Method for the specific determination of DNA sequences by means of parallel amplification

Method for the specific determination of DNA sequences by means of parallel amplification in a combined liquid-phase/solid-phase-DNA-microarray system by performing a nested polymerase chain reaction using x PCR primer sets each of at least 3 PCR primers P11, P21, P31, P12, P22, P32, P13, P23, P33, . . . P1x, P2x, P3x, whereby x signifies a positive integer and corresponds to the number of DNA sequences to be determined, and whereby for each of the x PCR primer sets

• (a) two outer PCR primers P1, P2 are chosen in such a way that they hybridise onto DNA sub-sequences lying upstream and downstream of the target DNA sequence that is to be amplified,
• (b) one inner PCR primer P3 is chosen in such a way that the one inner PCR primer P3 hybridises onto a DNA sub-sequence lying within the target DNA sequence that is to be determined, and is able to form a P3 extension product, and
• (c) the x outer PCR primers P11, P21, P12, P22, P13, P23, . . . P1x, P2x are present in liquid phase in excess relative to the x inner PCR primers P31, P32, P33, . . . P3x, and
• (d) the x inner PCR primers P31, P32, P33, . . . P3x are present irreversibly bonded to a solid phase at x spatially separated defined positions, forming a DNA microarray;
• the method comprising the steps of
• (1) providing the x PCR primer sets;
• (2) performing simultaneous amplification, involving a nested polymerase chain reaction, using said primers in both the liquid and solid phase in order to produce a P3 extension product irreversibly bonded to the solid phase, wherein the x outer PCR primers P11, P21, P12, P22, P13, P23, . . . P1x, P2x in the liquid phase are present in a 102 to 1012 -fold excess relative to the x inner PCR primers P31, P32, P33, . . . P3x in the solid phase;
• and
• (3) ascertaining the presence of the P3 extension product at one or more defined positions of the DNA microarray,
• wherein the solid phase, to which the x inner PCR primers P31, P32, P33, . . . P3x are irreversibly bonded, is a surface.

Method for the specific determination of DNA sequences by means of parallel amplification in a combined liquid-phase/solid-phase-DNA-micro array system by performing a nested polymerase chain reaction using x PCR primer sets each of at least 3 PCR primers P11, P21, P31, P12, P22, P32, P13, P23, P33, . . . P1x, P2x, P3x, whereby x signifies a positive integer and corresponds to the number of DNA sequences to be determined, and whereby for each of the x PCR primer sets

• (a) two outer PCR primers P1, P2 are chosen in such a way that they hybridise onto DNA sub-sequences lying upstream and downstream of the target DNA sequence that is to be amplified,
• (b) one inner PCR primer P3 is chosen in such a way that the one inner PCR primer P3 hybridises onto a DNA sub-sequence lying within the target DNA sequence that is to be determined, and is able to form a P3 extension product, and
• (c) the x outer PCR primers P11, P21, P12, P22, P13, P23, . . . P1x, P2x are present in solution in excess relative to the x inner PCR primers P31, P32, P33, . . . P3x, and
• (d) the x inner PCR primers P31, P32, P33, P3x are present irreversibly bonded to a solid phase at x spatially separated defined positions, forming a DNA microarray;
• the method comprising the steps of
• (1) providing the x PCR primer sets;
• (2) performing simultaneous amplification, involving a nested polymerase chain reaction, using said primers in both the solution and the solid phase in order to produce a P3 extension product irreversibly bonded to the solid phase, wherein the x outer PCR primers P11, P21, P12, P22, P13, P23,. . . P1x, P2x in the solution are present in a 102 to 102-fold excess relative to the x inner PCR primers P31, P32, P33, . . . P3x in the solid phase;
• and
• (3) ascertaining the presence of the P3 extension product at one or more defined positions of the DNA microarray,
• wherein the solid phase, to which the x inner PCR primers P31, P32, P33, . . . P3x are irreversibly bonded, is a non-porous surface.

Method for the specific determination of DNA sequences by means of parallel amplification in a combined liquid-phase/solid-phase-DNA-microarray system by performing a nested polymerase chain reaction using x PCR primer sets each of at least 3 PCR primers P11, P21, P31, P12, P22, P32, P13, P23, P33, . . . P1x, P2x, P3x, whereby x signifies a positive integer and corresponds to the number of DNA sequences to be determined, and whereby for each of the x PCR primer sets

• (a) two outer PCR primers P1, P2 are chosen in such a way that they hybridise onto DNA sub-sequences lying upstream and downstream of the target DNA sequence that is to be amplified,
• (b) one inner PCR primer P3 is chosen in such a way that the one inner PCR primer P3 hybridises onto a DNA sub-sequence lying within the target DNA sequence that is to be determined, and is able to form a P3 extension product, and
• (c) the x outer PCR primers P11, P21, P12, P22, P13, P23, P1x, P2x are present in solution in excess relative to the x inner PCR primers P31, P32, P33, P3x, and
• (d) the x inner PCR primers P31, P32, P33, . . . P3x are present irreversibly bonded to a solid phase at x spatially separated defined positions, forming a DNA microarray;
• the method comprising the steps of
• (1) providing the x PCR primer sets;
• (2) performing simultaneous amplification, involving a nested polymerase chain reaction, using said primers in both the solution and the solid phase in order to produce a P3 extension product irreversibly bonded to the solid phase, wherein the x outer PCR primers P11, P21, P12, P22, P13, P23, . . . P1x, P2x in the solution are present in a 102 to 1012-fold excess relative to the x inner PCR primers P31, P32, P33, . . . P3x in the solid phase;
• and
• (3) ascertaining the presence of the P3 extension product at one or more defined positions of the DNA microarray,
• wherein the solid phase, to which the x inner PCR primers P31, P32, P33, . . . P3x are irreversibly bonded, is a non-porous surface, wherein the non-porous surface is a glass surface and the x inner PCR primers P31, P32, P33, . . . P3x are irreversibly bonded to the glass surface via a bifunctional silane that has one, two or three hydrolysable atom(s) or group(s) on the silicon atom.