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==Cancer Padlock Probes + RT Primer== *The cancer padlock probes from Illumina are actually made to capture cDNA, not primary rolonies (probably by accident) **[[Media:Cancer_mutation_probes.docx | Cancer mutation probes]] ***BRAF looks correct but the mRNA sequence (gray highlight) is actually cDNA sequence ***EGFR has correct mRNA sequence but the padlock probe matches it, not reverse complementary ===New Padlock Probes=== *Use same barcode as old padlock probes **Each barcode contains 3 sites to hybridize decodingProbe *Padlock Probe design: H1 + Code1 + Code2 + CommonLinker + Code3 + H2 **CommonLinker (for ppRCA): CTTCAGCTTCCCGATATCCGACGG **[[Hosuk:FISSEQ_Primer_information#Agi26k_Probeset_Barcode-to-dcProbe_Key | Code sequences]] ***(10201) = dcProbe0-FAM, dcProbe2-Cy3, dcProbe4-FAM ====BRAF==== *mRNA sequence 1,821-1,900: 5β-ACCTCACAGTAAAAA(TAGGTGATTTTGGTCTAGCTACAG[T])GAAATCT(CGATGGAGTGGGTCCCATCAGTT)TGAACAGTTG-3β **() indicate where padlock probe hybridizes to **[] indicates SNP *H1: [A/T]CTGTAGCTAGACCAAAATCACCTA *H2: AACTGATGGGACCCACTCCATCG *BRAF_V600E_WT (10101): /5Phos/ACTGTAGCTAGACCAAAATCACCTA+ATGTATCGCGCTCGATTGGCAG+TAGAACTTGCGCGGATACACGG+CTTCAGCTTCCCGATATCCGACGG+TCGGAATACGTCGTTGACTGCG+AACTGATGGGACCCACTCCATCG *BRAF_V600E_MU (20202): /5Phos/TCTGTAGCTAGACCAAAATCACCTA+ACGTATCGGTAGTCGCAACGCA+TCTACTTCGTCGCGTCAGACCA+CTTCAGCTTCCCGATATCCGACGG+CTACCATTCGCGTGCGATTCCA+AACTGATGGGACCCACTCCATCG ====EGFR==== *mRNA sequence 2,591-2,660:5β-TCACCT(CCACCGTGCAGCTCATCA[C])GCAGCT(CATGCCCTTCGGCTGCCTCCT)GGACTATGTCCGGGAACA-3β **() indicate where padlock probe hybridizes to **[] indicates SNP *H1: [G/A]TGATGAGCTGCACGGTGG *H2: AGGAGGCAGCCGAAGGGCATG *EGFR_T790M_WT (01011): /5Phos/GTGATGAGCTGCACGGTGG+GTCTTGCGTGCGATACGGAGTA+CGAATTGTCCGCGCTCTACGAT+CTTCAGCTTCCCGATATCCGACGG+TCGGAATACGTCGTTGACTGCG+AGGAGGCAGCCGAAGGGCATG *EGFR_T790M_MU (02022): /5Phos/ATGATGAGCTGCACGGTGG+CAACGGTATTCGGTCGTCATCC+CCGTTTGATCGTTCGACCGAGA+CTTCAGCTTCCCGATATCCGACGG+CTACCATTCGCGTGCGATTCCA+AGGAGGCAGCCGAAGGGCATG ===RT Primer=== *>15nt length for specificity *Downstream of padlock probe capture sites *Not too many C's and G's with Tm >42C **Increase RT reaction temperature from 37C to 42C to increase specificity ***[http://www.enzymatics.com/products/m-mulv-reverse-transcriptase/ Enzyme is functional at 42C] *Include RCA primer /5Phos/TCTCGGGAACGCTGAAGA- **Can find region with sequence that also hybridizes a few bases with RCA primer (ie. TCTTC...) *Primer to stop RTase (May not stop M-MuLV because it has [http://www.jbc.org/content/273/16/9976.long Strand Displacement Activity]) **Try Short ~200nt & ~500nt cDNA by annealing primer "upstream" on mRNA of RT primer site **Don't include /5Phos/ so CircLigase will not circularize it **Tm slightly above 42C to keep specific **Try to avoid A's adjacent to primer in mRNA sequence since those could turn into aa-dUTP on cDNA and become fixed, thus decreasing the likelihood of circLigase to ligate ends together ====BRAF V600E: 1,900+ [http://www.ncbi.nlm.nih.gov/nuccore/NM_004333.4 BRAF, mRNA]==== *mRNA sequence 1,981-2,000: TGCATTTGGAATTGTTCTGT *RT primer: /5Phos/TCTCGGGAACGCTGAAGA+ACAATTCCAAATGCA **Hybridizes to "TGCATTTGGAATTGTTCT" Tm = 52.84 =====Short cDNA ~200nt Stop Primer===== *mRNA sequence 1,801-1,820: TAATATATTTCTTCATGAAG *Stop primer: CTTCATGAAGAAATATATTA Tm = 45.51 =====Long cDNA ~500nt Stop Primer===== *mRNA sequence 1,501-1,520: GGCAGTGAAAATGTTGAATG *Stop primer: ATTCAACATTTTCACT Tm = 44.65 **Hybridizes to "AGTGAAAATGTTGAAT" ====EGFR T790M: 2,660+ [http://www.ncbi.nlm.nih.gov/nuccore/NM_005228.3 EGFR transcript variant 1, mRNA]==== *Will use same RT primer as EGFR L858R since it's only 200nt downstream *No Stop primer to be used unless experiments show the BRAF stop primer to be effective ====EGFR L858R: 2,860+ [http://www.ncbi.nlm.nih.gov/nuccore/NM_005228.3 EGFR transcript variant 1, mRNA]==== *mRNA sequence 2,891-2,920: CATTGGAATCAATTTTACACAGAATCTATA *RT primer: /5Phos/TCTCGGGAACGCTGAAGA+TTCTGTGTAAAATTG **Hybridizes to "CAATTTTACACAGAATCT" Tm = 47.47
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