Heteroduplex Formation Research Articles

Porphyromonas gingivalis strain variability and periodontitis.

To determine if there is variability in virulence among strains of Porphyromonas gingivalis in human periodontitis, their distribution in a group of subjects with clear indicators of periodontitis and in a healthy, age-matched control group was examined. The presence of heteroduplex types of P. gingivalis in the two groups was determined with a PCR-based assay. This assay relied on detection of polymorphisms in the ribosomal internal spacer region (ISR). ISR fragments generated by PCR with P. gingivalis-specific primers were hybridized to fragments from reference strains, and the formation of heteroduplexes from the hybridization of nonidentical sequences was observed by polyacrylamide gel electrophoresis. Characteristic fingerprints from comparison with a panel of reference strains allowed the identification of heteroduplex types in clinical samples. One hundred thirty adults with periodontitis and 181 controls were sampled. With this approach, 11 heteroduplex types of P. gingivalis were detected in the population. Sufficient numbers were available for statistical analysis of six of these types. Heteroduplex type hW83 was found to be very strongly associated with periodontitis (P = 0.0000), and two additional types, h49417 and hHG1691, were also significantly associated with disease. The remaining types, h23A4, h381, and hA7A1, were detected more frequently in subjects with periodontitis than in healthy subjects, but the difference was not significant. These data indicate that virulence in human periodontitis varies among strains of P. gingivalis, and they identify an apparently highly virulent subgroup.

A RecG-independent nonconservative branch migration mechanism in Escherichia coli recombination.

To gain insight regarding the mechanisms that extend heteroduplex joints in Escherichia coli recombination, we investigated the effect of recG and ruv genotypes on heteroduplex strand polarity in intramolecular recombination products. We also examined the cumulative effect of mutational inactivation of RecG and single-strand-specific exonucleases on recombination proficiency and the role of Chi sites in RecG-independent recombination. All four strands of the two homologs were incorporated into heteroduplex structures in wild-type cells and in ruv mutants. However, in recG mutants heteroduplexes were generated almost exclusively by pairing the invasive 3'-ending strand with its complementary strand. To explain the dependence of strand exchange reciprocity on RecG activity, we propose that alternative mechanisms may extend the heteroduplex joints after homologous pairing: a reciprocal RecG-mediated mechanism and a nonreciprocal mechanism, mediated by RecA and single-strand-specific exonucleases. The cumulative effect of recG and recJ or xonA mutations on recombination proficiency and the inhibitory effect of recJ and xonA activities on heteroduplex formation by the 5'-ending strands are consistent with this proposal.

Rapid determination of apolipoprotein E genotype using a heteroduplex generator

The apoE gene exhibits two common polymorphisms that have been associated with both coronary artery disease and Alzheimer's disease. The polymorphisms create the three allelic isoforms E2, E3, and E4 which are encoded by Cys–Cys, Cys–Arg, and Arg–Arg at amino acid positions 112 and 158, respectively. Numerous methods have been described to identify these three apoE alleles although there are disadvantages and ambiguities associated with all of them. Here we describe a method by which the two common apoE polymorphisms can be identified simultaneously. The method involves PCR of the region containing the two polymorphic sites, followed by hybridization of this PCR product to a synthetic molecule called a universal heteroduplex generator (UHG). The UHG is used to induce heteroduplex formation which is visualized on a non-denaturing mini-gel using ethidium bromide staining. This technique which can also identify other rare mutations in the amplified region of DNA under investigation, is an unequivocal method of genotyping and is simpler and faster than many methods, including using restriction enzyme digestion.—Bolla, M. K., N. Wood, and S. E. Humphries. Rapid determination of apolipoprotein E genotype using a heteroduplex generator.

Minimizing the secondary structure of DNA targets with a modified deoxynucleoside and its implication for nucleic acid analysis by hybridization

Some regions of nucleic acid targets are not accessible to heteroduplex formation with complementary oligonucleotide probes because they are involved in secondary structure through intramolecular Watson-Crick pairing. In order to form a heteroduplex, we destabilize the secondary conformation of the target to assist its interactions with oligonucleotide probes. To achieve this goal, we modified the DNA target by replacing dC with N-4-ethyldeoxycytidine (d4EtC), which hybridizes specifically with natural dG to give a G4EtC base pair with reduced stability compared with the natural GC base pair. In contrast to its natural analogue, the use of d4EtC greatly minimized the formation of the target's secondary structure in preliminary solution studies. The lower level of secondary structure allowed hybridization with a complementary probe. To characterize further the influence of d4EtC on the stability of secondary structure, hybridization of the targets is currently being studied using an array of complementary oligonucleotides scanning the sequence.

