Patent ID: 7611871

Claim:
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 P 1 1, P 2 1, P 3 1, P 1 2, P 2 2, P 3 2, P 1 3, P 2 3, P 3 3, . . . P 1 x, P 2 x, P 3 x, 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 P 1 , P 2 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 P 3 is chosen in such a way that the one inner PCR primer P 3 hybridises onto a DNA sub-sequence lying within the target DNA sequence that is to be determined, and is able to form a P 3 extension product, and (c) the x outer PCR primers P 1 1, P 2 1, P 1 2, P 2 2, P 1 3, P 2 3, . . . P 1 x, P 2 x are present in liquid phase in excess relative to the x inner PCR primers P 3 1, P 3 2, P 3 3, . . . P 3 x, and (d) the x inner PCR primers P 3 1, P 3 2, P 3 3, . . . P 3 x 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 P 3 extension product irreversibly bonded to the solid phase, wherein the x outer PCR primers P 1 1, P 2 1, P 1 2, P 2 2, P 1 3, P 2 3, . . . P 1 x, P 2 x in the liquid phase are present in a 10 2 to 10 12 -fold excess relative to the x inner PCR primers P 3 1, P 3 2, P 3 3, . . . P 3 x in the solid phase; and (3) ascertaining the presence of the P 3 extension product at one or more defined positions of the DNA microarray.