Biomedical subjects

J Lundeberg

Publications and source records attributed to J Lundeberg.

At least 55 records · Page 3Linked to original sources

PubMed1999

Quantitative investigation of the modular primer effect for DNA and peptide nucleic acid hexamers.

The effect on oligonucleotide-template duplex stability upon cohybridization of adjacently annealing oligonucleotides, the modular primer effect, was studied with biosensor technology. DNA and peptide nucleic acid (PNA) hexamer modules and sensor chip-immobilized template DNA strands were designed for analysis of nick, overlap, and gap modular hybridization situations. The fast hybridization kinetics for such hexamer modules allowed for the determination of apparent duplex affinities from equilibrium responses. The results showed that the hybridizational stability of modular hexamer pairs is strongly dependent on the positioning, concentration, and inherent affinity of the adjacently annealing hexamer module. Up to 80-fold increases in apparent affinities could be observed for adjacent modular oligonucleotide pairs compared to affinities determined for single hexamer oligonucleotide hybridizations. Interestingly, also for coinjections of different module combinations where DNA hexamer modules were replaced by their PNA counterparts, a modular primer effect was observed. The introduction of a single base gap between two hexamer modules significantly reduced the stabilization effect, whereas a gap of two bases resulted in a complete loss of the effect. The results suggest that the described biosensor-based methodology should be useful for the selection of appropriate modules and working concentrations for use in different modular hybridization applications.

Biosensing Techniques↗

PubMed1999

Analyses of secondary structures in DNA by pyrosequencing.

A common problem in conventional DNA sequencing is the occurrence of DNA sequence compressions during gel electrophoresis, leading to misreading of the sequence. These compressions are usually due to secondary structures in the DNA fragment. In this study, we present a non-gel-based DNA sequencing technique that facilitates analysis of such DNA regions. A part of the polymorphic pertussis toxin promoter region in five different Bordetella species was successfully resolved by the new technique. The obtained sequence data revealed four related palindromic sequences. The ability of different DNA polymerases to read through such secondary structures is also described.

Base Sequence↗

PubMed1999

A high frequency of sequence alterations is due to formalin fixation of archival specimens.

Genomic analysis of archival tissues fixed in formalin is of fundamental importance in biomedical research, and numerous studies have used such material. Although the possibility of polymerase chain reaction (PCR)-introduced artifacts is known, the use of direct sequencing has been thought to overcome such problems. Here we report the results from a controlled study, performed in parallel on frozen and formalin-fixed material, where a high frequency of nonreproducible sequence alterations was detected with the use of formalin-fixed tissues. Defined numbers of well-characterized tumor cells were amplified and analyzed by direct DNA sequencing. No nonreproducible sequence alterations were found in frozen tissues. In formalin-fixed material up to one mutation artifact per 500 bases was recorded. The chance of such artificial mutations in formalin-fixed material was inversely correlated with the number of cells used in the PCR-the fewer cells, the more artifacts. A total of 28 artificial mutations were recorded, of which 27 were C-T or G-A transitions. Through confirmational sequencing of independent amplification products artifacts can be distinguished from true mutations. However, because this problem was not acknowledged earlier, the presence of artifacts may have profoundly influenced previously reported mutations in formalin-fixed material, including those inserted into mutation databases.

Base Sequence↗

PubMed1999

Solid-phase differential display and bacterial expression systems in selection and functional analysis of cDNAs.

Differential gene expression can be expected during activation and differentiation of cells as well as during pathological conditions, such as cancer. A number of strategies have been described to identify and understand isolated differentially expressed genes. The differential display methodology has rapidly become a widely used technique to identify differentially expressed mRNAs. In this chapter we described a variant of the differential display method based on solid-phase technology. The solid-phase procedure offers an attractive alternative to solution-based differential display because minute amounts of sample can be analyzed in considerably less time than previously. The employed solid support, monodisperse super paramagnetic beads, which circumvents precipitation and centrifugations steps, has also allowed for optimization of the critical enzymatic and preparative steps in the differential display methodology. We also described how bacterial expression can be used as a means to elucidate gene function. An efficient dual-expression system was presented, together with a basic concept describing how parallel expression of selected portions of cDNAs can be used for production of cDNA-encoded proteins as parts of affinity-tagged fusion proteins. The fusion proteins are suitable both for the generation of antibodies reactive to the target cDNA-encoded protein and for the subsequent affinity enrichment of such antibodies. Affinity-enriched antibodies have proved to be valuable tools in various assays, including immunoblotting and immunocytochemical staining, and can thus be used to localize the target cDNA-encoded protein to certain cells in a tissue section or even to a specific cell compartment or organelle within a cell. High-resolution localization of a cDNA-encoded protein would provide valuable information toward the understanding of protein function.

