Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-03-18
2001-10-16
Jones, W. Gary (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S005000, C435S006120, C435S091100, C435S091200, C536S023100, C536S024300, C536S032000, C536S033000, C422S068100
Reexamination Certificate
active
06303315
ABSTRACT:
BACKGROUND OF THE INVENTION
Brief Description of the Relevant Art
Organic solvents such as phenol and chloroform are traditionally used in techniques employed to isolate nucleic acid from prokaryotic and eukaryotic cells or from complex biological samples. Nucleic acid isolations typically begin with an enzymatic digest performed with proteases followed by cell lysis using ionic detergents and then extraction with phenol or a phenol/chloroform combination. The organic and aqueous phases are separated and nucleic acids which have partitioned into the aqueous phase are recovered by precipitation with alcohol. However, phenol or a phenol/chloroform mixture is corrosive to human skin and is considered as hazardous waste which must be carefully handled and properly discarded. Further, the extraction method is time consuming and laborious. Marmur, J. Mol. Biol., 3:208-218 (1961), describes the standard preparative procedure for extraction and purification of intact high molecular weight DNA from prokaryotic organisms using enzymatic treatment, addition of a detergent, and the use of an organic solvent such as phenol or phenol/chloroform. Chirgwin et al., Biochemistry, 18:5294-5299 (1979) described the isolation of intact RNA from tissues enriched in ribonuclease by homogenization in GnSCN and 2-mercaptoethanol followed by ethanol precipitation or by sedimentation through cesium chloride. Further developments of the methods are described by Ausubel et. al in Current Protocols in Molecular Biology, pub. John Wiley & Sons (1998).
Further, the use of chaotropic agents such as guanidine thiocyanate (GnSCN) is widely used to lyse and release nucleic acid from cells into solution, largely due to the fact that the chaotropic salts inhibit nucleases and proteases while at the same time facilitating the lysis of the cells.
Nucleic acid hybridisation is a known and documented method for identifying nucleic acids. Hybridization is based on base pairing of complementary nucleic acid strands. When single stranded nucleic acids are incubated in appropriate buffer solutions, complementary base sequences pair to form double stranded stable molecules. The presence or absence of such pairing may be detected by several different methods well known in the art.
In relation to the present invention a particular interesting technique was described by Dunn & Hassell in Cell, Vol.12, pages 23-36 (1977). Their assay is of the sandwich-type whereby a first hybridisation occurs between a “target” nucleic acid and a “capturing” nucleic acid probe which has been immobilized on a solid support. A second hybridisation then follows where a “signal” nucleic acid probe, typically labelled with a fluorophore, a radioactive isotope or an antigen determinant, hybridises to a different region of the immobilized target nucleic acid. The hybridisation of the signal probe may then be detected by, for example, fluorometry.
Ranki et al. in U.S. Pat. Nos. 4,486,539 and 4,563,419 and EP 0,079,139 describe sandwich-type assays which first require steps to render nucleic acids single stranded and then the single stranded nucleic acids are allowed to hybridise with a nucleic acid affixed to a solid carrier and with a nucleic acid labelled with a radioisotope. Thus, the Ranki et al. assay requires the nucleic acid to be identified or targeted in the assay to be first rendered single stranded.
One approach to dissolving a biological sample in a chaotropic solution and performing molecular hybridisation directly upon the dissolved sample is described by Thompson and Gillespie, “Analytical Biochemistry,” 163:281-291 (1987). See also WO 87/06621. Cox et al. have also described the use of GnSCN in methods for conducting nucleic acid hybridisation assays and for isolating nucleic acid from cells (EP-A-0-127-327).
Bresser, Doering and Gillespie, “DNA,” 2:243-254 (1983), reported the use of Nal, and Manser and Gefter, Proc. Natl. Acad. Sci. USA, 81:2470-2474 (1984) reported the use of NaSCN to make DNA or mRNA in biological sources available for trapping and immobilization on nitrocellulose membranes in a state which was suitable for molecular hybridisation with DNA or RNA probes.
The use of LNA as capturing-probes and detecting oligos has not been investigated until now. Due to the extraordinary features of LNA, it is possible to obtain efficient hybridisation under conditions where DNA and RNA cannot form stable hybrids e.g. pure water or buffers containing detergents and high concentrations of strong chaotropic agents. Thus, it is possible to perform the steps of catching-hybridisation, detection-hybridisation and cell-lysis in one step. This offers a substantial simplification to previous published methods.
SUMMARY OF THE INVENTION
This invention relates to compositions and assay methods for the hybridisation and extraction of nucleic acids. In particular, this invention relates to compositions and methods to release nucleic acids from cells in complex biological samples or specimens while simultaneously hybridising complementary nucleic acids released during lysis. A crucial component in the invention is LNA which is a novel class of DNA analogues that possess some extraordinary features that make it a prime candidate for improving in vitro DNA diagnostics. The LNA monomers are bi-cyclic compounds structurally very similar to RNA-monomers, see formula I. LNA shares most of the chemical properties of DNA and RNA, it is water-soluble, can be separated by gel electrophoreses, ethanol precipitated etc. Furthermore, LNA oligonucleotides are conveniently synthesised by standard phosphoramidite chemistry. The phosphoramidite chemistry allows chimeric oligos containing both LNA and DNA (or RNA) monomers to be synthesized. Thus, mixed LNA/DNA oligos with a predefined melting temperature (T
m
) can be prepared. The flexibility of the phosphoramidite synthesis approach furthermore facilitates the easy production of LNAs carrying all commercially available linkers, fluorophores and labelling-molecules available for this standard chemistry. Importantly, introduction of LNA monomers into either DNA or RNA oligos results in unprecedented high thermal stability of duplexes with complementary DNA or RNA while at the same time obeying the Watson-Crick base-pairing rules. In general the thermal stability of hetero-duplexes is increased 3-8° C. per LNA-monomer in the duplex. To the best of our knowledge LNA has the highest affinity towards complementary DNA or RNA ever to be reported (Tetrahedron, 54, 3607-3630 (1998)). The thermal stability of LNA/DNA and LNA/RNA heteroduplexes is sufficiently stable to allow efficient hybridisation to occur even in the presence of chaotropic agents such as guanidine thiocyanate (GnSCN).
This invention relates to novel methods for the release of nucleic acids from cells in complex biological samples or specimens to prepare and make available the nucleic acid material present for a hybridisation assay. Novel methods for hybridisation of nucleic acids are also presented. In particular, methods are described for the hybridisation of nucleic acids from a sample suspected of containing a target nucleic acid of interest wherein the sample is combined with a buffer comprising at least one strong chaotropic agent which promotes cell lysis and release of the cellular nucleic acid while at the same time allowing hybridisation with LNA. The extent of hybridisation of the complementary nucleic acid to the target nucleic acid is then determined.
One advantage of these hybridisation methods is that hybridisation may be carried out in one easy step with all reagents pre-combined.
DETAILED DESCRIPTION
This invention relates to a novel method for detecting nucleic acids released from a lysed complex biological mixture containing nucleic acids. The methods of the present invention enable one to readily assay for a nucleic acid suspected of being present in cells, parts of cells or virus, i.e. target nucleic acid(s). Such methods include lysing the cells in a hybridisation medium comprising a strong chaotropic agent, contacting the lysate
Corless Peter F.
Edwards & Angell LLP
Exiqon A/S
Jones W. Gary
Taylor Janell E.
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