Recombination and chimeragenesis by in vitro heteroduplex formation and in vivo repair.

We describe a simple method for creating libraries of chimeric DNA sequences derived from homologous parental sequences. A heteroduplex formed in vitro is used to transform bacterial cells where repair of regions of non-identity in the heteroduplex creates a library of new, recombined sequences composed of elements from each parent. Heteroduplex recombination provides a convenient addition to existing DNA recombination methods ('DNA shuffling') and should be particularly useful for recombining large genes or entire operons. This method can be used to create libraries of chimeric polynucleotides and proteins for directed evolution to improve their properties or to study structure-function relationships. We also describe a simple test system for evaluating the performance of DNA recombination methods in which recombination of genes encoding truncated green fluorescent protein (GFP) reconstructs the full-length gene and restores its characteristic fluorescence. Comprising seven truncated GFP constructs, this system can be used to evaluate the efficiency of recombination between mismatches separated by as few as 24 bp and as many as 463 bp. The optimized heteroduplex recombination protocol is quite efficient, generating nearly 30% fluorescent colonies for recombination between two genes containing stop codons 463 bp apart (compared to a theoretical limit of 50%).

Detection of a Deletion Polymorphism of the Human α2-Macroglobulin Gene (A2M-2) by a Semi-Automated PCR-Single-Stranded Conformational Polymorphism Method

Several genes such as the genes that code for apolipoprotein E4, amyloid β-precursor protein, and presenilin 1 and 2 have been linked to the development of Alzheimer disease (AD) (1). Recently, a polymorphism of the human α2-macroglobulin gene ( A2M ) was also identified as a risk factor for AD (2). The A2M gene is located on chromosome 12p13.3–p12.3 (3), and its product (α2-macroglobulin) is a serum pan-proteinase inhibitor occurring in many organs and tissues (4). Several studies have suggested that α2-macroglobulin may have protective effect against the deposition of β-amyloids (5)(6) and may stimulate their degradation (7)(8). A 5-nucleotide deletion mutation at the 5′ splice site of “exon II” of the bait region (exon 18; A2M-2 ) may lead to loss of this protection because of its location at the functional domain, and this loss confers increased risk for AD (2). Currently available methods based on DNA sequencing (2)(9), heteroduplex formation in nondenaturing gel (9), and PCR-restriction fragment length polymorphism …

Recombination and chimeragenesis by in vitro heteroduplex formation and in vivo repair

Recombination and chimeragenesis by in vitro heteroduplex formation and in vivo repair

Lack of allelic imbalance in APOE ϵ3/4 brain mRNA expression in Alzheimer's disease

Although the APOE ϵ4 allele is a strong risk factor for Alzheimer's disease (AD), it is not deterministic, as many APOE ϵ3/4 individuals do not develop AD. It has been hypothesized that this incomplete penetrance is due, in part, to an imbalance of allele expression in heterozygous individuals. In this regard, Lambert et al. (1998) reported that AD individuals have a higher APOE ϵ4/total APOE ratio than non-demented control subjects. We tested this hypothesis using radioactive RT-PCR to quantitate APOE ϵ3 and ϵ4 allele expression levels in AD and non-AD brain samples from APOE ϵ3/4 individuals. Quantitative analyses of amplified products within the linear range of amplification (18–20 cycles) revealed no difference from the expected 1:1 ratio in genomic DNA and in cDNA from AD and control brains. Using high PCR cycle numbers (∼30), we observed an artificial elevation of the APOE ϵ3/total APOE ratio in both DNA and cDNA samples, possibly due to DNA heteroduplex formation. Our results do not support the hypothesis that allelic imbalance contributes to the risk of developing AD among APOE ϵ3/4 heterozygote individuals.

