaria infections in Africa are commonly polyclonal, and standard PCRs cannot detect minority genotypes if present in <20% of the parasites in an individual host. We have developed a multiple site-specific heteroduplex tracking assay (MSS-HTA) that can detect pfcrt 76T mutant parasites consisting of as little as 1% of the parasite population. In clinical samples, no pfcrt 76T was detected in 87 pregnant Malawian women by standard PCR. However, 22 (25%) contained minority-variant resistant genotypes detected by the MSS-HTA. These results were confirmed by subcloning and sequencing. This finding suggests that the chloroquine-resistant genotype remains common in Malawians and that PCR-undetectable drug-resistant genotypes may be present in disease-endemic populations. Surveillance for minority-variant drug resistant mutations may be useful in making antimalarial drug policy.

Evaluation of a polymerase chain reaction-based heteroduplex assay for detecting the adulteration of processed orange juice with mandarin juice.

A polymerase chain reaction (PCR)-based heteroduplex assay was evaluated for the detection of mandarin juice in processed orange juice. PCR amplification of a fragment of the chloroplast trnT-trnL intergenic spacer derived from mixtures of DNA extracted from orange and mandarin juice resulted in heteroduplex formation. The heteroduplex resulted from the co-amplification of a fragment containing an 8 base-pair indel that distinguished mixtures of orange and mandarin juice from orange juice and mandarin juice alone. The heteroduplex assay was evaluated against authentic juices obtained from different citrus species and confirmed that the marker was homogeneous within Citrus. The data obtained demonstrated maternal inheritance of chloroplast type in Citrus sp. and allowed the identification and confirmation of the maternal parentage of unknown and known citrus hybrids. Analysis of the quantitative potential of the PCR and polyacrylamide gel electrophoresis (PAGE) analysis demonstrated good repeatability with a coefficient of variation of 7.5%. Greatest sources of variance in experimental results were attributable to species and varietal differences in the levels of the PCR target. Mandarin juice contained approximately 18% (w/v) less PCR target sequence than did orange juice. The assay was tested in a blind trial using processed juices and correctly identified 20/22 samples with no false-positive results.

Improved delivery in cell culture of radiolabeled antisense DNAs by duplex formation.

PURPOSE: Delivery remains an unresolved problem in applications requiring intravenous administration of DNAs. Recently improved antisense translation interruption in cells was reported for an antisense (AS) oligomer as a duplex compared to singlet AS oligomer presumably because of improved delivery. The unstable phosphodiester backbone of the sense (S) oligomer and its shorter chain length apparently encouraged intracellular dissociation and release of the AS oligomer. We have investigated the mechanism involved to evaluate whether the approach may be useful for antisense radionuclide imaging. PROCEDURES: Duplexes were formed between an AS phosphorothioate DNA against the mdr1 mRNA and the uniform phoshorothioate or uniform phosphodiester sense (S) DNAs with either four or six mismatches. RESULTS: Accumulations in KB-G2 (Pgp++) cells of radiolabeled AS DNA as duplex accumulated threefold higher compared to singlet. Accumulation was still antisense as shown by reduced accumulations with the radiolabel on the S DNA. However, the DNA backbone had no clear influence on accumulations. CONCLUSIONS: Targeting of mRNAs with radiolabeled AS DNAs may be improved in cell culture if duplexed with an S DNA engineered for low hybridization affinity to encourage dissociation in the presence of the target mRNA.

High-throughput alternative splicing quantification by primer extension and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Alternative splicing is a significant contributor to transcriptome diversity, and a high-throughput experimental method to quantitatively assess predictions from expressed sequence tag and microarray analyses may help to answer questions about the extent and functional significance of these variants. Here, we describe a method for high-throughput analysis of known or suspected alternative splicing variants (ASVs) using PCR, primer extension and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Reverse-transcribed mRNA is PCR amplified with primers surrounding the site of alternative splicing, followed by a primer extension reaction designed to target sequence disparities between two or more variants. These primer extension products are assayed on a MALDI-TOF mass spectrometer and analyzed automatically. This method is high-throughput, highly accurate and reproducible, allowing for the verification of the existence of splicing variants in a variety of samples. An example given also demonstrates how this method can eliminate potential pitfalls from ordinary gel electrophoretic analysis of splicing variants where heteroduplexes formed from different variants can produce erroneous results. The new method can be used to create alternative variant profiles for cancer markers, to study complex splicing regulation, or to screen potential splicing therapies.

