TripleHyb real time PCR for detection of single nucleotide polymorphisms in the VEGF promoter region

Transcription

1 TripleHyb real time PCR for detection of single nucleotide polymorphisms in the VEGF promoter region S. Fuessel 1, S. Unversucht 1, A. Lohse 1, S. Tomasetti 1, A. Rost 2, A. Edelmann 2, M.P. Wirth 1, A. Meye 1, T. Köhler 2 1 Department of Urology, Technical University of Dresden, 2 AJ Roboscreen GmbH, Leipzig, Germany Granted by technology support with financial sources of the European Regional Development Fund and the State of Saxony

2 Application of a new detection format establishment of a novel detection format for realtime PCR supporting quantitative applications suitability for genetic polymorphism detection improved or same sensitivity, specificity, flexibility, and robustness compared to available formats full compatibility with the most commonly used realtime instrument platforms no need for external licenses development of the TRIPLEHYB probe format

3 TRIPLEHYB probe format: basic principle upstream probe D2 3 5 D3 downstream probe 3 D1 stem structure D target sequence complementary probe subsequence D 1 D 4 : positions for the attachment of different dyes D1 fluorescent dye & D2 quencher

4 TRIPLEHYB probe format: probe design design of TaqMan primers and probes using a suited software (e.g. PrimerExpress,, Applied Biosystems) retaining of original TaqMan primers splitting of TaqMan probe into two target complementary subsequences (about 9159 bp length) appending of a targetunrelated, intermolecular stemforming subsequence to both the 3 end 3 of the upstream and the 5 end 5 of the downstream probe optimal stem length: 9 bp need for both up & do probes for high, robust signals

5 Proposed mechanism of TRIPLEHYB realtime fluorescence PCR format 1. POLYMERIZATION upstream primer upstream probe R downstream probe R = Reporter = uencher downstream primer 2. STRAND DISPLACEMENT R 3. CLEVAGE, TRIPLEX BODY DISPLACEMENT, COMPLETE POLYMERIZATION R

6 TRIPLEHYB : detection of point mutations TripleHyb particularly suited for detection of polymorphisms accidental finding during Hepatitis B virus genotyping! HBV genotype B no mismatch C G T A G C R (probe 1) A T CGCTGGATGTG...TCTGCGGCG...GCGACCTACAC...AGACGCCGC... HBV genotype F single G A exchange: no signal! C G T A G C R (probe 1) A T CG C TGGATGTG...TCTGCGGCG...GC A ACCTACAC...AGACGCCGC...

7 TRIPLEHYB : evaluation in SNP detection should be generally useful for SNP detection due to short hybridization length in upprobe probe known: base exchange in position 3 abolishes upprobe probe binding to template no signal unknown: at which positions it works too? model system: SNP C T C T at position 460 of the VEGF promoter known association to different diseases including predisposition to tumorigenesis

8 C460T polymorphism of the VEGF promoter probes containing the SNP at position 3 of the upstream probe mt R C G T A R1 G C A T AC A CCCTCAACC...CCACACGCACA...TG TGGGAGTTGG..GGTGTGCGTGT... R C G T A R2 G C A T AC GCCCTCAACC...CCACACGCACA...TG CGGGAGTTGG..GGTGTGCGTGT... R1 = FAM R2 = CFO or YY = BH1

9 Genotyping of VEGF C460T at position 3 homozygous mutant heterozygous homozygous wildtype R1 R1 R2 R1 R2 R2 R1 = FAM R2 = CFO or YY = BH1 RotorGene 3000 (Corbett Research)

10 SNP C460T at position of the upprobe probe system 1 system 2 system 3 R R R R system 4 system 5 R set of 3 probes for each of the 5 systems: upprobe for upprobe for mut doprobe systematic analysis of SNPs at positions 1 to 5 of the upstream probe

11 Optimization of assay performance systematic optimization of assay conditions and evaluation of system flexibility use of standard plasmids containing or mut probe ratios and concentrations primer concentrations MgCl 2 concentrations final aims: find suitable SNPpositions in upprobes probes validation using clinical samples relevance of VEGF C460T for prostate cancer?

