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Biomedical subjects

U Landegren

Publications and source records attributed to U Landegren.

At least 19 recordsLinked to original sources
PubMed1999

Inversion of in situ synthesized oligonucleotides: improved reagents for hybridization and primer extension in DNA microarrays.

Oligonucleotides synthesized in array format suffer from contamination by truncated species. We have developed a method to invert DNA molecules in situ after completed synthesis. Reactive functions at the 5'-ends of the oligonucleotides are permitted to react with functions on the support before the 3'-ends are released, in effect reversing the orientation of full-length oligonucleotides, while any 5'-truncated molecules are lost. This strategy serves both to purify in situ synthesized reagents and to reorient the oligonucleotides, causing them to expose free 3'-hydroxyls. In situ inverted oligonucleotides can be used in assays based on DNA polymerase-assisted extension of immobilized primers, and we demonstrate their utility in minisequencing and in pyrosequencing.

Base Sequence↗
PubMed1999

[A harvest time for genomic research].

The article consists in a review of the human genome project, launched a decade ago to characterise the entire human genome. The project has proved highly successful, due both to the economy of so large scale an endeavour and to the value of gaining access to such an abundance of biological information. Accordingly, similar approaches have also been adopted in efforts to characterise the entire range of genes expressed as mRNA and as protein. Genomic information has become an invaluable asset to biomedical research, and both the information obtained and the methodology developed are now important adjuncts of pharmaceutical research. Applications in clinical medicine follow, albeit at a slower rate.

Computational Biology↗
PubMed1999

Fifth International Mutation Detection Workshop, May 13-16, 1999, Vicoforte, Italy.

The Fifth International Mutation Detection Workshop brought together inventors and major users of mutation detection methodology in a freshly refurbished 17(th) century monastery in northern Italy. There were over 120 registrants from 22 nations, all of which gave either a poster or oral presentation, making it difficult to distill the meeting into a few pages. Here we review the meeting by method type and describe highlights within each. It was clear, however, that with the imminent completion of the Human Genome Project and the recent emphasis on the utility of single nucleotide polymorphisms (SNPs), many presenters emphasized the high-throughput aspect of their methods as well as cost.

DNA Mutational Analysis↗
PubMed1999

Accessing genomic information: alternatives to PCR.

The growing abundance of genomic sequence data invites increasingly large-scale genetic analyses. Studies of genetic variation in large sets of genes can illuminate important disease mechanisms and serve to identify novel drug targets or predict therapeutic responses. At present mostly a concern in extensive research projects, large-scale genetic analyses will gradually also find their way into clinical practice as an aid to the physician. It is timely, therefore, to take stock of methods that are becoming available for analyses of large sets of gene sequences. Clearly PCR remains the workhorse for molecular genetic analysis, and several modifications such as homogenous amplification assays and parallel detection on DNA microarrays further increase throughput. Recent developments, however, also offer hope that other methods will become available for genomic investigations, providing substantially increased analytical capacity.

Genetic Techniques↗
PubMed1998

Signal amplification of padlock probes by rolling circle replication.

Circularizing oligonucleotide probes (padlock probes) have the potential to detect sets of gene sequences with high specificity and excellent selectivity for sequence variants, but sensitivity of detection has been limiting. By using a rolling circle replication (RCR) mechanism, circularized but not unreacted probes can yield a powerful signal amplification. We demonstrate here that in order for the reaction to proceed efficiently, the probes must be released from the topological link that forms with target molecules upon hybridization and ligation. If the target strand has a nearby free 3' end, then the probe-target hybrids can be displaced by the polymerase used for replication. The displaced probe can then slip off the targetstrand and a rolling circle amplification is initiated. Alternatively, the target sequence itself can prime an RCR after its non-base paired 3' end has been removed by exonucleolytic activity. We found the Phi29 DNA polymerase to be superior to the Klenow fragment in displacing the target DNA strand, and it maintained the polymerization reaction for at least 12 h, yielding an extension product that represents several thousand-fold the length of the padlock probe.

Base Sequence↗
PubMed1998

Level of heteroplasmy for the mitochondrial mutation A3243G correlates with age at onset of diabetes and deafness.

The mitochondrial mutation A3243G has been shown to be associated with a syndrome of diabetes mellitus and sensorineural hearing loss. Using a solid-phase-based sequencing method we have investigated the relation between the proportion of mutant mitochondrial genomes and the time of disease onset among members of three families where the mutation segregates. A striking association was observed between the level of heteroplasmy and time of onset of disease, particularly hearing loss. Accordingly, this syndrome shares features of diseases caused by dynamic mutations in that variable transmission of the level of heteroplasmy between generations influences disease severity.

Adolescent↗
PubMed1997

Seven-color time-resolved fluorescence hybridization analysis of human papilloma virus types.

Identification of human papilloma virus (HPV) types is important in order to determine the risk of cervical carcinoma in women. This requires a technique to probe individual samples for multiple virus specificities. Here we describe simultaneous multicolor analysis of amplification products for any of seven amplified HPV types 16, 18, 31, 33, 35, 39, and 45, associated with cancer of the cervix. A seminested polymerase chain reaction was performed in a single tube using a biotinylated inner primer. Sets of amplification products, immobilized on a 96-pronged manifold solid support, were rendered single stranded and probed with a mix of seven type-specific, differentially labeled oligonucleotides. These probes contained 10 or 20 lanthanide chelates at the 5' ends with seven distinct combinations of europium, terbium, and samarium ions. The seven viral strains were correctly identified by time-resolved fluorescence measurement of the specifically hybridized probes. Using this assay format, simultaneous detection of any of seven or even more target variants is possible.

