Matt:LabNotes/2014-3-20
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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)
- 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
- Cancer mutation probes
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
- Code sequences
- (10201) = dcProbe0-FAM, dcProbe2-Cy3, dcProbe4-FAM
BRAF
- mRNA sequence 1,821-1,900: 5’-ACCTCACAGTAAAAA(TAGGTGATTTTGGTCTAGCTACAG[T])GAAATCT(CGATGGAGTGGGTCCCATCAGTT)TGAACAGTTG-3’
- () indicate where padlock probe hybridizes to
- [] indicates SNP
- H1: [A/T]CTGTAGCTAGACCAAAATCACCTA
- H2: AACTGATGGGACCCACTCCATCG
- BRAF_V600E_WT (10101): ACTGTAGCTAGACCAAAATCACCTA+ATGTATCGCGCTCGATTGGCAG+TAGAACTTGCGCGGATACACGG+CTTCAGCTTCCCGATATCCGACGG+TCGGAATACGTCGTTGACTGCG+AACTGATGGGACCCACTCCATCG
- BRAF_V600E_MU (20202): TCTGTAGCTAGACCAAAATCACCTA+ACGTATCGGTAGTCGCAACGCA+TCTACTTCGTCGCGTCAGACCA+CTTCAGCTTCCCGATATCCGACGG+CTACCATTCGCGTGCGATTCCA+AACTGATGGGACCCACTCCATCG
EGFR
- mRNA sequence 2,591-2,660:5’-TCACCT(CCACCGTGCAGCTCATCA[C])GCAGCT(CATGCCCTTCGGCTGCCTCCT)GGACTATGTCCGGGAACA-3’
- () indicate where padlock probe hybridizes to
- [] indicates SNP
- H1: [G/A]TGATGAGCTGCACGGTGG
- H2: AGGAGGCAGCCGAAGGGCATG
- EGFR_T790M_WT (01011): GTGATGAGCTGCACGGTGG+GTCTTGCGTGCGATACGGAGTA+CGAATTGTCCGCGCTCTACGAT+CTTCAGCTTCCCGATATCCGACGG+TCGGAATACGTCGTTGACTGCG+AGGAGGCAGCCGAAGGGCATG
- EGFR_T790M_MU (02022): 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 ~50?
- /5Phos/TCTCGGGAACGCTGAAGA-