The isolation and purification of nucleic acids from biological and clinical sample material is of crucial importance for fields of work in which operating techniques based on nucleic acids are employed, or in which technologies based on nucleic acids are actually the key to access. Examples include paternity analysis, tissue typing, identification of hereditary diseases, genome analysis, molecular diagnostics, determination of infectious diseases, animal and plant breeding, transgenic research, basic research in biology and medicine, as well as numerous related areas. In general, a difficulty is encountered in preparing biological or clinical sample materials in such a manner that the nucleic acids contained in them can be used directly in a desired analytical procedure.
The state of the art already includes many processes for the purification of DNA. For example, we know how to purify plasmid DNA for the purpose of cloning—and other experimental processes as well—according to the method of Birnboim (Methods in Enzymology, 100: 243 (1983)). In this process, a cleared lysate of bacterial origin is exposed to a cesium chloride gradient and centrifuged for a period of 4 to 24 hours. This step is usually followed by the extraction and precipitation of the DNA. This process is associated with the disadvantages that it is very apparatus-intensive, and it takes a great deal of time, is expensive to run and cannot be automated.
Other methods in which cleared lysates are used to isolate DNA are based on ion-exchange chromatography (e.g., Colpan et al., J. Chromatog., 296:339 (1984)) and gel filtration (e.g., Moreau et al., Analyt. Biochem., 166:188 (1987)). These processes are primarily alternatives to the cesium chloride gradients; however they require an extensive solvent supply system, and a precipitation of the DNA fractions is necessary, since these usually contain salts in high concentrations and are extremely diluted solutions.
Marko et al. Analyt. Biochem., 121:382 (1982), and Vogelstein et al., Proc. Nat. Acad. Sci., 76:615 (1979), have found that if the DNA from extracts containing nuclei acids is exposed to high concentrations of sodium iodide or sodium perchlorate, the DNA alone will adhere to small glass scintillation tubes, fiberglass membranes or fiberglass sheets that have been finely particulated by mechanical means, while RNA and proteins do not. The DNA that has been bound in this manner can be eluted, for example, with water.
For example, in international publication WO 87/06621, the immobilization of nucleic acids on a PVDF membrane is described. However, the nucleic acids bound to the PVDF membrane are not eluted in the next step; instead the membrane, together with all the bound nucleic acids is introduced directly into a PCR reaction. Finally, in this international patent application and in the other literature, it is stated that hydrophobic surfaces or membranes must in general be wetted beforehand with water or alcohol, in order to be able to immobilize the nucleic acids with yields that are satisfactory.
On the other hand, for a number of modern applications, such as, for example, the PCR, reversed transcription PCR, SunRise, LCR, branched-DNA, NASBA, or TaqMan technologies and similar real-time quantification methods for PCR, SDA, DNA and RNA chips and arrays for gene expression and mutation analyses, differential display analyses, RFLP, AFLP, cDNA synthesis or substractive hybridization, it is absolutely necessary to be able to release the nucleic acids directly from the solid phase. In this connection, WO 87/06621 teaches that, while the nucleic acids can indeed be recovered from the membranes used in the process, this recovery is fraught with problems and is far from suited to the quantitative isolation of nucleic acids. In addition, the nucleic acid obtained in this manner is, comparatively, extremely diluted, which makes subsequent isolation and concentration steps absolutely necessary.
For the reasons stated above, the processes known from the state of the art do not constitute—particularly with regard to automation of the process for obtaining nucleic acids—a suitable starting point for an isolation of nucleic acid that is as simple and productive as possible from the point of view of process engineering.
USPTO Backgrounds
USPTO Backgrounds