Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Heteroduplex Analysis”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 325 records · Page 18Linked to original sources
PubMed1987

A physical map of the chromosomal region determining O-antigen biosynthesis in Vibrio cholerae O1.

We have previously described the cosmid cloning of the genes determining the biosynthesis of the Inaba and Ogawa O-antigens of the lipopolysaccharides of Vibrio cholerae O1 (Manning et al., 1986). By Southern hybridization analysis of chromosomal and cosmid DNA, and heteroduplex analysis between the clones we have been able to precisely define the region of contiguous chromosomal DNA in the vicinity of the O-antigen-encoding region. These data and comparison of end points of clones and of deletion derivatives demonstrate that at least 16 kb of a 19-kb SstI fragment is required to encode O-antigen biosynthesis. Expression of O-antigen is independent of the orientation of this SstI fragment with respect to cloning vectors suggesting that its regulatory region has been cloned intact. No detectable differences were observed in the restriction patterns of the Inaba and Ogawa coding regions implying that only minor changes are involved when serotype conversion (Inaba to Ogawa or vice versa) occurs. Bhaskaran [Ind. J. Med. Res. 47 (1959) 253-260] originally defined this region associated with O-antigen biosynthesis oag; however, to be consistent with other organisms [Hitchcock et al., J. Bacteriol. 166 (1986) 699-705], it is suggested this be changed to rfb.

Antigens, Bacterial↗
PubMed1996

Genetic basis of Bart's syndrome: a glycine substitution mutation in type VII collagen gene.

Bart's syndrome was initially described as a genodermatosis characterized by congenital localized absence of the skin, together with blistering and nail abnormalities. Recent analysis of Bart's original kindred demonstrated ultrastructural abnormalities in the anchoring fibrils and linkage of the inheritance of the disease to the region of chromosome 3 near the type VII collagen gene (COL7A1). We have performed mutation analysis in this family by using electrophoretic heteroduplex analysis followed by direct nucleotide sequencing of DNA. These results disclosed a G-to-A transition within exon 73 of COL7A1, which results in a glycine-to-arginine substitution within the triple-helical domain of type VII collagen in affected individuals. In this family, these findings demonstrate that Bart's syndrome is a clinical variant of dominant dystrophic epidermolysis bullosa.

Amino Acid Sequence↗
PubMed1984

Cloning and characterization of DNA complementary to rat liver UDP-glucuronosyltransferase mRNA.

UDP-glucuronosyltransferase (transferase) clones were isolated from a cDNA bank constructed in pBR322 using transferase-enriched mRNA from the livers of phenobarbital-treated rats. The enrichment of mRNA was accomplished by polysome immunoadsorption with antibody to purified mouse liver transferase. This antibody was shown to bind specifically to rat transferase by Ouchterlony double diffusion analysis, immunoadsorption of glucuronidating activities, and selective inhibition of the immunoadsorption of in vitro synthesized enzyme by purified rat liver transferase. The isolated clones were verified to contain DNA complementary to transferase mRNA by hybrid translation-selection. Three classes of transferase cDNAs were characterized by restriction endonuclease mapping, and the largest insert-containing clone of each class was designated pUDPGTr-1, pUDPGTr-2, and pUDPGTr-3. Their insert sizes were approximately 2,400, 2,000, and 2,000 bp, respectively. All three cDNAs hybridized with a 2,300 +/- 150 bp mRNA, and each selected the translation of a 52,000-dalton polypeptide. Immunoadsorption of the 35S-labeled translation product could be competitively inhibited in each case by the addition of purified rat liver transferase. pUDPGTr-1 and pUDPGTr-3 inserts shared extensive sequence homology. This was demonstrated by Southern blot analysis using purified inserts and electron microscopic heteroduplex analysis. Southern blot analysis revealed that these cDNAs hybridized to overlapping genomic fragments. pUDPGTr-2 shared less sequence homology with the other two classes of cDNAs, based on the above criteria. In addition, mRNA corresponding to pUDPGTr-2 was elevated 5-fold by phenobarbital treatment, whereas the other mRNAs levels were unaffected. These studies demonstrate that in rat liver there are a minimum of three distinct transferase mRNAs, two of which may be associated with a common gene or gene family.

