Patent ID: 8551708

Claim:
A method for localized in situ detection of mRNA which codes for one or more mutations of the KRAS gene in a sample of cells that were formalin-fixed paraffin-embedded (FFPE) on a slide surface, comprising: (a) generating cDNA from mRNA in the sample using a primer; (b) digesting the mRNA hybridized to said cDNA; (c) contacting said sample with one or more padlock probes specific for mutations to the KRAS gene; (d) joining, directly or indirectly, the ends of said padlock probe(s) to form circularized padlock probe(s); (e) subjecting said circularized padlock probe(s) to rolling circle amplification (RCA) to generate rolling circle amplification products; and, (f) detecting the rolling circle amplification product(s), wherein the padlock probe comprises: (i) Y1-X1-Z1-A (ii) Y1-X1-Z1-T (iii) Y1-X1-Z1-C (iv) Y2-X1-Z2-A (v) Y2-X1-Z2-T (vi) Y2-X1-Z2-C (vii) Y3-X1-Z3-A; (viii) Y1-X2-Z1-G (ix) Y2-X2-Z2-G, or (x) Y3-X2-Z3-G where: X1 or X2 is from 16-50 nucleotides; Y1+Z1=20 to 29 nucleotides; Y2+Z2=20 to 29 nucleotides; Y3+Z3=20 to 29 nucleotides; Y1 is GTGGCGTAGGCAAGA (SEQ ID NO:1), GTGGCGTAGGCAAG (SEQ ID NO:2), GTGGCGTAGGCAA (SEQ ID NO:3), GTGGCGTAGGCA (SEQ ID NO:4), GTGGCGTAGGC (SEQ ID NO:5), GTGGCGTAGG (SEQ ID NO:6), GTGGCGTAG, GTGGCGTA, GTGGCGT, or GTGGCG; Y2 is TGGCGTAGGCAAGAG (SEQ ID NO:7), TGGCGTAGGCAAGA (SEQ ID NO:8), TGGCGTAGGCAAG (SEQ ID NO:9), TGGCGTAGGCAA (SEQ ID NO:10), TGGCGTAGGCA (SEQ ID NO:11), TGGCGTAGGC (SEQ ID NO:12), TGGCGTAGG, TGGCGTAG, TGGCGTA, or TGGCGT; Y3 is CGTAGGCAAGAGTGC (SEQ ID NO:13), CGTAGGCAAGAGTG (SEQ ID NO:14), CGTAGGCAAGAGT (SEQ ID NO:15), CGTAGGCAAGAG (SEQ ID NO:113), CGTAGGCAAGA (SEQ ID NO:114), CGTAGGCAAG (SEQ ID NO:115), CGTAGGCAA, CGTAGGCA, CGTAGGC, or CGTAGG; Z1 is TGGTAGTTGGAGCT (SEQ ID NO:27), GGTAGTTGGAGCT (SEQ ID NO:28), GTAGTTGGAGCT (SEQ ID NO:29), TAGTTGGAGCT (SEQ ID NO:30), AGTTGGAGCT (SEQ ID NO:31), GTTGGAGCT, TTGGAGCT, TGGAGCT, GGAGCT, or GAGCT; Z2 is GGTAGTTGGAGCTG (SEQ ID NO:16), GTAGTTGGAGCTG (SEQ ID NO:17), TAGTTGGAGCTG (SEQ ID NO:18), AGTTGGAGCTG (SEQ ID NO:19), GTTGGAGCTG (SEQ ID NO:20), TTGGAGCTG, TGGAGCTG, GGAGCTG, GAGCTG, or AGCTG; and Z3 is AGTTGGAGCTGGTG (SEQ ID NO:21), GTTGGAGCTGGTG(SEQ ID NO:22), TTGGAGCTGGTG(SEQ ID NO:23), TGGAGCTGGTG(SEQ ID NO:24), GGAGCTGGTG(SEQ ID NO:25), GAGCTGGTG(SEQ ID NO:26), AGCTGGTG, GCTGGTG, CTGGTG, or TGGTG.