ABSTRACT
The single-crystal structure of a DNA Holliday junction assembled from four unique sequences shows a structure that conforms to the general features of models derived from similar constructs in solution. The structure is a compact stacked-X form junction with two sets of stacked B-DNA-type arms that coaxially stack to form semicontinuous duplexes interrupted only by the crossing of the junction. These semicontinuous helices are related by a right-handed rotation angle of 56.5 degrees, which is nearly identical to the 60 degree angle in the solution model but differs from the more shallow value of approximately 40 degrees for previous crystal structures of symmetric junctions that self-assemble from single identical inverted-repeat sequences. This supports the model in which the unique set of intramolecular interactions at the trinucleotide core of the crossing strands, which are not present in the current asymmetric junction, affects both the stability and geometry of the symmetric junctions. An unexpected result, however, is that a highly wobbled A.T base pair, which is ascribed here to a rare enol tautomer form of the thymine, was observed at the end of a CCCC/GGGG sequence within the stacked B-DNA arms of this 1.9 A resolution structure. We suggest that the junction itself is not responsible for this unusual conformation but served as a vehicle for the study of this CG-rich sequence as a B-DNA duplex, mimicking the form that would be present in a replication complex. The existence of this unusual base lends credence to and defines a sequence context for the "rare tautomer hypothesis" as a mechanism for inducing transition mutations during DNA replication.
Subject(s)
Base Pairing , DNA, Cruciform/chemistry , Models, Molecular , Nucleic Acid Conformation , Nucleic Acid Heteroduplexes/chemistry , Base Pair Mismatch/genetics , Base Pairing/genetics , Base Sequence , Crystallization , DNA Replication/genetics , DNA, Cruciform/chemical synthesis , DNA, Cruciform/isolation & purification , Dinucleotide Repeats/genetics , Nucleic Acid Heteroduplexes/chemical synthesis , Nucleic Acid Heteroduplexes/isolation & purification , Recombination, Genetic , SolutionsABSTRACT
A new method to detect mutations based on the serial analysis of gene expression (SAGE) technique, ligation-mediated (LM) PCR, and recombinant nuclease CEL I named LM-SAGE assay is reported in the present study. Mismatched DNA heteroduplexes formed from wild-type and mutant DNA are fragmented with CEL I nuclease at the mutant site to produce a double-strand fragment with an overhanging base at the 3'-end. The fragment is ligated to a linker, and digested with MmeI and then ligated to another linker. PCR is performed to amplify the ligation products, and NlaIII is used to release 17-bp tags containing mutation sites followed by purification, concatemerization, cloning, and sequencing. The locations of mutations can be identified from the homology analysis of tags. This new LM-SAGE assay can detect both known and unknown mutations with a sensitivity of 1:50 (mutant:wild-type DNA ratio) in 2.4 x 10(6) copies starting DNA sample. Our results show that this method could be used as a potentially high-throughput assay for mutation detection, particularly for the discovery of unknown mutations in genomic DNA.
Subject(s)
DNA Mutational Analysis/methods , Base Pair Mismatch , Base Sequence , DNA/genetics , DNA/isolation & purification , DNA Mutational Analysis/statistics & numerical data , DNA Primers/genetics , Endonucleases , False Positive Reactions , Gene Expression , Heteroduplex Analysis/methods , Humans , Molecular Sequence Data , Nucleic Acid Heteroduplexes/genetics , Nucleic Acid Heteroduplexes/isolation & purification , Polymerase Chain Reaction/methods , Reproducibility of Results , Sensitivity and SpecificityABSTRACT
Most metazoan genes are alternatively spliced, and a large number of alternatively spliced isoforms are likely to be functionally significant and expressed at specific stages of pathogenesis or differentiation. Splicing changes usually only affect a small portion of a gene, and these changes may cause significant mRNA degradation. After RT-PCR, minor variants can form heteroduplexes with the major variants. Affinity purification of these heteroduplexes using immobilized Thermus aquaticus single-stranded DNA-binding protein allows purification of alternative splice forms in a 1:1 ratio, which makes it easy to sequence the rare form. This chapter provides a detailed protocol of the technique I have developed to identify spliced isoforms called enrichment of alternatively spliced isoforms or EASI.