Animals↗

PubMed1999

Selection analysis of single-stranded clones using solid-phase techniques.

An efficient selection analysis of single-stranded DNA fragments using subtractive hybridization on magnetic beads and fluorescence-based fragment analysis is described. This approach facilitates either direct cloning of captured fragments or removal of common clones from cDNA or shot-gun clone libraries.

Animals↗

PubMed1999

Mutational scanning of PCR products by subtractive oligonucleotide hybridization analysis.

Here, we describe a new approach for mutational scanning of PCR products through hybridization analysis between complementary oligonucleotides. Sets of overlapping probe oligonucleotides complementary to wild-type (WT) sequence are hybridized to microbead-immobilized PCR products under solution-like conditions. Mismatch-hybridization situations between a mutant sample and probe oligonucleotides result in higher remaining concentrations in solution of involved probe oligonucleotides. Post-hybridization supernatants are subsequently analyzed for their probe oligonucleotide compositions using surface plasmon resonance-based biosensor technology. Relative remaining probe oligonucleotide concentrations are monitored in real-time through hybridization analysis between probe oligonucleotides and their corresponding sensor-chip immobilized complementary counterparts. This allows for the construction of composition diagrams revealing the existence and approximate location of a mutation within an investigated sample DNA sequence. Applied on PCR products derived from clinical samples of microdissected tumor biopsies, single mutations in exons 6 and 7 of the human p53 tumor-suppressor gene were successfully detected and approximately localized.

Biosensing Techniques↗

PubMed1999

p53 Mutations in lung cancer associated with residential radon exposure.

Unusual mutation patterns in lung tumors among underground miners have been indicated, suggesting radon-specific alterations in the genome, but the data are not consistent. To investigate the association between residential radon exposure and p53 mutations in lung tumors, we performed a study on cases from a nation-wide population-based investigation in Sweden. Our study included 83 nonsmoking lung cancer cases and 250 smoking lung cancer cases, diagnosed 1980-1984, with a time-weighted average radon exposure over 140 Bq/m3 or up to 50 Bq/m3. Radon was measured in dwellings occupied by the study subjects at some time since 1947. Information on smoking habits and other risk factors was obtained from questionnaires. After exclusions because of the initiation of treatment or insufficient material, the p53-status of 243 tumors was determined using PCR-single-stranded conformation polymorphism analysis and sequencing determination of exons 5-8. The overall mutation prevalence was 23.9%. An increased mutation prevalence was suggested among those with high exposure to residential radon [odds ratio (OR), 1.4; 95% CI, 0.7-2.6], especially among nonsmokers (OR, 3.2; 95% CI, 0.7-15.5), but no specific mutational pattern was indicated. Furthermore, the mutation prevalence seemed to be higher among smoking lung cancer cases than among nonsmoking cases (OR, 2.1; 95% CI, 0.9-5.0), and particularly among those smoking less than 10 cigarettes per day. It may be concluded that residential exposure to radon seems to contribute to a higher mutation prevalence of the p53 gene in lung tumors.

Adenocarcinoma↗

PubMed1999

Recovery of upstream cDNA sequences by a PCR-based biotin-capture method.