Animals↗
PubMedDate not supplied

Von Hippel-Lindau tumour suppressor gene is not involved in sporadic human breast cancer.

OBJECTIVE: Mutations of the von Hippel-Lindau (vhl) gene, as well as allelic loss at the gene region (3p25-26) have been described in sporadic cases of the tumour types participating in VHL disease, but also in cancers not associated with the syndrome. In this study, we attempted mutation analysis of the vhl gene, as well as detection of allelic loss at 3p25-26 in sporadic human breast cancer. METHODS: Eighty-two tumour specimens were screened for loss of heterozygosity (LOH) at the vhl region, and compared to the adjacent, histologically normal tissue. Furthermore, mutations within the three exons of vhl in the same panel of tumours were detected using SSCP and heteroduplex analysis and direct sequencing. RESULTS: To our knowledge this is the first mutational analysis reported for the vhl gene in breast cancer, however we failed to reveal any mutations in the specimens examined. All the cases were informative for at least one of the microsatellite markers tested, 24 (29.2%) exhibited LOH at 3p25-26. Clinical and pathological data were available for all tumours examined, however no significant correlations were encountered. CONCLUSION: These results strongly indicate against a critical involvement of the tumour suppressor vhl in breast carcinogenesis.

Breast↗
PubMed1999

Novel mutations in the Wiskott-Aldrich syndrome protein gene and their effects on transcriptional, translational, and clinical phenotypes.

Wiskott-Aldrich syndrome (WAS) is an X-linked recessive immunodeficiency characterized by thrombocytopenia, eczema, and recurrent infections, and caused by mutations in the WAS protein (WASP) gene. WASP contains several functional domains through which it interacts with proteins involved in intracellular signaling and regulation of the actin cytoskeleton. In this report, 17 WASP gene mutations were identified, 12 of which are novel. DNA of affected males and obligate carriers was PCR amplified and analyzed by SSCA, heteroduplex analysis, and direct sequencing. The effects of the mutations at the mRNA and protein level were ascertained by RT-PCR and Western blot analyses. All missense mutations were located in exons 1-4. Most of the nonsense, frameshift and splice site mutations were found in exons 6-11. Mutations that alter splice sites led to the synthesis of several types of mRNAs, a fraction of which represented the normally spliced product. The presence of normally spliced transcripts was correlated with a milder phenotype. When one such case was studied by Western blotting, reduced amounts of normal-size WASP were present. In other cases as well, a correlation was found between the amount of normal or mutant WASP present and the phenotypes of the affected individuals. No protein was detected in two individuals with severe WAS. Reduced levels of a normal-size WASP with a missense mutation were seen in two individuals with XLT. It is concluded that mutation analysis at the DNA level is not sufficient for predicting clinical course. Studies at the transcript and protein level are needed for a better assessment.

Alternative Splicing↗
PubMed2002

An update on conformation sensitive gel electrophoresis.

Conformation-sensitive gel electrophoresis (CSGE) was developed as a method of heteroduplex analysis to screen large multi-exon genes for sequence variation. The novelty of the method was in the use of a non-proprietary acrylamide gel matrix that used 1,4-bis (acrolyl) piperazine (BAP) as a cross linker with ethylene glycol and formamide as mildly denaturing solvents. The denaturing environment enhances the conformation polymorphism present in DNA heteroduplexes containing variations as small as single nucleotide polymorphisms (SNPs). CSGE has also been adapted for use on a fluorescent platform (F-CSGE) that resulted in higher throughput and sensitivity. Variation in sensitivity of CSGE has been studied extensively. The results demonstrate that the nature of the mismatched base in a defined sequence context has the most profound effect on the conformation of the heteroduplex. Additionally, the size of the PCR product, as well as the location of the mismatch within the PCR product, are two important parameters that determine the resolution of the mismatch-containing heteroduplexes during CSGE. Like any other mutation scanning technique, CSGE can have limited resolution of two closely linked sequence variations. For specific genes, like BRCA1 and BRCA2 where multiple SNPs are present in the coding sequence, each CSGE shift has to be sequenced to define the exact nature of the sequence change. In conclusion, CSGE scanning provides a powerful, cost-efficient way to scan genes with high sensitivity and specificity.