Subject(s)
Alternative Splicing , RNA, Messenger/genetics , RNA, Messenger/isolation & purification , Bacterial Proteins/metabolism , Base Sequence , DNA Primers/genetics , DNA-Binding Proteins/metabolism , Humans , Male , Nucleic Acid Heteroduplexes/genetics , Nucleic Acid Heteroduplexes/isolation & purification , Nucleic Acid Heteroduplexes/metabolism , Polymerase Chain Reaction , RNA, Messenger/metabolism , Recombinant Proteins/metabolism , Testis/metabolism , Thermus/metabolismABSTRACT
Using optical microscopy, we have studied the phase behavior of mixtures of 12- to 22-bp-long nanoDNA oligomers. The mixtures are chosen such that only a fraction of the sample is composed of mutually complementary sequences, and hence the solutions are effectively mixtures of single-stranded and double-stranded (duplex) oligomers. When the concentrations are large enough, such mixtures phase-separate via the nucleation of duplex-rich liquid crystalline domains from an isotropic background rich in single strands. We find that the phase separation is approximately complete, thus corresponding to a spontaneous purification of duplexes from the single-strand oligos. We interpret this behavior as the combined result of the energy gain from the end-to-end stacking of duplexes and of depletion-type attractive interactions favoring the segregation of the more rigid duplexes from the flexible single strands. This form of spontaneous partitioning of complementary nDNA offers a route to purification of short duplex oligomers and, if in the presence of ligation, could provide a mode of positive feedback for the preferential synthesis of longer complementary oligomers, a mechanism of possible relevance in prebiotic environments.
Subject(s)
DNA/analysis , Nanostructures/analysis , Nucleic Acid Heteroduplexes/isolation & purification , Phase Transition , Crystallization , DNA/chemistry , DNA, Complementary/analysis , DNA, Complementary/chemistry , Microscopy, Fluorescence , Microscopy, Polarization , Nanostructures/chemistry , Nucleic Acid Heteroduplexes/chemistryABSTRACT
Uniform bimetallic nanowires, tunable in size, have been grown on artificial DNA templates via a two-step metallization process. Alkyne-modified cytosines were incorporated into 900-base-pair polymerase-chain-reaction fragments. The alkyne modifications serve as addressable metal-binding sites after conversion to a sugar triazole derivative via click chemistry. Reaction of the Tollens reagent with these sugar-coated DNA duplexes generates Ag0 metallization centers around the sugar modification sites of the DNA. After a subsequent enhancement step using gold, nanowires < or = 10 nm in diameter with a homogeneous surface profile were obtained. Furthermore, the advantage of this two-step procedure lies in the high selectivity of the process, due to the exact spatial control of modified DNA base incorporation and hence the confinement of metallization centers at addressable sites. Besides experiments on a membrane as a proof for the selectivity of the method, atomic force microscopy (AFM) studies of the wires produced on Si-SiO2 surfaces are discussed. Furthermore, we demonstrate time-dependent metallization experiments, monitored by AFM.
Subject(s)
Biomimetic Materials/chemistry , DNA/chemistry , Gold/chemistry , Nanowires/chemistry , Nucleic Acid Heteroduplexes/chemistry , Silver/chemistry , Base Pairing , Carbohydrates/chemistry , Microscopy, Atomic Force , Nucleic Acid Heteroduplexes/chemical synthesis , Nucleic Acid Heteroduplexes/isolation & purification , Nucleic Acid Heteroduplexes/ultrastructure , Saccharomyces cerevisiae , Time FactorsABSTRACT
Two-dimensional strandness-dependent electrophoresis (2D-SDE) separates nucleic acids in complex samples according to strandness, conformation and length. Under the non-denaturing conditions of the first electrophoretic step, single-stranded DNA, double-stranded DNA and RNA.DNA hybrids of similar length migrate at different rates. The second electrophoretic step is performed under denaturing conditions (7 mol l(-1) urea, 55 degrees C) so that all the molecules are single-stranded and separate according to length only. 2D-SDE is useful for revealing important characteristics of complex nucleic acid samples in manipulations such as amplification, renaturation, cDNA synthesis and microarray hybridization. It can also be used to identify mispaired, nicked or damaged fragments in double-stranded DNA. The protocol takes approximately 2 h and requires only basic skills, equipment and reagents.