Female↗
PubMed1997

Using PRINS for gene mapping in polytene chromosomes.

We have adapted the primed in situ labelling (PRINS) protocol for gene mapping in polytene chromosomes of two dipteran species. The method was used to localize the genes for the Balbiani ring (BR) 2.1 and the iron-regulatory protein 1A (IRP1A) in polytene salivary gland chromosomes of Chironomus tentans, and Drosophila melanogaster respectively. Two oligonucleotides, corresponding to the BR 2.1 and IRP1A genes, were used as primers and the whole procedure was performed within 3-4 h. The strong labelling with low background revealed the localization of the BR 2.1 gene in polytene chromosome IV of C. tentans and the IRP1A gene in polytene chromosome 3R83 of D. melanogaster. The results demonstrated that PRINS is a fast, sensitive and suitable approach for physical gene mapping in polytene chromosomes.

Animals↗
PubMed1997

Padlock probes reveal single-nucleotide differences, parent of origin and in situ distribution of centromeric sequences in human chromosomes 13 and 21.

Chromosome centromeres, composed of repeated DNA sequences, orchestrate the correct segregation of chromatids in cell division. We have examined the centromeres of human chromosomes 13 and 21 by studying the distribution, in situ, of two alpha satellite sequences that differ in a single nucleotide position. This was possible using padlock probes, oligo-nucleotides that can be ligated into circles upon target recognition. The segregation of individual 13 and 21 homologues in a family was followed by monitoring of the signals from two differentially labelled probes, specific for either sequence variant. A characteristic arrangement of the repeat motifs in three separate spots, oriented transverse to the length axis of the metaphase chromosomes and bilaterally symmetric, indicates that only parts of the detected regions are involved in the centromeric region, joining the sister chromatids before anaphase.

Base Sequence↗
PubMed1997

Locked on target: strategies for future gene diagnostics.

A rapidly expanding number of genetic mechanisms contributing to disease can be accessed via molecular analysis. In order to take full advantage of this potential for genetic diagnostics, and to monitor treatment by gene therapy and other emerging methods, a new generation of diagnostic techniques will be required. Of central importance in future analyses is the possibility to perform very large numbers of simultaneous genetic analyses in analytical devices of small linear dimensions. We have developed a new molecular probe design that is amenable to highly multiplex, specific analyses of total genomic DNA or of RNA molecules expressed in tissue samples. Here, a brief history of gene analytical procedures is presented, followed by a discussion of the properties, means of synthesis and applications of this new class of gene analytic reagents, padlock probes.

DNA↗
PubMed1997

X-linked recessive panhypopituitarism associated with a regional duplication in Xq25-q26.

We present a linkage analysis and a clinical update on a previously reported family with X-linked recessive panhypopituitarism, now in its fourth generation. Affected members exhibit variable degrees of hypopituitarism and mental retardation. The markers DXS737 and DXS1187 in the q25-q26 region of the X chromosome showed evidence for linkage with a peak LOD score (Zmax) of 4.12 at zero recombination fraction (theta(max) = 0). An apparent extra copy of the marker DXS102, observed in the region of the disease gene in affected males and heterozygous carrier females, suggests that a segment including this marker is duplicated. The gene causing this disorder appears to code for a dosage-sensitive protein central to development of the pituitary.

Alleles↗
PubMed1996

Synthesis of full-length oligonucleotides: cleavage of apurinic molecules on a novel support.

The synthesis of oligodeoxynucleotides is marred by several problems that contribute to the formation of defective molecules. This in turn seriously limits the usefulness of such reagents in DNA diagnostics, molecular cloning, DNA structural analysis and in antisense therapy. In particular, depurination reactions during the cyclical steps of synthesis lead to strand scission during cleavage of the completed molecules from the support. Here we present a remedy to this problem: a novel disiloxyl linkage that connects oligonucleotides to the support withstands reaction conditions that allow the removal of the 5' parts of any depurinated molecules. This ensures that all molecules that preserve the 5' protecting group when cleaved from the support will have both correct 3'- and 5'-ends. We demonstrate the application of the support for synthesis of padlock probe molecules.

Base Sequence↗
PubMed1996

Efficient detection of mutations in Wilson disease by manifold sequencing.

We have applied a solid support for parallel handling and direct loading of sequencing reactions--manifold sequencing--to analyze the coding sequence for the deficient copper transporting P-type ATPase in 24 families with Wilson disease. At least 100 different amplification reactions could be handled in parallel, with a minimal turnaround time of 12 h from isolated genomic DNA to identification of the mutations. Sixteen different mutations were found, accounting for 92% of the mutant genes. Ten of these mutations have not been previously described. Eleven were observed only in single families. Mutation His1069Gln, previously identified as the most prevalent mutation in Northern Europe, was found in one-third of the Northern European chromosomes in our material. Four patients were homozygous for this mutation, and three were homozygous for Thr977Met. The method allowed us to establish the diagnosis of Wilson disease in 24 h in a patient with acute hepatic failure.

Adenosine Triphosphatases↗
PubMed1996

Extremely sensitive, background-free gene detection using binary probes and beta replicase.

We have developed a specific and sensitive nucleic acid amplification assay that is suitable for routine gene detection. The assay is based on a novel molecular genetic strategy in which two different RNA probes are hybridized to adjacent positions on a target nucleic acid and then ligated to form an amplifiable reporter RNA. The reporter RNA is then replicated up to a hundred billion-fold in a 30-min isothermal reaction that signals the presence of the target. The assay can detect fewer than 100 nucleic acid molecules; it provides quantitative results over a wide range of target concentrations and it employs a universal format that can detect any infectious agent.

Base Sequence↗