Base Sequence↗
PubMed2003

Fluorescent heteroduplex assay for monitoring Bacillus anthracis and close relatives in environmental samples.

A fluorescent heteroduplex method was developed to assess the presence of 16S rRNA gene (rDNA) sequences from Bacillus anthracis and close relatives in PCR-amplified 16S rDNA sequence mixtures from environmental samples. The method uses a single-stranded, fluorescent DNA probe, 464 nucleotides in length, derived from a B. anthracis 16S rRNA gene. The probe contains a unique, engineered deletion such that all probe-target duplexes are heteroduplexes with an unpaired G at position 343 (deltaG343). Heteroduplex profiles of sequences >/=85% similar to the probe were produced using an ABI 377 sequencer in less than 3 h. The method divides strains of the Bacillus cereus-Bacillus thuringiensis-B. anthracis group into two subgroups. Each subgroup is defined by a specific 16S rRNA gene sequence type. Sequence type A, containing one mismatch with the probe, occurs in B. anthracis and a small number of closely related clonal lineages represented mostly by food-borne pathogenic isolates of B. cereus and B. thuringiensis. Sequence type B, containing two mismatches with the probe, is found in the majority of B. cereus and B. thuringiensis strains examined to date. Sequence types A and B, when hybridized to the probe, generate two easily differentiated heteroduplexes. Thus, from heteroduplex profiles, the presence of B. cereus-B. thuringiensis-B. anthracis subgroups in environmental samples can be inferred unambiguously. The results show that fluorescent heteroduplex analysis is an effective profiling technique for detection and differentiation of sequences representing small phylogenetic or functional groups in environmental samples.

Bacillus↗
PubMed2000

An analysis of ABCR mutations in British patients with recessive retinal dystrophies.

PURPOSE: Several reports have shown that mutations in the ABCR gene can lead to Stargardt disease (STGD)/fundus flavimaculatus (FFM), autosomal recessive retinitis pigmentosa (arRP), and autosomal recessive cone-rod dystrophy (arCRD). To assess the involvement of ABCR in these retinal dystrophies, the gene was screened in a panel of 70 patients of British origin. METHODS: Fifty-six patients exhibiting the STGD/FFM phenotype, 6 with arRP, and 8 with arCRD, were screened for mutations in the 50 exons of the ABCR gene by heteroduplex analysis and direct sequencing. Microsatellite marker haplotyping was used to determine ancestry. RESULTS: In the 70 patients analyzed, 31 sequence changes were identified, of which 20 were considered to be novel mutations, in a variety of phenotypes. An identical haplotype was associated with the same pair of in-cis alterations in 5 seemingly unrelated patients and their affected siblings with STGD/FFM. Four of the aforementioned patients were found to carry three alterations in the coding sequence of the ABCR gene, with two of them being in-cis. CONCLUSIONS: These results suggest that ABCR is a relatively polymorphic gene. Because putative mutations have been identified thus far only in 25 of 70 patients, of whom only 8 are compound heterozygotes, a large number of mutations have yet to be ascertained. The disease haplotype seen in the 5 patients carrying the same "complex" allele is consistent with the presence of a common ancestor.

ATP-Binding Cassette Transporters↗
PubMed1999

Analysis of DNA elements that modulate myosin VIIA expression in humans.