Subject(s)
DNA, Single-Stranded/isolation & purification , Electrophoresis, Gel, Two-Dimensional/methods , Nucleic Acid Heteroduplexes/isolation & purification , Base Pair Mismatch , DNA Breaks, Single-Stranded , DNA Damage , Research DesignABSTRACT
Efficient global scanning of single nucleotide variations in DNA sequences between related, complex DNA samples remains a challenge. In the present article we present an approach to this problem. We have used immobilized thymidine DNA glycosylases to capture and enrich DNA fragments containing internal mismatched base pairs and separate these fragments as a pool from perfectly base-paired fragments as another pool. Enrichments of up to several hundredfold were obtained with one cycle of treatment, and all of the four groups of single nucleotide mismatches were fully covered by combining use of two thymine DNA glycosylases generated here. We have used a heterohybrid-orientating strategy for selective amplification of duplexes with one strand derived from each of two input DNA samples, which can also be used for selective amplification of duplexes with both strands derived from one of two input samples when desired. By combining these methods, the single nucleotide variations either between two DNA pools or within one DNA pool can be obtained in one process. This approach has been applied to the total cDNA from a human cell line and has several potential applications in mapping genetic variations, particularly global scanning of cDNA single nucleotide variations or polymorphisms, and finally high-throughput mapping of complex genetic traits.
Subject(s)
Genetic Techniques , Polymorphism, Single Nucleotide , Base Pair Mismatch , Base Sequence , DNA, Complementary/genetics , DNA, Complementary/isolation & purification , Deoxyribonuclease (Pyrimidine Dimer) , Endodeoxyribonucleases/genetics , Enzymes, Immobilized , Humans , Nucleic Acid Heteroduplexes/genetics , Nucleic Acid Heteroduplexes/isolation & purification , Recombinant Proteins/geneticsABSTRACT
Mutation screening by the chemical-cleavage method is based on the fact that mismatched cytosine (C) and thymidine (T) are more reactive with the compounds hydroxylamine and osmium tetroxide, respectively, than are Watson and Crick-paired cytosine and thymidine bases. In this protocol, an excess of unlabeled target DNA is hybridized with labeled wild-type DNA probe and heteroduplexes are formed. One aliquot is treated with hydroxylamine, which reacts with mismatched C bases. Another aliquot is treated with osmium tetroxide, which reacts with mismatched T bases. The reactions are mixed with piperidine; the strands are then cleaved at the sites where hydroxylamine and osmium tetroxide react. Cleaved fragments are then electrophoresed and sized on polyacrylamide gels, identifying the point of cleavage (and hence the position of the mutation). Then only a small portion of the mutant gene needs to be sequenced to define a single change between two DNA sequences.
Subject(s)
DNA Mutational Analysis/methods , Nucleic Acid Heteroduplexes/genetics , Nucleic Acid Heteroduplexes/isolation & purification , Genetics, Medical , Heteroduplex Analysis/methods , Humans , MutationABSTRACT
Fluorescence-assisted mismatch analysis (FAMA) methodology uses bifluorescent double-stranded DNA substrates to maximize the reliability and sensitivity of mutation-scanning procedures that rely on cleavage of mismatches using chemical. This unit presents a nested PCR procedure to fluorescently label each DNA strand, followed by chemical cleavage to detect the point mutations. Fluorescent end labeling with strand-specific fluorophores, electrophoretic separation of cleavage products in an automated Perkin-Elmer ABI 373 or 377 DNA sequencer and analysis using the Perkin-Elmer GENESCAN software expands sensitivity by highlighting weak signals through superimposition of strand-specific fluorescence electropherograms for different samples.
