| 1. |
- Dahlén, P, et al.
(författare)
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Sensitive detection of genes by sandwich hybridization and time-resolved fluorometry
- 1987
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Ingår i: Molecular and Cellular Probes. - : Elsevier BV. - 0890-8508 .- 1096-1194. ; 1:2, s. 159-168
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Tidskriftsartikel (refereegranskat)abstract
- Europium has been used as a non-radioactive marker in immunoassays as this metal can be detected with high sensitivity by time-resolved fluorometry. In this work streptavidin labeled with europium was used to detect biotinylated probes in a sandwich nucleic-acid hybridization assay with microtitration strips as the solid phase. pBR 322 plasmids were detected with a sensitivity of 4 × 105 molecules. As the sample is added in solution in sandwich hybridization, fast and simple sample pre-treatment can be used without encountering background problems. The method was applied to test bacterial samples of uropathogenic Escherichia coli strains for the presence of the β-lactamase gene.
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| 2. |
- Jungell-Nortamo, A, et al.
(författare)
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Nucleic acid sandwich hybridization : enhanced reaction rate with magnetic microparticles as carriers
- 1988
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Ingår i: Molecular and Cellular Probes. - : Elsevier BV. - 0890-8508 .- 1096-1194. ; 2:4, s. 281-288
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Tidskriftsartikel (refereegranskat)abstract
- A method for the detection of nucleic acid hybrids using the sandwich hybridization technique with magnetic polystyrene microparticles as the solid support is described. The capture DNA is coupled to the polystyrene-hydroxy surface of the particles through p-toluenesulfonyl chloride activation. The use of microparticles results in a substantial increase in the reaction rate compared to filter hybridization, without decreasing the sensitivity of detection. Polyethylene glycol additionally enhances the reaction rate. The use of magnetic microparticles allows rapid and convenient collection of the formed hybrids.
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| 3. |
- Parkkinen, S, et al.
(författare)
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Sandwich hybridization in solution : a rapid method to screen HPV 16 DNA in cervical scrapes
- 1989
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Ingår i: Molecular and Cellular Probes. - : Elsevier BV. - 0890-8508 .- 1096-1194. ; 3:1, s. 1-11
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Tidskriftsartikel (refereegranskat)abstract
- A solution hybridization method is introduced as a rapid diagnostic method for demonstration of papillomavirus DNA in cervical scrapes. 32P-Labelled detector probe and the biotinylated capture probes were hybridized with DNA of the specimen after pretreatment by boiling in alkaline SDS. After 4 h of hybridization the hybrids were collected onto avidin coated beads and measured. The sensitivity of the method was 1–5 × 105 HPV 16 DNA molecules. Cervical carcinoma cell lines CaSki and SiHa were informative as to the sensitivity of the solution hybridization and the in situ hybridization methods. CaSki cells containing about 700 HPV 16 DNA copies per cell were positive by both methods. SiHa cells with one HPV 16 DNA copy per cell were positive by the sandwich assay but remained negative in the in situ test. A series of 126 cervical scrapes collected from consecutive patients participating in a follow-up study for cervical HPV infection were tested for HPV 16 DNA by both methods. The detection rate of the sandwich test was 19/126 (15%) and that of the in situ method 21/126 (17%) yielding 26 diagnoses altogether. Twelve of these were obtained by one method only. The results obtained by studying the cervical cell lines and repeated specimens taken from constantly HPV 16 positive patients suggested that the two methods can measure different types of infections and thus complement each other in the diagnosis of cervical HPV infections.
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| 4. |
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| 5. |
- Syvänen, Ann-Christine, et al.
(författare)
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A complex of single-strand binding protein and M13 DNA as hybridization probe
- 1985
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Ingår i: Nucleic Acids Research. - 0305-1048 .- 1362-4962. ; 13:8, s. 2789-2802
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Tidskriftsartikel (refereegranskat)abstract
- Single-stranded DNA was complexed to the single-strand binding protein (SSB) of Escherichia coli in a mass ratio of 30:1. The protein moiety of this complex can be labelled by a number of methods of which we have chosen radio-iodination and biotinylation as examples. The SSB-M13 DNA complexes, labelled to high specific activities, were used as probes in hybridization experiments in which 1.6 X 10(-18) moles of immobilized target DNA were detected. The stability of the hybrids was not severely decreased by the binding of SSB. Analysis of hybrids by electron microscopy showed that complexing of DNA with SSB could be used to allow its subsequent identification in the hybrids.
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| 6. |
- Syvänen, Ann-Christine, et al.