Usher syndromeIb (USH1B), an autosomal recessive disorder caused by mutations in myosin VIIa (MYO7A), is characterized by congenital profound hearing loss, vestibular abnormalities and retinitis pigmentosa. Promoter elements in the 5 kb upstream of the translation start were identified using adult retinal pigment epithelium cells (ARPE-19) as a model system. A 160 bp minimal promoter within the first intron was active in ARPE-19 cells, but not in HeLa cells that do not express MYO7A. A 100 bp sequence, 5' of the first exon, and repeated with 90% homology within the first intron, appeared to modulate expression in both cell lines. Segments containing these elements were screened by heteroduplex analysis. No heteroduplexes were detected in the minimal promoter, suggesting that this sequence is conserved. A -2568 A>T transversion in the 5' 100 bp repeat, eliminating a CCAAT element, was found only in USH1B patients. However, in all 5 families, -2568 A>T was in cis with the same missense mutation in the myosin VIIa tail (Arg1240Gln), and 4 of the 5 families were Dutch. These observations suggest either 1) linkage disequilibrium or 2)that a combination of a promoter mutation with a less active myosin VIIa protein results in USH1B.

Cells, Cultured↗
PubMed2001

High-throughput methods for detection of genetic variation.

Understanding human genetic variation is currently believed to reveal the cause of individual susceptibility to disease and the large variation observed in response to treatment. In this review, we will focus on different approaches to identify and visualize genetic alterations. The various approaches for allele discrimination are formally systematically divided into (i) enzymatic approaches, in which the properties of different enzymes to discriminate between nucleotides are used (restriction enzymes type II, Cleavase and Resolvase, DNA polymerase, and ligase); (ii) electrophoretic methods, in which the allele discrimination is based on the difference in mobility in polymeric gels or capillaries (single- and double-stranded conformation assays, heteroduplex analysis, and DNA sequencing); (iii) solid-phase determination of allelic variants, including high-density oligonucleotide arrays for hybridization analysis, minisequencing primer extension analysis, and fiberoptic DNA sensor array; (iv) chromatographic methods such as denaturing high-performance liquid chromatography (DHPLC); (v) other physical methods of discrimination of allelic variants such as mass spectrometry (mass and charge) or fluorescence exchange-based techniques; and (vi) in silico methods such as high-throughput analysis of expressed sequence tag data. The most frequently used techniques and instrumental settings applied in different combinations are described, and other methods that are less broadly used but have interesting potentials are discussed.

Alleles↗
PubMed1999

Characterization of APC exon 15 germ-line mutation in FAP family with severe phenotype showing extracolonic symptoms.

The adenomatous polyposis coli (APC) gene plays a crucial role in colorectal carcinogenesis. Germ-line mutations of APC gene give rise to familial adenomatous polyposis coli (FAP) - autosomal dominant syndrome manifesting hundreds to thousands of colorectal polyps, if untreated with malignant progression. We have used the techniques of heteroduplex analysis (HDA), protein truncation test (PTT), single strand conformation polymorphism (SSCP) and DNA sequencing for the identification and detailed positional analysis of mutations in IFAP family with the expressive phenotype characterized by polyposis and extracolonic lesions. Detailed analysis revealed a 5bp deletion in a mutation cluster region (MCR) in exon 15 of APC gene in codon 1308. Two screened members of the FAP family exhibited this novel mutation.

Adenomatous Polyposis Coli↗
PubMed2000

Erratum: analysis of DNA elements that modulate myosin VIIa expression in humans.

Usher syndromeIb (USH1B), an autosomal recessive disorder caused by mutations in myosin VIIa (MYO7A), is characterized by congenital profound hearing loss, vestibular abnormalities and retinitis pigmentosa. Promoter elements in the 5 kb upstream of the translation start were identified using adult retinal pigment epithelium cells (ARPE-19) as a model system. A 160 bp minimal promoter within the first intron was active in ARPE-19 cells, but not in HeLa cells that do not express MYO7A. A 100 bp sequence, 5' of the first exon, and repeated with 90% homology within the first intron, appeared to modulate expression in both cell lines. Segments containing these elements were screened by heteroduplex analysis. No heteroduplexes were detected in the minimal promoter, suggesting that this sequence is conserved. A -2568 A>T transversion in the 5' 100 bp repeat, eliminating a CCAAT element, was found only in USH1B patients. However, in all 5 families, -2568 A>T was in cis with the same missense mutation in the myosin VIIa tail (Arg1240Gln), and 4 of the 5 families were Dutch. These observations suggest either 1) linkage disequilibrium or 2)that a combination of a promoter mutation with a less active myosin VIIa protein results in USH1B.