Subject(s)
Base Pair Mismatch , DNA Mutational Analysis/methods , Fluorescent Dyes , Genetics, Medical , Humans , Nucleic Acid Heteroduplexes/genetics , Nucleic Acid Heteroduplexes/isolation & purification , Polymerase Chain Reaction/methods , Sequence Analysis, DNAABSTRACT
Deficiencies in DNA mismatch repair (MMR) have been found in hereditary colon cancers (hereditary non-polyposis colon cancer, HNPCC) as well as in sporadic cancers, illustrating the importance of MMR in maintaining genomic integrity. We have examined the interactions of specific mismatch repair proteins in human nuclear extracts. Western blot and co-immunoprecipitation studies indicate two complexes as follows: one consisting of hMSH2, hMSH6, hMLH1, and hPMS2 and the other consisting of hMSH2, hMSH6, hMLH1, and hPMS1. These interactions occur without the addition of ATP. Furthermore, the protein complexes specifically bind to mismatched DNA and not to a similar homoduplex oligonucleotide. The protein complex-DNA interactions occur primarily through hMSH6, although hMSH2 can also become cross-linked to the mismatched substrate when not participating in the MMR protein complex. In the presence of ATP the binding of hMSH6 to mismatched DNA is decreased. In addition, hMLH1, hPMS2, and hPMS1 no longer interact with each other or with the hMutSalpha complex (hMSH2 and hMSH6). However, the ability of hMLH1 to co-immunoprecipitate mismatched DNA increases in the presence of ATP. This interaction is dependent on the presence of the mismatch and does not appear to involve a direct binding of hMLH1 to the DNA.
Subject(s)
Base Pair Mismatch , Cell Nucleus/metabolism , DNA Repair , DNA-Binding Proteins/metabolism , DNA/metabolism , Neoplasm Proteins/metabolism , Nucleic Acid Heteroduplexes/metabolism , Proto-Oncogene Proteins/metabolism , Adaptor Proteins, Signal Transducing , Carrier Proteins , DNA/isolation & purification , DNA-Binding Proteins/isolation & purification , HeLa Cells , Humans , MutL Protein Homolog 1 , MutL Proteins , MutS Homolog 2 Protein , Neoplasm Proteins/isolation & purification , Nuclear Proteins , Nucleic Acid Heteroduplexes/isolation & purification , Protein Binding , Proto-Oncogene Proteins/isolation & purification , Ultraviolet RaysABSTRACT
GB virus C/hepatitis G virus (GBV-C/HGV) is a positive-sense RNA virus belonging to the Flaviviridae family identified recently. Reverse transcription polymerase chain reaction (RT-PCR) was used to detect GBV-C/HGV RNA using nested primers designed to amplify 245 bp of the 5'-untranslated region (UTR). GBV-C/HGV RNA was detected in 20.7% of 101 HCV-RNA positive and 6.8% of 44 HCV-RNA negative specimens. Sequencing of the PCR products demonstrated they had between 84.3 and 100% nucleotide identity. Most of the diversity corresponded to two variable regions identified within the 5'-UTR. Phylogenetic analysis indicated that GBV-C/HGV subtypes present in Australia belonged to group 2 and were closest in evolutionary terms to isolates from the USA and Europe. All isolates were analysed using single-strand conformation polymorphism (SSCP) and heteroduplex mobility analysis (HMA) on 8% non-denaturing polyacrylamide gels. SSCP of the isolates identified a number of distinct conformation polymorphisms that corresponded with sequence-determined genetic diversity. HMA was developed to assess the amount of genetic diversity between isolates without the need for sequencing. The average difference between the predicted divergence of two isolates calculated from the mobility of the heteroduplex and the actual value (based on nucleotide sequence) was 2.3% in this sample of isolates, where the mean sequence divergence was 8.52%.