Heteroduplex Formation by Human Rad51 Protein: Effects of DNA End-structure, hRP-A and hRad52

Purified human Rad51 protein (hRad51) catalyses ATP-dependent homologous pairing and strand transfer reactions, characteristic of a central role in homologous recombination and double-strand break repair. Using single-stranded circular and partially homologous linear duplex DNA, we found that the length of heteroduplex DNA formed by hRad51 was limited to approximately 1.3 kb, significantly less than that observed with Escherichia coli RecA and Saccharomyces cerevisiae Rad51 protein. Joint molecule formation required the presence of a 3′ or 5′-overhang on the duplex DNA substrate and initiated preferentially at the 5′-end of the complementaryx strand. These results are consistent with a preference for strand transfer in the 3′-5′ direction relative to the single-stranded DNA. The human single-strand DNA-binding protein, hRP-A, stimulated hRad51-mediated joint molecule formation by removing secondary structures from single-stranded DNA, a role similar to that played by E. coli single-strand DNA-binding protein in RecA-mediated strand exchange reactions. Indeed, E. coli single-strand DNA-binding protein could substitute for hRP-A in hRad51-mediated reactions. Joint molecule formation by hRad51 was stimulated or inhibited by hRad52, dependent upon the reaction conditions. The inhibitory effect could be overcome by the presence of hRP-A or excess heterologous DNA.

Determining the influence of structure on hybridization using oligonucleotide arrays.

We have studied the effects of structure on nucleic acid heteroduplex formation by analyzing hybridization of tRNAphe to a complete set of complementary oligonucleotides, ranging from single nucleotides to dodecanucleotides. The analysis points to features in tRNA that determine heteroduplex yield. All heteroduplexes that give high yield include both double-stranded stems as well as single-stranded regions. Bases in the single-stranded regions are stacked onto the stems, and heteroduplexes terminate at potential interfaces for coaxial stacking. Heteroduplex formation is disfavored by sharp turns or a lack of helical order in single-stranded regions, competition from bases displaced from a stem, and stable tertiary interactions. The study is relevant to duplex formation on oligonucleotide microarrays and to antisense technologies.

Assembly of the Escherichia coli RuvABC resolvasome directs the orientation of holliday junction resolution.

Genetic recombination can lead to the formation of intermediates in which DNA molecules are linked by Holliday junctions. Movement of a junction along DNA, by a process known as branch migration, leads to heteroduplex formation, whereas resolution of a junction completes the recombination process. Holliday junctions can be resolved in either of two ways, yielding products in which there has, or has not, been an exchange of flanking markers. The ratio of these products is thought to be determined by the frequency with which the two isomeric forms (conformers) of the Holliday junction are cleaved. Recent studies with enzymes that process Holliday junctions in Escherichia coli, the RuvABC proteins, however, indicate that protein binding causes the junction to adopt an open square-planar configuration. Within such a structure, DNA isomerization can have little role in determining the orientation of resolution. To determine the role that junction-specific protein assembly has in determining resolution bias, a defined in vitro system was developed in which we were able to direct the assembly of the RuvABC resolvasome. We found that the bias toward resolution in one orientation or the other was determined simply by the way in which the Ruv proteins were positioned on the junction. Additionally, we provide evidence that supports current models on RuvABC action in which Holliday junction resolution occurs as the resolvasome promotes branch migration.

Prenatal diagnosis of JAK3 deficient SCID

The JAK3 gene, encoding a tyrosine kinase functionally coupled to cytokine receptors which share the common gamma chain, has been identified as the defective gene for autosomal recessive severe combined immunodeficiency (SCID). Thus, specific mutational diagnosis has become possible. We screened all exons with a combined single strand conformational polymorphism and hetero-duplex formation assay followed by sequence analysis to identify specific mutations in two families. This assay was used on chorionic villus sampling derived DNA in two fetuses from two unrelated families, where we found mutations in both parents. We were able to exclude the mutations in both fetuses by the 12th week of gestation. The described method for first-trimester prenatal diagnosis of autosomal recessive T-B+SCID provides a valid tool to aid in genetic counselling and possibly prenatal therapy in this disease.

