WO2001092568A2 - Method for producing dna-arrays - Google Patents

Abstract

The invention relates to a method for analyzing nucleic acids. A surface is provided with islands of DNA of the same variety, i.e. tertiary nucleic acids; the tertiary nucleic acids are brought into contact with a probe or a mixture of several probes to enable hybridization to occur between the tertiary nucleic acids and the probe or probes; the tertiary nucleic acids and/or probes which are localized at points where a differential hybridization event occurs are separated from other nucleic acids and/or probes where no differential hybridization event is localized.

Claims

- 39 - Patent claims Method for nulein acid analysis, whereby (a) a surface is provided which has islands of number acids of the same type, tertiary number acids; (b) the tertiary nuclear acids are brought into contact with a probe or a mixture of several probes in such a way that hybridization between the tertiary Nucleic acids and the probe or probes can take place; (c) the tertiary nucleic acids and/or probes located at locations where a differential hybridization event is located, from the other nucleic acids and/or probes located at locations where no differential hybridization event is located, located. Method according to claim 1, wherein in step (a) (al) a surface is provided to which at least primer molecules of a first primer and a second primer and optionally a nucleic acid mixture comprising the nucleic acid molecules with which both primers can hybridize have been irreversibly immobilized, both primers forming a primer pair; (a2) nucleic acid molecules of the nucleic acid mixture are hybridized with one or with both primers of the same primer pair; (a3) the irreversibly immobilized primer molecules are extended complementary to the counterpart strand to form secondary nucleic acids; (a4) the surface is provided in a form freed from nuclear acid molecules that are not bound to the surface by irreversible immobilization; (a5) the secondary nuclear acids are amplified to form tertiary nuclear acids. - 40 - Method according to claim 2, wherein in step (al) a surface to which at least one primer pair forming primer molecules have been irreversibly immobilized is provided. Method according to claim 2, characterized in that according to Sliritt (a5) the following steps are carried out: (a6) The surface of step (a5), the first surface, is with a second Surface brought into contact on which primer molecules, which form at least one primer pair, are immobilized, under conditions in which the primer molecules of the second surface can hybridize with the tertiary nucleic acids of the first surface; (a7) the immobilized primer molecules of the second surface are extended complementary to the counterpart strand to form secondary nucleic acids; (a8) the nucleic acid molecules not bound to the second surface by the immobilization in step (a6) are removed from the second surface, if necessary; (a9) the secondary nucleic acids are amplified to obtain tertiary nucleic acids; (alO) ^" Steps (a6) to (a9) are repeated analogously until the desired number of prepared surfaces has been produced; (al 1) the surfaces produced in step (alO) are subjected to steps (b) and (c). . Method according to one of claims 1 to 4, characterized in that in step (c) the probes and/or nucleic acids which have caused a differential hybridization event are detached from the surface. Method according to claim 5, characterized in that in step (c) the probes and/or NuMeic acids that have caused a differential hybridization event are released from the surface by photolytic cleavage of the hybridization partner. Method according to claim 5, characterized in that in step (c) the probes and/or NuMeic acids which have caused a differential hybridization event, can be detached from the surface by a local increase in temperature. 41. Method according to claim 5, characterized in that in step (c) the probes and/or nucleic acids which have not caused a differential hybridization event are bound to the surface either as a result of thermal action or by cross-linking. . Compound of general formula I

where n and m independently mean 1 to 30, R ¹ means dimethoxytrityloxy (DMTO) or a group which allows immobilization to a surface or which allows coupling to an immobilizable compound, and R ² has the meaning -OP(OC ₂ H ₄ CN)N(CH(CH ₃ ) ₂ ) ₂ or -OP(OCH ₃ )N(CH(CH ₃ ) ₂ ) ₂₅

carries, as well as their salts. 10. A compound according to claim 9, characterized in that R ¹ has the meaning dimethoxytrityloxy (DMTO), -NH ₂ , -OH, -OPO ₃ H ₂ , -SH, -NCS, -NGO or N-succinimidyloxycarbonyl. 11. Use of the compounds according to claim 9 or 10 as a photochemically cleavable group in the immobilization of nuclear acids. 12. Device for carrying out a method according to one of claims 1 to 8, comprising (a) a reaction and hybridization chamber in the form of a flow cell, - 42 - (b) at least one light source for stimulating fluorophores used for marble formation, (c) at least one light source for photolysis to cleave bonds intended, (d) optionally at least one light source for photochemical or thermal modification of nuclear acid or probe molecules, (e) at least one photodeteMor for measuring the emitted fluorescent radiation, (f) optionally a device for scanning the surface in the flow cell in the XY direction, (g) optionally at least one storage vessel for fresh reaction solutions and at least one vessel for used reaction solutions, as well as at least one Device for liquid conveyance, (h) if necessary an electronic computer, which stores the measured fluorescence intensity depending on the position on the surface, if necessary further processes the data sets obtained in this way and if necessary makes an automatic selection of the areas of interest on the surface as well as the intended effect of electromagnetic devices Radiation controls.