Amino Acid Substitution↗
PubMed2005

The usefulness of clonality for the detection of cases clinically and/or histopathologically not recognized as cutaneous T-cell lymphoma.

BACKGROUND: The determination of clonality has proven to be a useful adjunct to the diagnosis of cutaneous lymphocytic infiltrates. It is considered particularly helpful for the distinction of mycosis fungoides (MF) and inflammatory dermatoses. OBJECTIVES: To verify the sensitivity of the polymerase chain reaction (PCR)-heteroduplex analysis of T-cell receptor gamma-chain gene (TCRgamma) rearrangements in patients with MF and to establish whether a clinicopathological re-evaluation of lesions previously unclassified or considered to be non-neoplastic entities but found to be monoclonal allowed the recognition of additional cases of MF. METHODS: Included in the study were 116 patients, seen at our Institute from April 2002 to September 2003 and tested for TCRgamma rearrangements. Thirty-six patients were affected by clinically and histopathologically proven MF, while the remaining 80 cases had not been classified or had been classified as non-neoplastic entities. The sensitivity of the molecular analysis was determined on the basis of the results obtained in the 36 patients with MF. The 29 cases of the second series of patients found to be monoclonal were clinically and histopathologically re-evaluated. RESULTS: Clonal rearrangements were found in 87.5% of patients with plaque stage MF and in 20% of those with patch stage MF. The clinicopathological re-evaluation allowed us to reclassify 15 of 29 monoclonal cases of the second series of patients as MF. CONCLUSIONS: The study showed that the PCR-heteroduplex technique can determine a high percentage of monoclonality only in plaque stage MF. However, in spite of the low sensitivity of the method, several cases previously unrecognized could be reclassified as MF when their clinical and histopathological features were re-evaluated taking into account the clonality of the lymphocytic infiltrate.

Diagnosis, Differential↗
PubMed1999

Mitochondrial phylogeography of the European pond turtle, Emys orbicularis (Linnaeus 1758).

The phylogeny and phylogeography of Emys orbicularis was inferred from mitochondrial nucleotide sequences of the cytochrome b gene analysed by DNA sequencing and RNA heteroduplex analysis. Within the family Emydidae the monotypic genus Emys is affiliated with the nearctic taxa Emydoidea blandingii and Clemmys marmorata. The analysis of 423 individuals of E. orbicularis, originating throughout its distribution range, revealed a remarkable intraspecific differentiation in 20 different haplotypes with distinct geographical ranges. Maximum parsimony analysis produced a star-like phylogeny with seven main lineages which may reflect separations in the late Pliocene. The haplotype distribution examined by partial Mantel tests and analysis of molecular variance revealed a substantial effect of glacial periods. This historical perspective suggests the existence of multiple glacial refugia and considerable Holocene range expansion which was modulated by climatic traits. Further support is gained for the occurrence of long-term parapatry in glacial refugia.

Animals↗
PubMed2001

Sequence requirements for interaction of human herpesvirus 7 origin binding protein with the origin of lytic replication.

As do human herpesvirus 6 variants A and B (HHV-6A and -6B), HHV-7 encodes a homolog of the alphaherpesvirus origin binding protein (OBP), which binds at sites in the origin of lytic replication (oriLyt) to initiate DNA replication. In this study, we sought to characterize the interaction of the HHV-7 OBP (OBP(H7)) with its cognate sites in the 600-bp HHV-7 oriLyt. We expressed the carboxyl-terminal domain of OBP(H7) and found that amino acids 484 to 787 of OBP(H7) were sufficient for DNA binding activity by electrophoretic mobility shift analysis. OBP(H7) has one high-affinity binding site (OBP-2) located on one flank of an AT-rich spacer element and a low-affinity site (OBP-1) on the other. This is in contrast to the HHV-6B OBP (OBP(H6B)), which binds with similar affinity to its two cognate OBP sites in the HHV-6B oriLyt. The minimal recognition element of the OBP-2 site was mapped to a 14-bp sequence. The OBP(H7) consensus recognition sequence of the 9-bp core, BRTYCWCCT (where B is a T, G, or C; R is a G or A; Y is a T or C; and W is a T or A), overlaps with the OBP(H6B) consensus YGWYCWCCY and establishes YCWCC as the roseolovirus OBP core recognition sequence. Heteroduplex analysis suggests that OBP(H7) interacts along one face of the DNA helix, with the major groove, as do OBP(H6B) and herpes simplex virus type 1 OBP. Together, these results illustrate both conserved and divergent DNA binding properties between OBP(H7) and OBP(H6B).

