Mats NilssonDepartment of Genetics & Pathology, Uppsala University. Home Page
ResearchIn the post-genome era it has become evident that the methods available for analysis of individual macromolecules are not adequate for the analysis of the tens of thousand different genes, transcripts and proteins that are necessary to explain human health and disease. My research aims at developing some of the required analysis tools. The developed techniques will allow analysis at the ultimate level of single nucleic acid molecules, through padlock probing and rolling-circle replication, enabling determination of quantities and location of specific nucleic acids in situ; quantities and identity of specifically reacted probes in solution; and sequence composition of DNA samples. The basic molecular device that will be used is the padlock probe (Nilsson et al. 1994), a reagent with specificity matching that of PCR. Unlike PCR, the padlock probe allows large numbers of probes to be combined in a single reaction, which has allowed the company ParAllele that I have co-founded to genotype 20 000 single nucleotide polymorphisms in single reactions using pools of padlock probes. In an analogous reaction, large sets of genomic DNA fragments can specifically be circularized and amplified using so called selector probes (Dahl et al. submitted).
Single-molecule analysis in situ. Rolling-circle replicated padlock probes spontaneously form micron-sized coils that can be identified by hybridization of fluorescence labeled tag-oligonucleotides (Blab et al. 2004), and be observed in a microscope both in situ and in vitro. Therefore, individual padlock probes that have been circularized on individual target sequences turn into easily observable rolling-circle replication product that can be counted and positioned within cells and tissue sections (Larsson et al. 2004). The technique will further be developed to allow studies of allelic imbalances in expressed genes. The digital mode of quantification will require improved image analysis tools which will be developed in collaboration with the Centre for Image Analysis, Uppsala University.
Single-molecule analyses in vitro. In a FOI-supported project, integrated microfluidic devices are developed, suitable for use in field or at the bed-side. The device will be custom designed to identify and count individual rolling-circle products in a simple and rapid analysis format, with high dynamic range, enhanced quantitative precision compared to real-time PCR assays, and enabling intermediary levels of multiplexing. A high-density single-molecule array analysis platform will further be developed in an Uppsala BioX financed project, in which reacted probes or genomic fragments can be analysed in an ultra high-throughput format. Extreme sensitivity can be obtained by applying a circle-dependent DNA amplification method that we recently have developed (Dahl et al. 2004).
Five Selected PublicationsNilsson, M., Malmgren, H., Samiotaki, M., Kwiatkowski, M.,
Chowdhary, B.P. & Landegren, U. Padlock probes: Circularizing
oligonucleotides for localized DNA detection. Science 265, 2085-2088
Nilsson, M., Barbany, G., Antson, D.-O., Gertow, K. & Landegren, U. Enhanced detection and distinction of RNA by enzymatic probe ligation. Nature Biotechnol. 18, 791-793 (2000).
Dahl, F., Baner, J., Gullberg, M., Mendel-Hartvig, M., Landegren, U. & Nilsson, M. Circle-to-circle amplification for precise and sensitive DNA analysis. Proc. Natl. Acad. Sci. U S A 101, 4548-4553 (2004).
Blab, G.A., Schmidt, T. & Nilsson, M. Sensitive and homogenous detection of single rolling-circle replication products. Anal. Chem. 76, 495-498 (2004).
Larsson, C., Koch, J., Nygren, A., Janssen, G., Raap, A.K., Landegren, U. & Nilsson, M. In situ genotyping individual DNA molecules by target-primed rolling-circle amplification of padlock probes. Nature Meth. 1, 227-232 (2004).