Scorpions Technology for Real Time PCR and Genotyping

Transcription

1 Scorpions Technology for Real Time PCR and Genotyping

2 Outline Origins of Scorpions Principles of Scorpions Performance Applications Conclusions

3 Origins of Scorpions

4 A Bit of History At Zeneca Diagnostics, we made products for human gene based diagnostics Initial technology- PCR + Gels Customers (educated, research-oriented, smart ) customised the tests(!) Split the reagents Added too much DNA Wanted to remove customer from the equation Homogeneous (closed tube) Simplicity

5 Benefits of Homogeneous Simple to Use Automatable Closed tube minimises contamination issues Suitable for both endpoint and Real time

6 Homogeneous options Intercalation Easy to use but not suitable for diagnostic applications Cannot be multiplexed Fluorescence Polarisation Difficult to use with real time PCR Instrumentation (in 1998)

7 Fluorophore/Quencher Technologies Taqman Excellent technology Not commercially available for Diagnostic Use Molecular Beacons Neat Idea Commercially available Amplifluor/Sunrise Neat and effective No third level specificity so not suitable for Diagnostics

8 Molecular Beacons CT G C A A C A C G T T A A T GG G C C T G CG AT GC GC CG F Beacon Q F Q C G G C G A T C C G C G CCTTTCACCTGACAGAAGGGT AACAGTCTAAAGGAAAGTGGACTGTCTTCCCAGCTGACC Beacon + target

9 Issues With Bimolecular Probe Systems Kinetics are slow Energy cost to open stem/loop of probe Bimolecular collision Competition for Molecular Beacon Target site from: PCR product synthesis Target/complement reannealing Target strand folding These problems also apply to other bimolecular (Eclipse) and tri-molecular systems (LightCycler Probes)

10 Principles of Scorpions

11 If the Target Molecule Folds Efficiently Why not stick the probe to the amplicon? Q F

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13 What s the Blocker for? F Q F Q

14 Prevention of Non-Specific Signals F Q F Q

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16 Benefits of dual oligo Scorpions Linear molecules for easier synthesis Single labels for more cost effective synthesis Greater separation of fluorophore and quencher gives even stronger signals

17 Summary Scorpions combines probe and primer into a single molecule Two simple formats for presenting quencher Quenched probe ensures low backgrounds The probe sees the extension product of its own primer- unimolecular rearrangement Probing is predicted to be fast and efficient leading to high signals

18 Performance of Scorpions

19 Does it work? Amplicon Detection FAM fluorescecne intensity cycles

20 Strong signals, low backgrounds Scorpion Beacon TaqMan

21 Details of signal generation Intercalation TaqMan FAM Time (s) FAM Time Molecular Beacons Scorpions FAM Time (s) FAM Time

22 10 minute PCR Scorpions vs TaqMan, fast cycling Fluorescence TaqMan B TaqMan B neg Scorpion B Scorpion B neg Cycles

23 PCR Limiting? Very Fast Cycling Cycles FAM Cycles FAM

24 Sensitivity and Specificity 20,000 copies 2 copies

25 Sensitivity and Specificity

26 Short Probes/Quenchers Annealing

27 Summary Scorpions detect amplicons efficiently High signals, low backgrounds Reliable Quantitative data Shorter probes allow greater allelic specificity Scorpions probing is fast PCR is probably the limiting factor

28 Applications Genotyping Q-PCR Others

29 Genotyping Reaction Quencher/Probe Variant A Q F Quencher/Probe Variant B Q R

30 Genotyping Products F Q F Q R F R

31 ARMS Single Tube Genotyping Reaction Scheme A Rox Fam Allele A primer Allele B primer B

32 Genotyping Data 70:30 Polymorphism Processed F/R aa ab bb Sample Number

33 Genotyping data aa ab bb aa+4sd ab+4sd bb+4sd aa-4sd ab-4sd bb-4sd

34 Real Time Genotyping Wild type Sample Cycles Fluorescence WT MUT No Template Control Cycles Fluorescence WT MUT Heterozygote Sample Cycles Fluorescence WT MUT Mutant Sample Cycles Fluorescence WT MUT

35 Reverse Transcription/PCR Tth system (Mn Buffer) In vitro transcript RT for 7 min at 60 C PCR: 95 C 2s, 60 C 10s, 72 C 3s

36 Reverse Transcription & PCR N N 20 NTC N

37 Haplotyping Other Applications Pooled Samples

38 Haplotyping A B F Q

39 Haplotyping Data Rpost-Rpre

40 Accuracy Improves with Larger Pools Observed Allele Frequency Pools of 12 Pools of 48 Pools of 96 Pools of 192 Pools of % accuracy Predicted Allele Frequency

41 Summary Scorpions has proved to be very applicable to Genotyping Endpoint and Real Time ARMS or hybridisation QPCR (+/- reverse transcription) Multiplexing to at least 3 achieved on RT-PCR Haplotyping Determining allele frequency in pooled samples

42 One last thing Scorpions are easy molecules to design (after bit of practice!); but Software for probe design now available from DNA Software Inc

43 Conclusions Scorpions are linked primer/probe molecules with a unimolecular mode of action Benefits of this MOA include Speed Efficiency Reliability Stoichiometry: 1 molecule gives one signal Applications include Genotyping Haplotyping Pooled Genotyping Q-PCR +/- RT