A competitive reverse transcription-polymerase chain reaction assay for quantitation of GB virus C/hepatitis G virus RNA that circumvents heteroduplex artifact

The role of GB virus C (GBV-C)/hepatitis G virus (HGV) in hepatitis has been controversial. To investigate its possible pathogenicity and site(s) of replication, it is important to develop an accurate quantitative assay for both positive and negative strand GBV-C/HGV RNA. In this study, a competitive reverse transcription-polymerase chain reaction (RT-PCR) assay for both positive and negative strand GBV-C/HGV RNA quantitation was developed. In developing the quantitative assay, heteroduplex formation was repeatedly observed. A heterologous competitor RNA with GBV-C/HGV primer-binding sequences was introduced, and heteroduplex artifact was circumvented successfully. Two-hundred thirty-seven serum specimens were screened by RT-PCR for GBV-C/HGV RNA. Two of the 62 patients infected with chronic hepatitis C virus (HCV) were found to be positive for GBV-C/HGV RNA. None of the 50 other patients with no evidence of HCV infection and none of the 125 normal individuals were positive for GBV-C/HGV RNA. The sensitivity of RT-PCR was 3000 gE/ml (30 gE in RT-PCR). Alternate methods for residual DNA removal and its detection in synthetic RNA were introduced. A RT control containing no primer before PCR is necessary to evaluate the trace amounts of template DNA remaining in synthesized RNA. The method will differentiate reliably between positive and negative strand RNAs up to a 10 4-fold difference in titer. The positive and negative strand GBV-C/HGV RNAs were detected in one patient by RT-PCR and hybridization analysis, and the strand titer was determined by RT-PCR.

Roles of the DNA mismatch repair and nucleotide excision repair proteins during meiosis.

Numerous proteins are involved in the nucleotide excision repair (NER) and DNA mismatch repair (MMR) pathways. The function and specificity of these proteins during the mitotic cell cycle has been actively investigated, in large part due to the involvement of these systems in human diseases. In contrast, comparatively little is known about their functioning during meiosis. At least three repair pathways operate during meiosis in the yeast Saccharomyces cerevisiae to repair mismatches that occur as a consequence of heteroduplex formation in recombination. The first pathway is similar to the one acting during postreplicative mismatch repair in mitotically dividing cells, while two pathways are responsible for the repair of large loops during meiosis, using proteins from MMR and NER systems. Some MMR proteins also help prevent recombination between diverged sequences during meiosis, and act late in recombination to affect the resolution of crossovers. This review will discuss the current status of DNA mismatch repair and nucleotide excision repair proteins during meiosis, especially in the yeast S. cerevisiae.

Prenatal molecular diagnosis of Wiskott–Aldrich syndrome by direct mutation analysis

We have performed prenatal diagnosis for Wiskott–Aldrich syndrome (WAS) in two unrelated families by direct gene analysis. Using a combined non-radioactive analysis of single-strand conformational polymorphism (SSCP) and heteroduplex formation (HD), followed by automated sequencing, we studied DNA from chorionic villus sampling (CVS), allowing the diagnosis of one affected and one healthy male at the 12th week of gestation. Copyright © 1999 John Wiley & Sons, Ltd.

Rapid DNA mutation identification and fingerprinting using base excision sequence scanning.

Base excision sequence scanning (BESS) is a new polymerase chain reaction (PCR)-based mutation scanning method that locates and identifies all DNA mutations. The BESS method consists of two procedures that generate "T" (BESS T-Scan) and "G" ladders (BESS G-Tracker) analogous to T and G ladders of dideoxy sequencing. The BESS procedures are simple to perform and require no special equipment or gels, no reaction optimization beyond PCR, and no heteroduplex formation. The samples are analyzed on standard sequencing gels or on automated DNA sequencers, and the data produced are easy to interpret, requiring a simple comparison of the sequence of normal and mutant DNA. The BESS method is versatile, having applications not only for mutation detection, but also single nucleotide polymorphism (SNP) discovery and analysis, DNA fingerprinting (including viral and bacterial typing), and clone identification. In this study, we utilize BESS in two of these applications: detection of a point mutation in BRCA1, and DNA typing of human papilloma virus (HPV).

Prenatal molecular diagnosis of Wiskott-Aldrich syndrome by direct mutation analysis

We have performed prenatal diagnosis for Wiskott Aldrich syndrome (WAS) in two unrelated families by direct gene analysis. Using a combined non-radioactive analysis of single-strand conformational polymorphism (SSCP) and heteroduplex formation (HD), followed by automated sequencing, we studied DNA from chorionic villus sampling (CVS), allowing the diagnosis of one affected and one healthy male at the 12th week of gestation.