12 TRIPLEHYB : ratio of probes probe matrix: molar excess of downstream probe required upstream : downstream 1:1 / 1:2 / 1:3 / 1:4 1:4 at different concentrations: similar performance 0.2µM : 0.8µM (1:4) 0.2µM : 0.6µM (1:3) 0.2µM : 0.4µM (1:2) 0.2µM : 0.2µM (1:1) 0.2µM : 0.8µM 0.3µM : 1.2µM 0.4µM : 1.6µM 3step PCR (45 cycles; LC480): 95 C/15s; 45 C/1s; 59 C/40s, system 3; primers: 0.5µM each; probes: CFOup do, varying ratios and concentrations; MgCl 2 : 5mM; templates: 10 4 molecules plasmid

13 TRIPLEHYB : primer matrix primer matrix 1: 50 / 300 / 900nM ratios 1:1 / 1:3 / 3:1 promising medium concentration range primer matrix 2: nM equimolar best medium concentration range 900/50 (18:1) 300/900 (1:3) 300/300 (1:1) 900/300 (3:1) 50/900 (1:18) 300/50 (6:1) 50/50 (1:1) 900/900 (1:1) 50/300 (1:6) 500/500 (1:1) 600/600 (1:1) 300/900 (1:3) 400/400 (1:1) 300/300 (1:1) 200/600 (1:3) 900/300 (3:1) 600/200 (3:1) 3step PCR (45 cycles; LC480): 95 C/15s; 45 C/1s; 59 C/40s, system 3; 3 primers: varying; probes: 0.3µM CFOup/0.4µM FAMup 1.2µM do; MgCl 2 : 7.5mM; templates: 10 4 molecules plasmid (detection of CFO)

14 TRIPLEHYB : dependence on MgCl 2 relatively high MgCl 2 concentrations required system 3: upcfo do system 3: upfam do 5mM 6mM 7mM 8mM 9mM 10mM 10mM 9mM 8mM 7mM 6mM 5mM 3step PCR (45 cycles; LC480): 95 C/15s; 45 C/1s; 59 C/40s, system 3; primers: 0.5µM each; probes: 0.3µM CFOup 1.2µM do; MgCl 2 : varying between 510mM; 5 templates: 10 4 molecules plasmid

15 Evaluation of SNPpositions in upprobes probes detection of reliable reaction curves with the right template? after further optimization for each system: YES but not for system 1 & 2 with upcfo () system upfam mut upcfo

16 Evaluation of SNPpositions in upprobes probes detection of reliable reaction curves with the right template? after further optimization for each: YES but not for system 1 & 2 with upcfo () system upfam mut upcfo upfam n.t. n.t. n.t. n.t. n.t. = not tested

17 Evaluation of SNPpositions in upprobes probes detection of reliable reaction curves with the right template? after further optimization for each: YES but not for system 1 & 2 with upcfo () quenching by G? system upfam mut upcfo upfam n.t. n.t. n.t. n.t. upyy n.t. = not tested

18 Systems 15: 1 YYup()/FAM up()/famup(mut) do best performance for systems 3 & 4 no or very weak detection of curves in the other channel detection of template (YY) detection of muttemplate (FAM) system 5 systems 3 & 4 system 5 systems 3 & 4 system 2 system 2 system 1 system 1 3step PCR (37 cycles; LC480): 95 C/15s; 45 C/1s; 59 C/40s, systems 15; 1 primers: 0.5µM each; probes: 0.3µM YYup/0.4µM FAMup 1.2µM do; MgCl 2 : 5mM; templates: 10 5 molecules or mutplasmid

19 Systems 35: 3 CFOup()/FAM up()/famup(mut) do best performance for systems 3 & 4 system 3 system 4 detection of CFOsignal: wildtype (10 6 /10 5 /10 4 mol.) heterozygous (5x10 5 /10 4 /10 3 each) mut mut mut mut detection of FAMsignal: mutant (10 6 /10 5 /10 4 mol.) mut mut mut mut heterozygous (5x10 5 /10 4 /10 3 each) 3step PCR (37 cycles; LC480): 95 C/15s; 45 C/1s; 59 C/40s, systems 35; 3 primers: 0.5µM each; probes: 0.3µM CFOup/0.4µM FAMup 1.2µM do; MgCl 2 : 5mM; templates: 10 4 / 10 5 / 10 6 molecules or mutplasmid or 1:1 mixtures

20 Conclusion SNP at position 3 works best (HBV, VEGF), also at positions 4 & 5 using plasmid templates only preliminary data on clinical samples: so far VEGF C460T detectable in tumor DNA by systems 3 & 4 (CFOup) further optimization needed (background elimination, other dyes, use of genomic DNA, evaluation of other positions and models) TRIPLEHYB format as promising genotyping tool with more flexibility regarding SNP position

21 Advantages: TRIPLEHYB in summary universal format: broad applicability for both genotyping and quantitative target analysis open format: may be run on several real time platforms e.g. tested with LightCyler, ABI 7000 SDS, RotorGene 3000 safe format: intercalating dyes which are frequently used for genotyping may be replaced fast format: usable with fast cycling, SNP analysis does not require subsequent melting point analysis reliable format: since the detector probe hybridization area may be very short (912 bases), analysis particularly of targets with short conserved subsequences e.g. RNA viruses multiplexing capability Disadvantages: assay setup more complex particularly multiplex assays