Detection of the P35S promoter in transgenic maize via various isothermal amplification techniques: a practical approach

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1 Analytical and Bioanalytical Chemistry Electronic Supplementary Material Detection of the P35S promoter in transgenic maize via various isothermal amplification techniques: a practical approach C. Zahradnik, C. Kolm, R. Martzy, R.L. Mach, R. Krska, A.H. Farnleitner, K. Brunner Fig.. Alignment of the P35S promoter sequences for RoundUp Ready TM soy (EU ) and transgenic maize MON810 (JX13971). Forward outer primer (F3) and backward outer primer (B3) as published by [26] in red and as designed by the authors in green. The alignment shows that the primer set designed for RoundUp Ready TM soy cannot be used for amplification of the P35S promotor in transgenic maize, since this sequence is shorter and the F3 primer lies outside of the target sequence. Therefore, efficient amplification cannot be expected, since LAMP relies on the hybridization of all primers to the target in order to amplify the desired DNA sequence (Fig. )

2 Fig.. Amplification plot with primers and reaction conditions as previously published and performed [26, 25] for GM maize, resulting in poor amplification efficiency as well as low fluorescence intensity. n = 5 Table.. Various primer sets for NEAR, SDA and RCA were tested, including different nicking and restriction enzymes (, Nt.BspQI, HincII, BsrI). *Primer and oligo nucleotide sequences for NEAR (A1-8), SDA (-4) and RCA (C1-7) used in feasibility tests as shown in figure 1. Recognition sites of restriction enzymes are underlined. Method Primer Sequence 5' 3' NEAR* CAAAAAAAAAAAAGCGAGTCTAGCACTCCACTGACGTAAGGGATG CAAAAAAAAAAAAGCGAGTCTAGCAAAGGGTCTTGCGAAGGATAG CAAAAAAAAAAAAGCGAGTCTAGCATCTCCACTGACGTAAGGGA CAAAAAAAAAAAAGCGAGTCTAGCAGAAGGGTCTTGCGAAGGAT CAAAAAAAAAAAAGCGAGTCTAGCAC CAAAAAAAAAAAAGCGAGTCTAGCAAGGATAGTGGGATTGTGCG CGAGGAGCATCGTGGA GTCCTCTCCAAATGAAATGAAC CAAAGTCTGAGTCTGGGAC CAAAGTCTGAGTCTGGGAAGGATAGTGGGATTGTGCG CGAGGAGCATCGTGGA GTCCTCTCCAAATGAAATGAAC CAAAAAAAAAAAAGCGAGTCTAGCACTCCACTGACGTAAGGGATG

3 Nt.Bsp QI SDA_Bs ri SDA_Hi ncii SDA_Hi ncii CAAAAAAAAAAAAGCGAGTCTAGCAAAGGGTCTTGCGAAGGATAG CAAAAAAAAAAAAGCGAGTCTAGCAACGTAAGGGATGACGCA CAAAAAAAAAAAAGCGAGTCTAGCAGAGGAAGGGTCTTGCGA CAAAAAAAAAAAAGCGAGTCTAGCACCACTGACGTAAGGGATGA CAAAAAAAAAAAAGCGAGTCTAGCAGGGTCTTGCGAAGGATAGT CAAAAAAAAAAAAAAGCTCTTCTCCAACCACGTCTTC CAAAAAAAAAAAAAAGCTCTTCTGGGATTGTGCGTC CCACGAGGAGCATCG GAAGGGTCTTGCGA CCGAGACTTAGGATTCACTCTACAACCAGTGGATGACGCACAATCCCAC CCGAGACTTAGGATTCACTCTACAACCAGTAGAGGAAGGGTCTTGCGA GCCGACAGTGGTCCCAAAGAT AGCGTGTCCTCTCCAAATGAAATG CAAAAAAAAAAAAGTTGACCGTCTTCAAAGC CAAAAAAAAAAAAGTTGACTGGGATTGTGC TCGTGGAAAAAGAA GTCTTGCGAAGG CAAAAAAAAAAAGTTGACAAGGGATGACG CAAAAAAAAAAAGTTGACGAGGAAGGGTC ACCACGTCTTCA CCAAATGAAATGAAC SDA* TAGGATGAGCATTCTGCGGTTGCCAGTCGTTCCAACCACGTCTTCAAAGCAAGT TAGGATGAGCATTCTGCGGTTGCCAGTAGGAAGGGTCTTGCGAAGGATAGTGGG GCCGACAGTGGTCCCAAAGAT AGCGTGTCCTCTCCAAATGAAATG RCA* hybridization TTTTCCACTATCTTCACAATAAAGTTTTGATGCCGTATGCCTAGCACGGAATTAACTTGCTAG CCGTCCAGGTTAGCCACCTTCC Primer1 GCCGTATGCCTAGCA Primer2 TTGCTAGCCGTCCAG RCA2 hybridization GCCTCTGCCGACAGTGTTTGATGCCGTATGCCTAGCACGGAATTAACTTGCTAGCCGTCCA GGTTATCTTCAACG ATCATTGCGATAAAGGAAAGGCCATCGTTGAAGAT Primer1 GCTAGGCATACGGCATCAAA Primer2 AACTTGCTAGCCGTCCAGGTT DNA TCACTTAGGACGTAGTGAAGCAGGAAACACCTATGCC RCA3 hybridization TCTATATAAGGAAGTTCATTTCATTTTTGATGCCGTATGCCTAGCACGGAATTAACTTGCTAG CCGTCCAGGTTCCTTCCCAGAACGCACAATCCCACTATCCTTCGCAAGACCCTTCC Primer1 TGC TAG GCA TAC GGC ATC AAA Primer2 AAC TTG CTA GCC GTC CAG GTT RCA4 hybridization GAATCCGAGGAGGTTTCCGTTTGATGCCGTATGCCTAGCACGGAATTAACTTGCTAGCCGT CCAGGTTCTGGGCAATG Primer1 AAACGGAAACCTCCTCGGATTCCATT Primer2 TTGATGCCGTATGCCTAGCACGGA RCA5 hybridization TTTTCCACTATCTTCACAATAAAGTTTTGATGCCGTATGCCTAGCACGGAATTAACTTGCTAG CCGTCCAGGTTAGCCACCTTCC Primer1 GCCGTATGCCTAGCA Primer2 TTGCTAGCCGTCCAG RCA6 hybridization AGTGGAAAAGGAAGGTGGCTCTTTGATGTAGTATGCCTAGTACTGAATTAACTTGCTAGCCG