Amino Acid Sequence↗
PubMed1995

A homozygous nonsense mutation in the alpha 3 chain gene of laminin 5 (LAMA3) in lethal (Herlitz) junctional epidermolysis bullosa.

The inherited mechanobullous disorder, junctional epidermolysis bullosa (JEB), is characterized by extensive blistering and erosions of the skin and mucous membranes. The diagnostic hallmarks of JEB include ultrastructural abnormalities in the hemidesmosomes of the cutaneous basement membrane zone, as well as an absence of staining with antibodies against the anchoring filament protein, laminin 5. Therefore, the three genes encoding alpha 3, beta 3 and gamma 2 chains of laminin 5, known as LAMA3, LAMB3 and LAMC2, are candidate genes for JEB. We have previously demonstrated mutations in the LAMB3 and LAMC2 genes in several families with JEB. We initiated mutation analysis from an affected child by PCR amplification of individual LAMA3 exons, followed by heteroduplex analysis. Nucleotide sequencing of heteroduplexes identified a homozygous nonsense mutation within domain I/II of the alpha 3 chain. These findings provide the first evidence that nonsense mutations within the LAMA3 gene are also involved in the pathogenesis of JEB, and indicate that mutations of all three genes of laminin 5 can result in the JEB phenotype.

Base Sequence↗
PubMed2001

"Second hit" in sebaceous tumors from Muir-Torre patients with germline mutations in MSH2: allele loss is not the preferred mode of inactivation.

Muir-Torre syndrome is an autosomal-dominant inherited disorder predisposing to both sebaceous skin tumors and internal neoplasms. In a significant proportion of Muir-Torre syndrome patients skin tumors exhibit microsatellite instability as a hallmark of hereditary nonpolyposis colorectal cancer. Most individuals predisposed to hereditary nonpolyposis colorectal cancer harbor a germline mutation in the DNA mismatch repair genes MSH2 or MLH1. In Muir-Torre syndrome the vast majority of germline mutations have been identified in MSH2. Microsatellite instability in tumor tissue develops after somatic inactivation of the corresponding second mismatch repair allele ("second hit"). So far, the mechanisms of somatic inactivation of the second allele in microsatellite instability positive tumors from patients with known mismatch repair germline mutations are not well understood. We examined whether allele loss (loss of heterozygosity) is a frequent mechanism for inactivation of the second MSH2 allele in a sample of nine microsatellite instability positive skin tumors from eight unrelated Muir-Torre patients with known MSH2 germline mutations. Loss of heterozygosity was determined using microsatellite markers or heteroduplex analysis, respectively. Only one of the nine skin tumors exhibited loss of heterozygosity at the MSH2 locus. Thus, we could show in a sample of sebaceous tumors from patients with genetically proven Muir-Torre syndrome that loss of heterozygosity most probably is not the preferred mode of somatic inactivation of the second MSH2 allele.

Adult↗
PubMed2000

Screening of AP endonuclease as a candidate gene for amyotrophic lateral sclerosis (ALS).

DNA extracted from CNS tissue of 84 patients was screened by single-stranded conformation polymorphism (SSCP) and heteroduplex analysis for mutations in the apurinic/apyrimidinic endonuclease (APE) gene. One mutation was identified and characterized as a 4bp deletion in the 3'UTR. A rare polymorphism was identified in exon 3 and a common polymorphism in the coding region of exon 5. These results suggest that APE mutations do not account for a large number of ALS cases.

3' Untranslated Regions↗