Biomedical subjects

A Kurg

Publications and source records attributed to A Kurg.

7 recordsLinked to original sources

PubMed2000

Arrayed primer extension: solid-phase four-color DNA resequencing and mutation detection technology.

The technology and application of arrayed primer extension (APEX) is presented. We describe an integrated system with DNA chip and template preparation, multiplex primer extension on the array, fluorescence imaging, and data analysis. The method is based upon an array of oligonucleotides, immobilized via the 5' end on a glass surface. A patient DNA is amplified by PCR, digested enzymatically, and annealed to the immobilized primers, which promote sites for template-dependent DNA polymerase extension reactions using four unique fluorescently labeled dideoxy nucleotides. A mutation is detected by a change in the color code of the primer sites. The technology was applied to the analysis of 10 common beta-thalassemia mutations. Nine patient DNA samples, each of which carries a different mutation, and four wild-type DNA samples were correctly identified. The signal-to-noise ratio of this technology is, on the average, 40:1, which enables the identification of heterozygous mutations with a high confidence level. The APEX method can be applied to any DNA target for efficient analysis of mutations and polymorphisms.

DNA↗

PubMed2000

Unravelling genetic data by arrayed primer extension.

We have developed a method for arrayed primer extension (APEX) on an oligonucleotide microchip together with the 4-color fluoresence imaging equipment and supporting software, that allows analysis of the DNA sequence and changes in it. Mutation analysis of BRCA1 gene and single nucleotide polymorphism (SNP) chip for genotyping were used as a model system. Chip surface chemistry, template preparation and APEX reaction conditions were optimised and the assay is ready to be implemented in variety of DNA analysis from SNP testing to DNA resequencing.

Alleles↗

PubMed1997

Provirus variants of the bovine leukemia virus and their relation to the serological status of naturally infected cattle.

Infection of cattle with the bovine leukemia virus (BLV) results in a strong permanent antibody response to the BLV antigens some weeks after infection. However, cattle may carry provirus and not have detectable antibody titers. To prove the occurrence of different BLV provirus variants in German cattle and to study the influence of special BLV variants on the immunoreaction, a 444-bp fragment of the env gene of 35 naturally BLV infected animals was analyzed. Seven different groups of BLV provirus variants were found on the basis of restriction fragment length polymorphism. Three BLV provirus variant groups and five additionally sequenced BLV isolates showed a high similarity to BLV provirus isolates from other geographical areas. The variation in nucleotide sequence of the five BLV isolates compared with nine previously sequenced BLV isolates ranged up to 5. 3%. While BLV provirus variant groups A, C, D, E, F, and G were clearly related to agar-gel immunodiffusion test (AGID)- and enzyme-linked immunosorbent assay (ELISA)-positive animals, BLV provirus variant group B was solely found in permanent AGID- and ELISA-negative or in transient ELISA-positive animals. Altogether, these results indicate that special BLV provirus variants may be responsible for atypical forms of BLV infection in cattle.

Amino Acid Sequence↗

PubMed1997

Minisequencing: a specific tool for DNA analysis and diagnostics on oligonucleotide arrays.

We describe a method for multiplex detection of mutations in which the solid-phase minisequencing principle is applied to an oligonucleotide array format. The mutations are detected by extending immobilized primers that anneal to their template sequences immediately adjacent to the mutant nucleotide positions with single labeled dideoxynucleoside triphosphates using a DNA polymerase. The arrays were prepared by coupling one primer per mutation to be detected on a small glass area. Genomic fragments spanning nine disease mutations, which were selected as targets for the assay, were amplified in multiplex PCR reactions and used as templates for the minisequencing reactions on the primer array. The genotypes of homozygous and heterozygous genomic DNA samples were unequivocally defined at each analyzed nucleotide position by the highly specific primer extension reaction. In a comparison to hybridization with immobilized allele-specific probes in the same assay format, the power of discrimination between homozygous and heterozygous genotypes was one order of magnitude higher using the minisequencing method. Therefore, single-nucleotide primer extension is a promising principle for future high-throughput mutation detection and genotyping using high density DNA-chip technology.