A high-throughput mutation detection method based on heteroduplex analysis using graft copolymer matrixes: application to Brca1 and Brca2 analysis.

We present here a new approach to electrophoretic heteroduplex analysis (EHDA) based on improved matrixes. EHDA is an appealing technique for the detection of unknown point mutations because of its simplicity and high throughput. We present here a new matrix for electrophoretic heteroduplex analysis much more sensitive for insertions, deletions, and substitutions than reported for previous EHDA separations and also superior to DHPLC. This separation matrix is based on a copolymer with a comb architecture, poly(acrylamide-g-polydimethylacrylamide), made of a high molecular weight polyacrylamide backbone grafted with poly(dimethylacrylamide) side chains. The effect of operational parameters on electrophoretic resolution and sensitivity to single-nucleotide mismatches was studied using a collection of samples from patients bearing mutations in the breast cancer predisposition genes BRCA1 and BRCA2. Seventeen fragments (10 mutations), implying mostly substitutions on fragments with sizes ranging from 200 to 600 bp, were analyzed using a single set of separation conditions. A success rate of 94% was achieved with a qualitative analysis in terms of number of peaks, and 100% identification of mutations was obtained with a more quantitative test using peak width analysis. This strong improvement of performance with regard to previous HDA methods is attributed to a composite mechanism of separation, combining steric and chromatographic effects. It opens the route to a significant reduction of development time and operation cost for diagnostic and genomic applications.

Highly sensitive detection of GG mismatched DNA by surfaces immobilized naphthyridine dimer through poly(ethylene oxide) linkers.

Naphthyridine dimer is a unique molecule that strongly, and selectively, binds to the guanine-guanine mismatch in duplex DNA. We have synthesized naphthyridine dimers possessing a different length of poly(ethylene oxide) (PEO) linker, and immobilized them to CM5 sensor chip to carry out a surface plasmon resonance (SPR) assay of DNA duplexes containing a single base mismatch. The sensitivity of the sensor remarkably increased with increasing numbers of PEO units incorporated into the linker. With the sensor surface immobilized naphthyridine dimer for 1.5 x 10(3) response unit (RU) through three PEO units, the distinct SPR signal was observed at a concentration of 1 nM of the 27-mer G-G mismatch.

Spectroscopic analysis of DNA duplexes including T-T mismatches.

The DNA duplex with tandem T x T mismatches has been studied by NMR spectroscopy. For this study, we synthesized decamer duplex with two successive T x T mismatches, TT10:d(CGCGTTGTCC) x d(GGACTTCGCG). From two-dimensional (2D) 1H-1H NOESY spectrum, we were able to trace sequential NOE walks between base protons and anomeric protons (H1'), and assigned all the base protons and anomeric protons. These results indicate that the overall structure of the duplex is a right-handed duplex, with two successive T x T mismatches stacked in the helix.

Electrophoretic analysis of ITS from Piscirickettsia salmonis Chilean isolates.

Piscirickettsia salmonis is the most important pathogen in salmonid mariculture in Chile. Since it was reported numerous piscirickettsiosis outbreaks have occurred differing in virulence and mortality. Genetic variability of P. salmonis isolates has been suggested as one factor to explain this. However until now isolates obtained from outbreaks have not been analyzed. Knowledge of genetic variability of P. salmonis is very limited and also a useful screening method for genetic variations in isolates without sequencing is not available. Here we report an electrophoretic analysis of internal transcribed spacer region (ITS) of eleven P. salmonis isolates obtained from different salmon species and places in southern Chile. When PCR products were submitted to polyacrylamide gel electrophoresis (PAGE) a characteristic electrophoretic pattern was observed, distinguishable from ITS of other bacteria, including fish pathogens. Even though this pattern is conserved in all isolates, a difference in ITS electrophoretic mobility was observed, determining clearly two groups: ITS with higher or with lower electrophoretic mobility, including LF-89 and EM-90 isolates, respectively. A higher ITS sequence homology inside each group was shown by heteroduplex mobility assay (HMA). Our results show that genetic variability between Chilean P. salmonis isolates allows the differentiation of two groups with similar behavior observed previously when six P. salmonis isolates from three geographic origins were analyzed by 16S, 23S and ITS sequencing. PAGE analysis of ITS and HMA could be a basis to develop an assay for screening genetic variability between P. salmonis isolates.

