Matt:LabNotes/2014-3-20

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Cancer Padlock Probes + RT Primer[edit]

  • 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

New Padlock Probes[edit]

  • 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[edit]

  • 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): /5Phos/ACTGTAGCTAGACCAAAATCACCTA+ATGTATCGCGCTCGATTGGCAG+TAGAACTTGCGCGGATACACGG+CTTCAGCTTCCCGATATCCGACGG+TCGGAATACGTCGTTGACTGCG+AACTGATGGGACCCACTCCATCG
  • BRAF_V600E_MU (20202): /5Phos/TCTGTAGCTAGACCAAAATCACCTA+ACGTATCGGTAGTCGCAACGCA+TCTACTTCGTCGCGTCAGACCA+CTTCAGCTTCCCGATATCCGACGG+CTACCATTCGCGTGCGATTCCA+AACTGATGGGACCCACTCCATCG

EGFR[edit]

  • 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): /5Phos/GTGATGAGCTGCACGGTGG+GTCTTGCGTGCGATACGGAGTA+CGAATTGTCCGCGCTCTACGAT+CTTCAGCTTCCCGATATCCGACGG+TCGGAATACGTCGTTGACTGCG+AGGAGGCAGCCGAAGGGCATG
  • EGFR_T790M_MU (02022): /5Phos/ATGATGAGCTGCACGGTGG+CAACGGTATTCGGTCGTCATCC+CCGTTTGATCGTTCGACCGAGA+CTTCAGCTTCCCGATATCCGACGG+CTACCATTCGCGTGCGATTCCA+AGGAGGCAGCCGAAGGGCATG

RT Primer[edit]

  • >15nt length for specificity
  • Downstream of padlock probe capture sites
  • Not too many C's and G's with Tm >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 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+ BRAF, mRNA[edit]

  • mRNA sequence 1,981-2,000: TGCATTTGGAATTGTTCTGT
  • RT primer: /5Phos/TCTCGGGAACGCTGAAGA+ACAATTCCAAATGCA
    • Hybridizes to "TGCATTTGGAATTGTTCT" Tm = 52.84
Short cDNA ~200nt Stop Primer[edit]
  • mRNA sequence 1,801-1,820: TAATATATTTCTTCATGAAG
  • Stop primer: CTTCATGAAGAAATATATTA Tm = 45.51
Long cDNA ~500nt Stop Primer[edit]
  • mRNA sequence 1,501-1,520: GGCAGTGAAAATGTTGAATG
  • Stop primer: ATTCAACATTTTCACT Tm = 44.65
    • Hybridizes to "AGTGAAAATGTTGAAT"

EGFR T790M: 2,660+ EGFR transcript variant 1, mRNA[edit]

  • 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+ EGFR transcript variant 1, mRNA[edit]

  • mRNA sequence 2,891-2,920: CATTGGAATCAATTTTACACAGAATCTATA
  • RT primer: /5Phos/TCTCGGGAACGCTGAAGA+TTCTGTGTAAAATTG
    • Hybridizes to "CAATTTTACACAGAATCT" Tm = 47.47