Inversion/dimerization of plasmids mediated by inverted repeats

In contrast with earlier studies on the lambda and Escherichia coli genomes, recombination between inverted repeats on plasmids is highly efficient and shown to be recA-independent. In addition, the recombination product is exclusively a head-to-head inverted dimer. Here, we show that this recombination/rearrangement event can occur on different plasmid replicons and is not specific to the particular sequence within the inverted repeats. Transcription readthrough into the inverted repeats has little effect on this event. Genetic analysis has also indicated that most known recombination enzymes are not involved in this process. Specifically, single or double mutants defective in Holliday junction resolution systems (RuvABC and/or RecG/RusA) do not abolish this recombination/rearrangement event. This result does not support the previous models (i.e. the reciprocal-strand-switching and the cruciform-dumbbell models) in which intermediates containing Holliday junctions are proposed. Further analysis has demonstrated that the recombination/rearrangement frequency is dramatically (over 1000-fold) reduced if mismatches (2.8%) are present within the inverted repeats. Mutations in dam, mutH and mutL genes partially or completely restored the recombination/rearrangement frequency to the level exhibited by the perfect inverted repeats, suggesting the formation of heteroduplexes during recombination/rearrangement. Sequencing analysis of the recombination/rearrangement products have indicated that the majority of the products do not involve crossing-over. We discuss a possible mechanism in which blockage of the lagging strand polymerase by a hairpin triggers recombination/rearrangement mediated by inverted repeats.

The reason of non-specific background of methodologies for random mutation identification: X-structures formation in purified PCR products solutions

Chemical or Enzyme Mismatch Cleavage represent the methodologies directed to point random mutation detection. They are based on the formation of heteroduplexes consisting of one wild-type and one mutant strand from PCR-products. During mutation detection analysis, high nonspecific background prompted a detailed investigation of the DNA used in the study. The phenomenon of diverse DNA structures of double size of PCR product approximately and of higher molecular weight appearing in concentrated solutions of purified DNA fragments was demonstrated for 5 PCR products differing both by size and sequence (various genes PCR products from 118 bp to 1249 bp). The present investigation on the model of p53 cDNA 1249 bp fragment confirm that the diverse DNA structures are formed by double stranded DNA fragments via Sl-nuclease analysis: treated by the enzyme DNA fragments retain the ability to form higher molecular weight structures. Using the number of following purification procedures it was demonstrated, that the phenomenon is due to interaction of purified ds DNA fragments and is not connected with the any contamination of standard PCR products. The electroellution of twice the size DNA structures from agarose gel followed by electron microscopy reveals the presence of X-structure in this fraction of DNA. In our investigation the ability of DNA to interact with formation of Holliday junction, the intermediate structures of the recombination process, without participation of cell proteins was demonstrated as it own phisico-chemical characteristic. Our data are significant for optimization of methodologies of CMC or EMC or other analysis using concentrated PCR product solutions via preventing of formation of complex DNA structures.

Molecular Genetic Study of Autosomal Dominant Retinitis pigmentosa in Lithuanian Patients

Lithuanian patients with visual problems were clinically examined for retinitis pigmentosa (RP). A total of 33 unrelated families with autosomal dominant RP (adRP) were identified. Screening for mutations in the rhodopsin (RHO) and peripherin/RDS (RDS) genes was performed using DNA heteroduplex analysis. Direct DNA sequencing in the cases of heteroduplex formation showed the presence of the following mutations and polymorphisms in 14 adRP patients: RHO gene – Lys248Arg (1 case), and Pro347Leu (2 cases); RDS gene – Glu304Gln (12 cases), Lys310Arg (5 cases), and Gly338Asp (12 cases). The presence of these mutations (except Lys248Arg in the RHO gene) was confirmed by relevant restriction enzyme digestion. The frequency of the RDS gene mutations Glu304Gln and Gly338Asp was estimated to be 36.4%, while mutation Lys310Arg was less frequent (15.2%). These 3 RDS gene mutations appear to be polypeptide polymorphisms not related to adRP.