The world-wide, large-scale sequencing efforts have generated an abundance of partial cDNA sequences, i.e., expressed sequence tags (ESTs), accessible in the public databases. To enable functional characterization of these partial cDNA sequences, general and robust methods for recovery of upstream full-coding cDNA sequences are needed. Here, a novel biotin- and PCR-assisted capture method was used directly on poly(A)+ RNA for the purpose of generating a full-coding sequence of a gene with only partially known sequence and for which a full-length clone of the gene was not found in existing cDNA libraries. The presented method involves linear extension by reverse transciptase from a biotinylated primer annealing in a region with known sequence. After capture of the generated single-stranded cDNA onto paramagnetic beads, unspecifically annealing primers, i.e., arbitrary primers, were used to generate cDNA fragments that could be amplified by PCR and thereafter directly sequenced without subcloning. By using the presented strategy, which is to be seen as a complement to rapid amplification of cDNA ends (RACE)-related methods, we were able to recover full-coding sequence versions of two potential splice variants of the target gene. The general applicability of the novel method for recovery and sequencing of cDNA sequences is discussed.

Animals↗

PubMed1999

Forensic evidence based on mtDNA from dog and wolf hairs.

In six forensic cases involving murder, bank robbery, theft and poaching, evidence material comprising shed hairs supposedly originating from dogs or wolves was analyzed by mitochondrial (mt) DNA sequencing. A 79 bp segment of the control region was amplified, sequenced, and compared with an established database of the domestic dog and wolf populations. In three murder cases exclusions of all eight suspects could be made. Furthermore, two of the murders could be linked to each other by a rare sequence variant, and the breed of the dog was indicated. In a theft case and a bank robbery a link could be established between the evidence material and the suspects. In a case of suspected wolf poaching, it could be established that the evidential material was of dog rather than wolf origin. We conclude that single hairs from common pets are suitable for DNA analysis and that the described method has proved to be a valuable tool for forensic investigations.

Animals↗

PubMed1998

Gene discovery in the wood-forming tissues of poplar: analysis of 5, 692 expressed sequence tags.

A rapidly growing area of genome research is the generation of expressed sequence tags (ESTs) in which large numbers of randomly selected cDNA clones are partially sequenced. The collection of ESTs reflects the level and complexity of gene expression in the sampled tissue. To date, the majority of plant ESTs are from nonwoody plants such as Arabidopsis, Brassica, maize, and rice. Here, we present a large-scale production of ESTs from the wood-forming tissues of two poplars, Populus tremula L. x tremuloides Michx. and Populus trichocarpa 'Trichobel.' The 5,692 ESTs analyzed represented a total of 3,719 unique transcripts for the two cDNA libraries. Putative functions could be assigned to 2,245 of these transcripts that corresponded to 820 protein functions. Of specific interest to forest biotechnology are the 4% of ESTs involved in various processes of cell wall formation, such as lignin and cellulose synthesis, 5% similar to developmental regulators and members of known signal transduction pathways, and 2% involved in hormone biosynthesis. An additional 12% of the ESTs showed no significant similarity to any other DNA or protein sequences in existing databases. The absence of these sequences from public databases may indicate a specific role for these proteins in wood formation. The cDNA libraries and the accompanying database are valuable resources for forest research directed toward understanding the genetic control of wood formation and future endeavors to modify wood and fiber properties for industrial use.

Arabidopsis↗

PubMed1998

Genetic instability in the 9q22.3 region is a late event in the development of squamous cell carcinoma.

Squamous cell carcinoma (SCC) of the skin represents a group of neoplasms which is associated with exposure to UV light. Recently, we obtained data suggesting that invasive skin cancer and its precursors derive from one original neoplastic clone. Here, the analysis were extended by loss of heterozygosity (LOH) analysis in the chromosome 9q22.3 region. A total of 85 samples, taken from twenty-two sections of sun-exposed sites, corresponding to normal epidermis, morphological normal cells with positive immuno-staining for the p53 protein (p53 patches), dysplasias, cancer in situ (CIS) and squamous cell carcinomas (SCC) of the skin were analysed. Overall, about 70% of p53 patches had mutations in the p53 gene but not LOH in the p53 gene or 9q22.3 region. Approximately 70% of the dysplasias showed p53 mutations of which about 40% had LOH in the p53 region but not in the 9q22.3 region. In contrast, about 65% of SCC and CIS displayed LOH in the 9q22.3 region, as well as frequent (80%) mutations and/or LOH in the p53 gene. These findings strongly suggest that alterations in the p53 gene is an early event in the progression towards SCC, whereas malignant development involves LOH and alterations in at least one (or several) tumor suppressor genes located in chromosome 9q22.3.