Subject(s)
5' Untranslated Regions , Flaviviridae/genetics , Genetic Variation , Virology/methods , Base Sequence , DNA Primers/genetics , Evaluation Studies as Topic , Flaviviridae/isolation & purification , Hepacivirus/isolation & purification , Humans , Molecular Sequence Data , Nucleic Acid Heteroduplexes/isolation & purification , Phylogeny , Polymorphism, Single-Stranded Conformational , RNA, Viral/genetics , RNA, Viral/isolation & purification , Reverse Transcriptase Polymerase Chain Reaction , Sequence Homology, Nucleic AcidSubject(s)
DNA Damage , DNA Glycosylases , DNA Repair , DNA/chemistry , Oocytes/metabolism , Animals , Apurinic Acid , Base Sequence , DNA/genetics , DNA/isolation & purification , Electrophoresis, Agar Gel/methods , Female , N-Glycosyl Hydrolases/metabolism , Nucleic Acid Heteroduplexes/chemistry , Nucleic Acid Heteroduplexes/genetics , Nucleic Acid Heteroduplexes/isolation & purification , Oligodeoxyribonucleotides/chemistry , Oligodeoxyribonucleotides/metabolism , Tissue Extracts/metabolism , Uracil-DNA Glycosidase , Xenopus laevisSubject(s)
Base Pair Mismatch/genetics , DNA Repair , Animals , Bacteriophages/genetics , Cell Line , Cell Nucleus/metabolism , DNA/genetics , DNA/isolation & purification , DNA, Viral/genetics , DNA, Viral/isolation & purification , Electrophoresis, Agar Gel/methods , Humans , Mammals , Nucleic Acid Heteroduplexes/genetics , Nucleic Acid Heteroduplexes/isolation & purification , Restriction Mapping/methodsABSTRACT
A specialized form of ion-pair reversed-phase high-performance liquid chromatography is gaining widespread application in mutation detection for single nucleotide polymorphisms (SNP). The technique relies on temperature-modulated heteroduplex analysis (TMHA) by chromatographic separation of partially denatured DNA heteroduplexes from homoduplexes. Here, we demonstrate that fluorescent labeling is compatible with mutation analysis by this form of DNA chromatography and offers advantages over the use of unlabeled DNA fragments. Uniform labeling of wild-type and mutant alleles for TMHA yields peak patterns identical to unlabeled fragments. However, fluorescent labels increase retention times but do not influence resolution of heteroduplexes from homoduplexes. They increase sensitivity and decrease the amount of DNA required for analysis; e.g., in the case presented here, one allele can be detected in the presence of a 500-fold excess of another allele. Furthermore, allele-specific wild-type probes, fluorescently labeled on one strand only, make it possible to selectively monitor specific homoduplexes and wild-type/mutant heteroduplexes. This, in combination with an internal homoduplex standard, greatly reduces the complexity of fluorescence chromatograms compared with chromatograms recorded in the UV. These simplified chromatograms, in which only the internal homoduplex standard and the labeled heteroduplex are detected in the presence of a mutation, greatly facilitate the detection and identification of mutant alleles.
Subject(s)
DNA/genetics , Polymorphism, Single-Stranded Conformational , Y Chromosome/genetics , Base Sequence , DNA/isolation & purification , DNA Mutational Analysis/methods , Fluorescent Dyes , Genetic Markers , Humans , Male , Nucleic Acid Amplification Techniques , Nucleic Acid Denaturation , Nucleic Acid Heteroduplexes/genetics , Nucleic Acid Heteroduplexes/isolation & purification , Polymerase Chain Reaction/methodsABSTRACT
Capillary affinity gel electrophoresis was applied to sequence-specific and base composition-specific recognition of oligodeoxynucleotides, utilizing the formation of heteroduplexes between a nucleic acid analogue immobilized into the capillary gel and soluble oligodeoxynucleotides with different sequences. Capillary affinity gel electrophoresis using capillaries filled with a conjugated gel of polyacrylamide and a synthetic nucleic acid analogue [poly(9-vinyladenine)] was effective for the selective separation of hexathymidylic acid from a mixture of four homopolymers of A6, C6, G6, and T6 and for the complete resolution of five heteropolymers of hexadeoxynucleotides (TAAAAA, TTAAAA, TTTAAA, TTTTAA, TTTTTA). We also demonstrated that capillary affinity gel electrophoresis was useful for the selective and the sensitive sequence-specific recognition of sequence isomers of DNA (TTTTAA, TTTTAAT, TTTATA, and TTTTAA).
