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
- Cancer mutation probes
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
- Increase RT reaction temperature from 37C to 42C to increase specificity
- 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