| 1. |
- Göransson, Jenny, et al.
(författare)
-
Rapid Identification of Bio-Molecules Applied for Detection of Biosecurity Agents Using Rolling Circle Amplification
- 2012
-
Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 7:2, s. e31068-
-
Tidskriftsartikel (refereegranskat)abstract
- Detection and identification of pathogens in environmental samples for biosecurity applications are challenging due to the strict requirements on specificity, sensitivity and time. We have developed a concept for quick, specific and sensitive pathogen identification in environmental samples. Target identification is realized by padlock- and proximity probing, and reacted probes are amplified by RCA (rolling-circle amplification). The individual RCA products are labeled by fluorescence and enumerated by an instrument, developed for sensitive and rapid digital analysis. The concept is demonstrated by identification of simili biowarfare agents for bacteria (Escherichia coli and Pantoea agglomerans) and spores (Bacillus atrophaeus) released in field.
|
|
| 2. |
- Howell, W. Mathias, et al.
(författare)
-
Glycosylases and AP-cleaving enzymes as a general tool for probe-directed cleavage of ssDNA targets
- 2010
-
Ingår i: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 38:7, s. e99-
-
Tidskriftsartikel (refereegranskat)abstract
- The current arsenal of molecular tools for site-directed cleavage of single-stranded DNA (ssDNA) is limited. Here, we describe a method for targeted DNA cleavage that requires only the presence of an A nucleotide at the target position. The procedure involves hybridization of a complementary oligonucleotide probe to the target sequence. The probe is designed to create a deliberate G:A mismatch at the desired position of cleavage. The DNA repair enzyme MutY glycosylase recognizes the mismatch structure and selectively removes the mispaired A from the duplex to create an abasic site in the target strand. Addition of an AP-endonuclease, such as Endonuclease IV, subsequently cleaves the backbone dividing the DNA strand into two fragments. With an appropriate choice of an AP-cleaving enzyme, the 3'- and 5'-ends of the cleaved DNA are suitable to take part in subsequent enzymatic reactions such as priming for polymerization or joining by DNA ligation. We define suitable standard reaction conditions for glycosylase/AP-cleaving enzyme (G/AP) cleavage, and demonstrate the use of the method in an improved scheme for in situ detection using target-primed rolling-circle amplification of padlock probes.
|
|
| 3. |
- Weibrecht, Irene, et al.
(författare)
-
Proximity ligation assays : a recent addition to the proteomics toolbox
- 2010
-
Ingår i: Expert Reviews of Proteomics. - 1478-9450. ; 7:3, s. 401-409
-
Forskningsöversikt (refereegranskat)abstract
- An essential skill for every researcher is to learn how to select and apply the most appropriate methods for the questions they are trying to answer. With the extensive variety of methods available, it is increasingly important to scrutinize the advantages and disadvantages of these techniques prior to making a decision on which to use. In this article, we describe an approach to evaluate methods by reducing them into subcomponents. This is exemplified by a brief description of some commonly used proteomics methods. The same approach can also be used in method development by rearranging subcomponents in order to create new methods, as demonstrated with the development of proximity ligation assays (PLAs). PLA is a method as designed in our laboratory for detection of proteins, protein-protein interactions and post-translational modifications. Fundamentally, protein-recognition events are converted into detectable DNA molecules. The technique uses protein DNA conjugates as binders for the targets of interest. Binding of two or more conjugates to the target results in assembly of an assay-specific DNA molecule. Subsequent amplification of the DNA molecule generates a signal that can be detected using PCR, for detection of minute amounts of proteins in serum, or standard fluorescence microscopy for detection of protein protein interactions in tissue sections. Lastly, we apply the approach of recombining subcomponents to develop a few novel hypothetical methods hoping this might stimulate the readers to utilize this approach themselves.
|
|