Subject(s)
Electrophoresis, Capillary/methods , Oligodeoxyribonucleotides/isolation & purification , Affinity Labels , Base Composition , Base Sequence , Gels , Nucleic Acid Heteroduplexes/chemistry , Nucleic Acid Heteroduplexes/isolation & purification , Oligodeoxyribonucleotides/chemistry , SolubilitySubject(s)
Polymerase Chain Reaction/methods , RNA, Catalytic/metabolism , Binding Sites , Binding, Competitive , In Vitro Techniques , Nucleic Acid Heteroduplexes/isolation & purification , RNA/genetics , RNA/isolation & purification , RNA/metabolism , Substrate Specificity , Transcription, GeneticSubject(s)
Carrier Proteins/genetics , Polymorphism, Genetic , Receptors, Cell Surface , Swine/genetics , Animals , Base Sequence , Crosses, Genetic , DNA/isolation & purification , DNA Primers , Electrophoresis, Polyacrylamide Gel/methods , Female , Genetic Variation , Humans , Male , Molecular Sequence Data , Nucleic Acid Heteroduplexes/isolation & purification , Pedigree , Polymerase Chain Reaction/methods , Receptors, Leptin , Species SpecificitySubject(s)
DNA/isolation & purification , Nucleic Acid Conformation , Nucleic Acid Heteroduplexes/isolation & purification , RNA/isolation & purification , DNA, Bacterial/chemistry , DNA, Single-Stranded/isolation & purification , DNA, Viral/chemistry , RNA, Double-Stranded/isolation & purification , Silicon DioxideABSTRACT
A 37-year old AIDS patient presented with foreign body sensation. Microsporidia were detected in smears from a conjunctival swab and urine sediment stained with calcofluor and a modified trichrome blue stain and by indirect fluorescent-antibody staining with murine polyclonal antiserum raised against Encephalitozoon hellem. This antiserum cross-reacted with other Encephalitozoon species, so PCR was performed to amplify the microsporidian ribosomal DNA (rDNA) with pan-Encephalitozoon primers. The PCR DNA products from the urine and conjunctival clinical specimens, along with the tissue culture-derived microsporidian controls, were assayed by Southern analysis with oligonucleotide probes specific for Encephalitozoon cuniculi, E. hellem, and Encephalitozoon (Septata) intestinalis. The PCR product amplified from the urine specimen hybridized with the E. hellem probe only, while insufficient DNA was amplified from the conjunctiva specimen for detection by Southern analysis. For corroboration of the PCR-Southern analysis results, aliquots of the urine and conjunctiva specimens were seeded onto RK-13 cell monolayers. The rDNA extracts of the cultured microsporidia were amplified by PCR with pan-Encephalitozoon primers, and the PCR DNA products were subjected to digestion with restriction endonuclease FokI. The amplified rDNA of both the urine and conjunctiva isolates generated digestion patterns that were identified to the E. hellem PCR rDNA digestion pattern. In addition, double-stranded heteroduplex mobility shift analysis with these PCR products indicated that the urine and conjunctiva isolates were identical to each other and to E. hellem. The patient was treated with albendazole and topical fumagillin and responded rapidly, with no recurrence of ophthalmologic signs. The results of this study demonstrate that PCR-Southern analysis provides a basis for distinguishing E. cuniculi, E. hellem, and E. intestinalis in clinical specimens.