(författare)
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Direct sequencing of affinity-captured amplified human DNA application to the detection of apolipoprotein E polymorphism
- 1989
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Ingår i: FEBS Letters. - : Wiley. - 0014-5793 .- 1873-3468. ; 258:1, s. 71-74
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Tidskriftsartikel (refereegranskat)abstract
- We describe a method for the direct sequencing of DNA amplified by the polymerase chain reaction (PCR). Biotin is introduced into one strand of the amplified DNA using a 5'-biotinylated PCR primer. The synthesized fragment is captured on an avidin-matrix and rendered single stranded, whereafter the nucleotide sequence of the immobilized strand is determined by the chain termination method. The method involves few and simple operations and is thus applicable to the analysis of human genes for routine diagnostic purposes. Here we applied the method for determination of the three-allelic polymorphism of the apolipoprotein E (apo E) gene. We were able to correctly identify the alleles in both homozygous and heterozygous samples.
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| 7. |
- Syvänen, Ann-Christine, et al.
(författare)
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Fast quantification of nucleic acid hybrids by affinity-based hybrid collection
- 1986
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Ingår i: Nucleic Acids Research. - 0305-1048 .- 1362-4962. ; 14:12, s. 5037-5048
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Tidskriftsartikel (refereegranskat)abstract
- A hybridization technique for the quantification of nucleic acids is described. In the method a probe pair is allowed to form hybrids with the target nucleic acid in solution. One of the probes has been modified with an affinity label, by which the formed hybrids can be isolated after the reaction. Streptavidin-agarose was used to capture hybrids containing biotinylated DNA. The hybrids were measured using radioiodine as label on the second probe. The rate of the hybridization reaction in solution is fast, allowing the whole procedure to be carried out in 3 h. The method is quantitative with a detection limit of 4 X 105molecules (0.67 attomoles) target DNA. The test is insensitive to impurities in biological samples, which are analyzed without purification of the target DNA. Non-isotopic measurement of the hybrids can also be applied. In this case the hybrids are bound to microtitration wells and detected spectrophotometrically by peroxidase-catalyzed colour development.
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| 8. |
- Syvänen, Ann-Christine
(författare)
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Nucleic acid hybridization : from research tool to routine diagnostic method
- 1986
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Ingår i: Medical biology. - 0302-2137. ; 64:6, s. 313-324
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Tidskriftsartikel (refereegranskat)abstract
- The nucleic acid hybridization reaction is extremely specific and thus a valuable tool for the identification of genes or organism of interest. The increasing use of nucleic acid hybridization in applied fields like diagnostic medicine has led to the development of more convenient hybridization assays than those originally used in basic research. In conventional nucleic acid hybridization methods immobilized nucleic acids are detected on a filter by a radiolabelled probe. Sandwich hybridization is a simple test format for the analysis of unpurified biological material, but has the disadvantage of a slow reaction rate. Solution hybridization methods are fast and easy to perform provided that a method to separate the formed hybrids from the reaction mixture is available. In non-isotopic detection the nucleic acid probe is modified with a chemical group, which is identified with a labelled detector molecule after hybridization. The low sensitivity of detection is the main problem in nucleic acid hybridization methods. Procedures to amplify the detectable signal or the amount of detectable nucleic acid sequences are potential solutions to this problem. The new hybridization methods have successfully been used for some applications, but still need to be combined into well performing tests to be applicable to any desired purpose.
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| 9. |
- Syvänen, Ann-Christine, et al.
(författare)
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Quantification of polymerase chain reaction products by affinity-based hybrid collection
- 1988
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Ingår i: Nucleic Acids Research. - 0305-1048 .- 1362-4962. ; 16:23, s. 11327-11338
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Tidskriftsartikel (refereegranskat)abstract
- We have used oligonucleotides modified with biotin in the 5'-end as primers in the polymerase chain reaction (PCR)-amplification. This results in the synthesis of 5'-biotinylated DNA molecules, which are detected by hybridization to a labelled probe in solution. The formed hybrids are collected on an avidin-matrix by mediation of the biotin residue of the target molecules. The affinity-based hybrid collection method is quantitative and makes it possible to measure the amount of DNA produced in the PCR-amplification. At low concentrations of template the efficiency of the process is close to 100%, making it possible to detect the presence of a few molecules of target DNA in 25 cycles. With high template concentrations the efficiency of the process is low.
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| 10. |
- Syvänen, Ann-Christine, et al.
(författare)
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Time-resolved fluorometry : a sensitive method to quantify DNA-hybrids
- 1986
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Ingår i: Nucleic Acids Research. - 0305-1048 .- 1362-4962. ; 14:2, s. 1017-1028
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Tidskriftsartikel (refereegranskat)abstract
- Europium and other lanthanides can be excitated with UV-radiation, whereafter the energy is released as fluorescence, delayed in time up to 1 ms after the excitation. Eu can be used as a sensitive label in biological assays. Here we report on the application of time-resolved fluorometry to detect nucleic acid hybrids. The probe DNA was tagged with a hapten, either a fluorene or a sulfone group. After hybridization the probe DNA was detected by a two-step immunological assay with the second antibody labelled with Eu. The method is quantitative with a detection limit of 0.3 pg of actual target regions of immobilized adenovirus genomic DNA. The label was also used in sandwich hybridization, which allowed analyzing nasopharyngeal mucus for the presence of adenovirus.
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