Carcinoma in Situ↗

PubMed1998

Cloning and characterization of ZNF189, a novel human Krüppel-like zinc finger gene localized to chromosome 9q22-q31.

A 3-kb-long cDNA encoding a Krüppel-like human zinc finger protein was isolated and mapped to chromosome 9q22-q31. The ZNF189 gene encodes a protein with 16 zinc fingers at its C-terminus and belongs to the Krüppel-associated box (KRAB)-containing group of zinc finger proteins. Four differently spliced cDNA transcripts, differing at the 5' coding region where a KRAB A repressor domain is encoded, were isolated. In addition, Northern blot analysis indicates the presence of two additional unidentified splice variants. Comparison of cDNA and genomic sequences shows that the ZNF189 gene spans approximately 11 kb and is organized into at least four exons, the large 3'-end exon coding for the complete zinc finger domain and the 3' untranslated region. ZNF189 is expressed in all tissues and cell types currently investigated, at varying levels, but with a tissue- or cell-type-restricted expression pattern for the different splice variants. ZNF189 is conserved in the genome of several mammalian species. Direct sequencing of the ZNF189 gene in microdissected tumor biopsies of sporadic basal cell carcinoma and squamous cell carcinoma reveals no mutations in the coding sequence or at exon/intron boundaries.

Amino Acid Sequence↗

PubMed1998

Clones of normal keratinocytes and a variety of simultaneously present epidermal neoplastic lesions contain a multitude of p53 gene mutations in a xeroderma pigmentosum patient.

A patient with xeroderma pigmentosum group C was extensively examined for mutations in the p53 gene in normal skin exposed to varying degrees of sunlight and in excisional biopsies of basal cell cancer, squamous cell cancer, and squamous cell dysplasia. Seventy-three samples were analyzed by microdissection of small cell clusters, followed by PCR and direct DNA sequencing. In skin taken from areas that most likely had never been exposed to the sun, no mutations were found. However, in skin exposed to the sun, we observed a multitude of mutations in the p53 gene. UV light-induced mutations were found in all types of lesions, as well as in clusters of morphologically normal epidermal cells. Twenty-nine distinct mutations were found in exons 5-8, all missense or nonsense, of which 27 (93%) were UV-specific C --> T or CC --> TT transitions at dipyrimidine sites of the nontranscribed strand. Two types of normal skin areas containing p53 mutations were observed: areas that stain strongly with p53 antibody (p53 patches) and those that do not stain. Because no silent or intron mutations were found in these cell clusters, the alterations in the p53 gene of morphologically normal cells are likely to have resulted in a selective growth advantage. The poor correlation between mutations and morphological phenotypes demonstrates that p53 mutations alone do not determine the phenotypes observed.

Adolescent↗

PubMed1998

Direct sequencing of bacterial artificial chromosomes (BACs) and prokaryotic genomes by biotin-capture PCR.

Determination of unknown DNA sequences adjacent to known segments is an important task in genome-related research. We have applied the methodology of biotin-capture PCR for direct sequencing of bacterial artificial chromosomes (BACs) and bacterial genomes. The strategy involves extension of a biotinylated primer from a known locus into unknown regions of the template to yield single-stranded DNA, which is immobilised onto paramagnetic beads. An arbitrary primer initiates extension from the unknown region and back towards the known locus. The arbitrary primer contains a universal primer 'handle', which is utilised for subsequent amplification. The PCR products are then directly sequenced by solid-phase or cycle sequencing. The fact that BACs or bacterial chromosomes can be sequenced without prior purification or subcloning might be useful in numerous applications, such as gap-filling, sequencing of regulatory regions upstream known genes and determination of intron/exon-boundaries.

Base Sequence↗

PubMed1998

Capture of single-stranded DNA assisted by oligonucleotide modules.