4 RCA7 Primer1 Primer2 DNA hybridization Primer1 Primer2 DNA TCCAGGTTATCTTCACATATCTGTCACTTTATTGTGAAGAT ACCTTCCTTTTCCACTATC TAGTATGCCTAGTACTGAATTAACTT TCACTTAGGACGTAGTGAAGCAGGAAACACCTATGCC ATCTTCAACGATGGCCTTTCTGAACGACGAATCTGTACCATGCTAATGCGGCGTGATGTATT ATGCGTATGACGGCAGAGGC TGCTAATGCGGCGTGATGT GGAATCCGAGTGAACGACGA TGAACGACGAATCTGTACCATGCTAATGCGGCGTGATGTATTATGCGTATGGA Table. Reaction conditions and buffer s tested for Rolling Circle Amplification, resulting in a total number of tested combinations of 3481 Ligation Denaturation step at 95 Ligation reaction time Ligases used padlock probe IDs melting temperatures Tm of padlock probe 5'arms Tm pf padlock probe 3'arms padlock probe s DNA applied to ligation reaction Exonuclease I Exonuclease III Incubation time of exonucleases Amplification Denaturation step Polymerases tested 3 min. at 95 C none 30 min. 60 min. 90 min. 120 min. 180 min. 240 min. Ampligase at T4 DNA ligase 65 C at 37 C RCA RCA2 RCA3 RCA4 RCA5 RCA6 RCA7 70 C 68.3 C 63.9 C 69.5 C 50 C 70 C 70.2 C 50 C 77.7 C 68.3 C 32.6 C 70 C 61.1 C 49.6 C 100pM 10pM 1pM 10 ng/μl 1 ng/μl 0.1 ng/μl 1 Unit 10 Units 20 Units 1 Unit 10 Units 20 Units 10 min. 30 min. 120 min. 3 min. at 95 C none Bst (65 C) phi29 Divalent cations 6 mm 4 mm 2 mm 1 mm s buffer ph Table S3. Reaction conditions and buffer s tested for Nicking Enzyme Amplification Reaction Tm of primer target 37 C 40 C 45 C 55 C 60 C 65 C Tm of primer 5 end 37 C 40 C 45 C 55 C 60 C 65 C Nicking Enzymes Temp: 37 C Temp: 55 C (recognition site) Nt.BbvCI Nb.BtsI Nt.BspQI Assay time 15 min 30 min 60 min Buffer ph Buffer 20 mm 40 mm 80 mm KCl 25 mm 50 mm 100 mm MgCl mm 5 mm 10 mm dntps 0.2 mm 0.4 mm 0.8 mm Additives Formamid (5%) Betain (1M) DMSO (5%) Et.SSB Glycerin (5%) BSA (4 ng/µl) 100 µg/µl Nicking Enzyme 2.5 units 5 units 10 units 20 units Polymerase Phi29 Bst (55-65 C) Polymerase conc. 2.5 units 5 units 10 units

5 Table S4. Reaction conditions and buffer s tested for Strand Displacement Amplification. Tm of primer target 37 C 40 C 45 C 60 C 65 C Tm of primer 5 end 37 C 40 C 45 C 60 C 65 C 70 C Restriction Enzymes Temp: Temp: 65 C (recognition site) 37 C BsrI BsmI HincII Assay time 30 min 60 min 90 min Buffer ph Buffer 20 mm 40 mm 80 mm KCl 25 mm 50 mm 100 mm MgCl mm 5 mm 10 mm Protection dntp datp S dctp S dntps 0.2 mm 0.4 mm 0.8 mm Restriction Enzyme 2.5 units 5 units 10 units 20 units Polymerase Exo - Bst Klenow (55-65 C) Polymerase conc. 2.5 units 5 units 10 units