Identification of signature and primers specific to genus Pseudomonas using mismatched patterns of 16S rDNA sequences.

BACKGROUND: Pseudomonas, a soil bacterium, has been observed as a dominant genus that survives in different habitats with wide hostile conditions. We had a basic assumption that the species level variation in 16S rDNA sequences of a bacterial genus is mainly due to substitutions rather than insertion or deletion of bases. Keeping this in view, the aim was to identify a region of 16S rDNA sequence and within that focus on substitution prone stretches indicating species level variation and to derive patterns from these stretches that are specific to the genus. RESULTS: Repeating elements that are highly conserved across different species of Pseudomonas were considered as guiding markers to locate a region within the 16S gene. Four repeating patterns showing more than 80% consistency across fifty different species of Pseudomonas were identified. The sub-sequences between the repeating patterns yielded a continuous region of 495 bases. The sub-sequences after alignment and using Shanon's entropy measure yielded a consensus pattern. A stretch of 24 base positions in this region, showing maximum variations across the sampled sequences was focused for possible genus specific patterns. Nine patterns in this stretch showed nearly 70% specificity to the target genus. These patterns were further used to obtain a signature that is highly specific to Pseudomonas. The signature region was used to design PCR primers, which yielded a PCR product of 150 bp whose specificity was validated through a sample experiment. CONCLUSIONS: The developed approach was successfully applied to genus Pseudomonas. It could be tried in other bacterial genera to obtain respective signature patterns and thereby PCR primers, for their rapid tracking in the environmental samples.

Computational and in vitro analysis of destabilized DNA regions in the interferon gene cluster: potential of predicting functional gene domains.

Recent evidence adds support to a traditional concept according to which the eukaryotic nucleus is organized into functional domains by scaffold or matrix attachment regions (S/MARs). These regions have previously been predicted to have a high potential for stress-induced duplex destabilization (SIDD). Here we report the parallel results of binding (reassociation) and computational SIDD analyses for regions within the human interferon gene cluster on the short arm of chromosome 9 (9p22). To verify and further refine the biomathematical methods, we focus on a 10 kb region in the cluster with the pseudogene IFNWP18 and the interferon alpha genes IFNA10 and IFNA7. In a series of S/MAR binding assays, we investigate the promoter and termination regions and additional attachment sequences that were detected in the SIDD profile. The promoters of the IFNA10 and the IFNA7 genes have a moderate approximately 20% binding affinity to the nuclear matrix; the termination sequences show stronger association (70-80%) under our standardized conditions. No comparable destabilized elements were detected flanking the IFNWP18 pseudogene, suggesting that selective pressure acts on the physicochemical properties detected here. In extended, noncoding regions a striking periodicity is found of rather restricted SIDD minima with scaffold binding potential. By various criteria, the underlying sequences represent a new class of S/MARs, thought to be involved in a higher level organization of the genome. Together, these data emphasize the relevance of SIDD calculations as a valid approach for the localization of structural, regulatory, and coding regions in the eukaryotic genome.

Quantification of PCR bias caused by a single nucleotide polymorphism in SMN gene dosage analysis.

Approximately 94% of patients with spinal muscular atrophy lack both copies of SMN1 exon 7, and most carriers have only one copy of SMN1 exon 7. We described previously the effect of SMN1/SMN2 heteroduplex formation on SMN gene dosage analysis, which is a multiplex quantitative PCR assay to determine the copy numbers of SMN1 and SMN2 using DraI digestion to differentiate SMN2 from SMN1. We describe herein the quantification of PCR bias between SMN1 exon 7 and SMN2 exon 7, which differ by only one nucleotide that is not present in either primer binding site. Using samples from 272 individuals with various SMN genotypes, we found that the amplification efficiency of SMN2 was consistent only approximately 80% that of SMN1. Thus, even a single nucleotide polymorphism, not in primer binding sites, can cause reproducible PCR bias. The precision and accuracy of our SMN gene dosage analysis are high because our assay design and controls take advantage of the consistency of the PCR bias. As additional clinically significant single nucleotide polymorphisms (SNPs) are discovered, assessment of PCR bias, and judicious selection of standards and controls, will be increasingly important for quantitative PCR assays.