DNA-Directed DNA Polymerase↗

PubMed1996

Evaluation of polymerase chain reaction (PCR) application in diagnosis of bovine leukaemia virus (BLV) infection in naturally infected cattle.

The practical application of polymerase chain reaction (PCR) for the diagnosis of bovine leukaemia virus (BLV) infections in naturally infected cattle was evaluated. Compared to serological tests the PCR was definitely found to be a more sensitive method, yielding the highest number of positive results (10% more compared to enzyme-linked immunosorbent assay, (ELISA), and 17.7% more compared to agar-gel immunodiffusion, (AGID)). In testing cattle from herds with BLV incidence under 5%, out of 52 provirus positive cattle only 43 were correctly identified by ELISA. When compared to AGID only 37 of the 52 PCR positive animals were correctly identified. Of 18 cattle imported from the Slovak Republic and kept in a quarantine stable, four were found to be BLV provirus positive by PCR, while serological tests indicated one animal positive and three negative. Therefore, it is impossible to prevent the spread of the infection from one country to another by serological testing only. Moreover, it is feasible to identify animals with changing antibody titres correctly by PCR. Using PCR we were also able to distinguish BLV infected from uninfected calves that were serologically positive due to colostral antibodies. Higher sensitivity of BLV provirus detection by PCR was achieved using env gene rather than tax gene specific primers. Negative results by PCR in cases of positive serological reactions are still possible, as shown in case of one adult animal. These findings indicate that PCR is a highly sensitive method and might be successfully used and economically advantageous for different practical applications in detection of BLV infection in naturally infected cattle.

Animals↗

PubMed1995

An RNA stem-loop structure involved in the packaging of bovine leukemia virus genomic RNA in vivo.

An RNA secondary structure of the bovine leukemia virus (BLV) 5'-terminal RNA sequence was constructed by computer-assisted RNA secondary structure analysis. Mutations were created in the noncoding region (NCR) of BLV, which contains a conserved consensus sequence, to disrupt predicted secondary structure of this region. After transfection of these constructs into FLK-BLV cells and analysis of viral particles a reduction in mutant RNA content was observed relative to that of unmutated vector RNA. The packaging efficiency of the mutant with a substitution in the consensus sequence was reduced threefold and that of the mutant with a deleted 5' NCR was reduced fivefold. We conclude that predicted RNA secondary structure and/or nucleotide sequence of the BLV noncoding region is essential for BLV RNA packaging in vivo.

Animals↗

PubMedDate not supplied

Direct use of cell lysates in PCR-based diagnosis of bovine leukemia virus infection.

Polymerase chain reaction (PCR) has been used for direct detection of bovine leukemia virus (BLV) proviral DNA in cattle, but it is still mainly used for experimental research. One bottleneck for routine diagnosis of BLV by PCR has always been the isolation and purification of DNA. We compare the use of not purificated with highly-purified DNA in the PCR-based diagnosis of BLV infection. DNA extracted from whole blood by chloroform extraction (CP-DNA) and DNA prepared only by osmotic shock, washing, heating and freezing procedures (RPoS-DNA), were utilized. Fifteen cattle well characterized serologically were investigated for BLV-provirus with PCR using this different DNA preparations. With both methods all but one investigated animal were correctly identified. It was estimated that in case of CP-DNA PCR 10 BLV-provirus copies were sufficient to obtain a positive result. The sensitivity of RPoS-DNA PCR was similar. Because of the greater practicability of the latter technique we used it in a small field study with ten cattle. All serologically positive animals were correctly identified by the PCR. In addition one seronegative animal was found to carry BLV-provirus. Therefore RPoS-DNA PCR might be a good tool for the routine diagnosis of BLV-infected cattle.

Animals↗