Real-time biospecific interaction analysis was employed to monitor direct capture of a hepatitis C virus (HCV) derived polymerase chain reaction (PCR) product by nucleic acid hybridization. Different formats for hybridization were used to study the interaction between a single-stranded HCV PCR product and capture oligonucleotides immobilized on a sensor chip via streptavidin-biotin chemistry. By employing a prehybridization step in solution with nonbiotin oligonucleotides complementary to the single-stranded target and adjacent to the immobilized probe, a significant capture was achieved in comparison to the low capture efficiency obtained using single immobilized probes (9-36 mer). High capture efficiencies were also observed when shorter immobilized probes were used in combination with strings of adjacently positioned prehybridized probes (i.e., modules). Interestingly, the introduction of single nucleotide gaps between prehybridized and/or immobilized probes dramatically reduced the capture efficiency. These results suggest that flexible systems for capture could be designed from libraries of short oligonucleotides (9 mers) used in module fashion, taking advantage of stacking interactions between the oligonucleotides. The potential applications of such oligonucleotide-assisted capture systems are discussed.

DNA Primers↗

PubMed1998

Variation in the hepatitis C virus NS5a region in relation to hypervariable region 1 heterogeneity during interferon treatment.

The putative interferon sensitivity determining region (ISDR) in the NS5a region of the hepatitis C virus (HCV) was analyzed in 13 interferon alpha (IFN-alpha) treated patients representing genotypes 1a, 1b, and 2b. These patients had previously been followed longitudinally during treatment with respect to viral load and to virus heterogeneity using the hypervariable region 1 (HVR1) sequence as a marker. In the present study, the NS5a region was analyzed for nonresponders and sustained responders using direct DNA sequencing. While the previous results of analyzing viral composition and load showed evidence of selection, no corresponding selection of specific NS5a ISDR sequences was observed in the nonresponders, and identical ISDR sequences were observed among both sustained responders and nonresponders. Thus, we cannot verify a correlation between ISDR sequence and the observed selection of IFN-alpha-resistant quasispecies demonstrated as a restriction of HVR1 heterogeneity. This indicates that the potential for using ISDR as a diagnostic or prognostic marker during IFN-alpha treatment is limited.

Amino Acid Sequence↗

PubMed1998

Use of multiple competitors for quantification of human immunodeficiency virus type 1 RNA in plasma.

Quantification of human immunodeficiency virus type 1 (HIV-1) RNA in plasma has rapidly become an important tool in basic HIV research and in the clinical care of infected individuals. Here, a quantitative HIV assay based on competitive reverse transcription-PCR with multiple competitors was developed. Four RNA competitors containing identical PCR primer binding sequences as the viral HIV-1 RNA target were constructed. One of the PCR primers was fluorescently labeled, which facilitated discrimination between the viral RNA and competitor amplicons by fragment analysis with conventional automated sequencers. The coamplification of known amounts of the RNA competitors provided the means to establish internal calibration curves for the individual reactions resulting in exclusion of tube-to-tube variations. Calibration curves were created from the peak areas, which were proportional to the starting amount of each competitor. The fluorescence detection format was expanded to provide a dynamic range of more than 5 log units. This quantitative assay allowed for reproducible analysis of samples containing as few as 40 viral copies of HIV-1 RNA per reaction. The within- and between-run coefficients of variation were <24% (range, 10 to 24) and <36% (range, 27 to 36), respectively. The high reproducibility (standard deviation, <0.13 log) of the overall procedure for quantification of HIV-1 RNA in plasma, including sample preparation, amplification, and detection variations, allowed reliable detection of a 0.5-log change in RNA viral load. The assay could be a useful tool for monitoring HIV-1 disease progression and antiviral treatment and can easily be adapted to the quantification of other pathogens.

Binding, Competitive↗

PubMed1998

Cooperative oligonucleotides mediating direct capture of hepatitis C virus RNA from serum.

A novel method for direct capture of hepatitis C virus (HCV) RNA from clinical samples has been developed. This approach takes advantage of the cooperative interactions between adjacently hybridized oligonucleotides. Here, this cooperative effect was combined with solid-phase technology, whereby a capture probe was covalently coupled to magnetic beads and a second probe, which anneals adjacent to the capture probe site, was prehybridized in solution to the target. When these contiguously hybridized probes were used for the extraction of HCV RNA from clinical samples, the capture efficiency was increased up to 25-fold in comparison to capture with a single probe. The applicability of this sample preparation assay was further investigated by performing a comparative study with both a conventional guanidinium extraction method and a commercial quantitative assay.

Biosensing Techniques↗