Patent EP-2706124-A1 (EP)
Inventor: WEISHAUPT SONJA
IPC: C12Q1/68
Filed: 2013-08-30
Abstract: Differentiated detection of at least two different first and second RNA molecules, comprises: e.g. providing a sample solution which has at least two different first and second RNA molecules, primer complementary-Zip (cZip) x-polynucleotide and a primer complementary-Zip y-polynucleotide, and a nucleotide microarray; adenylating first and second RNA molecules; carrying out hybridization of first and second adenylated RNA molecule; hybridizing primer-cZip x- and primer-cZip y-polynucleotide with first and second cZip; and detecting first and second RNA molecules through fluorescence detection. Differentiated detection of at least two different first and second RNA molecules, which are present in sample solution, and respectively comprise identical polynucleotide sequence, comprises: (a) providing a sample solution which has at least two different first and second RNA molecules, where the first RNA molecule with first polynucleotide sequence, and the second RNA molecule with the second polynucleotide sequence has a nucleotide element immediately at closing 5' direction; (b) providing primer complementary-Zip (cZip) x-polynucleotide having a primer nucleotide sequence and a complementary-Zip x-nucleotide sequence primer having complementary-Zip (cZip) x-polynucleotide, where the primer nucleotide sequence is complementary to a terminal 5' portion of the polynucleotide sequence, and a primer complementary-Zip y-polynucleotide having primer nucleotide sequence and complementary-Zip y-nucleotide sequence is provided; (c) providing a nucleotide microarray comprising at least two different first and second complementary-Zips immobilized on a nucleotide microarray substrate at different positions, where the first cZip is complementary to the x-nucleotide sequence, and the second cZip is complementary to the nucleotide sequence; (d) adenylating the first and second RNA molecules present in the solution from the step (a), where a solution containing first and second adenylated RNA molecules is obtained; (e) separating the solution comprising first and second adenylated RNA molecules obtained in the step (d) in a first solution and a second solution; (f) placing the first solution with the primer-cZip x-polynucleotide and carrying out hybridization with the first and second adenylated RNA molecule provided in step (b), where a third solution (A1) is obtained; (g) placing the second solution with the primer-cZip y-polynucleotide and carrying out hybridization with the first and second adenylated RNA molecule provided in step (b), where a fourth (B1) solution is obtained; (h) placing the third solution obtained in the step (f) with at least a first nucleoside triphosphate which is labeled with a fluorescent dye, where the first nucleoside triphosphate is deoxythymidine triphosphate or deoxyuridine triphosphate and carrying out an enzymatic strand elongation of the hybridized primer-cZip x-polynucleotide to obtain fifth solution (A2); (i) placing the fourth solution obtained in the step (g) with at least a second nucleoside triphosphate which is labeled with a fluorescent dye, where the first nucleoside triphosphate is deoxyguanosine triphosphate, deoxyadenosine triphosphate and deoxycytidine triphosphate and carrying out an enzymatic strand elongation of the hybridized primer-cZip y-polynucleotide to obtain sixth solution (B2); (j) contacting the fifth solution A2 and sixth solution obtained in steps (h) and (i) with nucleotide micro array provided in the step (c) and performing a hybridization of the primer-cZip x- and primer-cZip y-polynucleotide with the first and second cZip; and (k) detecting of at least two different first and second RNA molecules through a fluorescence detection. An independent claim is also included for a kit for differentiated detection of at least two different first and second RNA molecules, which are present in sample solution, comprising a nucleotide microarray comprising at least two different first and second complementary-Zips, having primer complementary-Zip (cZip) x-polynucleotide having a primer nucleotide sequence and a c-Zip x-nucleotide sequence, and a primer complementary-Zip y-polynucleotide having primer nucleotide sequence and complementary Zip y-nucleotide sequence.