TINA - Twisted Intercalating Nucleic Acid

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2 Welcome to the webinar: TINA - Twisted Intercalating Nucleic Acid Improve the efficiency of your stressed PCR

3 TINA - Twisted Intercalating Nucleic Acid Improve the efficiency of your stressed PCR Dr. Jutta Huber Managing Director Eurofins MWG GmbH

4 Introduction Eurofins MWG Operon is serving a large global customer base as provider for oligonucleotides which are used in Research and development Kit components Diagnostic assays As a member of the Eurofins Scientific group we have an intensive exchange on performance criteria for PCR assays and assay developments GMO and food testing Forensic and regulatory requirements

5 Our Focus in DNA Synthesis Bridging know-how in chemistry to applications. Adapting synthetic procedures to best performance results in specific applications Optimised Application Oligos. Finding new interesting molecules which can contribute to better results and efficiency in a diversity of applications.

6 Cooperation with QuantiBact Inc. QuantiBact is developing novel amplification and quantification procedures utilising nucleic acid intercalators, such as TINA. Eurofins MWG Operon is happy to be the preferred supplier for TINA modified oligonucleotides. We are pleased to work with QuantiBact and our customers to identify interesting applications where TINA can make a difference.

7 TINA - Twisted Intercalating Nucleic Acid Improve the efficiency of your stressed PCR Gorm Lisby, MD PhD Senior Vice President, Founder QuantiBact, Inc.

8 PCR - frequent challenges.

9 - primer-limiting situations - complex sample material - multiplexing

10 PCR with TINA-modified Primers

11 TINA primers 5 modification Re-design of primers not necessary Works with end-point as well as Real-time PCR Increases on-time in primer/template hybridization Protects primers against exonucleases Stability and storage as conventional DNA primers Tested successfully with many different buffer/enzymes Available from Eurofins MWG Operon

12 Twisted Intercalating Nucleic Acid TINA

13 TINA-PCR PCR components TINA intercalator Forward primer 5 z ATAATCGACGTACGAGTC3 3 TATTAGCTGCATGCTCAG CGCATTACGTGACTCATC 5 5 ATAATCGACGTACGAGTC GCGTAATGCACTGAGTAG 3 Target Plus: PCR buffer Nucleotides DNA polymerase 3 CGCATTACGTGACTCATC z 5 Reverse primer TINA intercalator

14 TINA-PCR PCR amplicons TINA intercalator TINA intercalator zataatcgacgtacgagtc GCGTAATGCACTGAGTAG TATTAGCTGCATGCTCAG CGCATTACGTGACTCATC z PCR amplicon

15 TINA-modified Primers Impact on Annealing Temperature Primer Concentration

16 Effect of annealing temperature and different primer concentrations

17 TINA-modified Primers Impact on Design Flexibility

18 TINA PCR Preliminary singleplex PCR test of xxx primers Annealing gradient from 55 to 70 C. TINA primers increase annealing: HPLC DNA primers HPLC TINA primers HPLC TINA primers (2bp shorter)

19 TINA-modified Primers Impact on PCR Efficiency

20 Streptomyces detection Comparison of amplification curves with TINA-modified primers (high fluorescence) and non-modified primers (low fluorescence), detection with SYBR Green to 50 target copies. All negative controls were negative (data not shown). 50 target copies, TINA primers Cq = target copies, DNA primers Cq = 35 5 target copies, TINA primers 5 target copies, DNA primers

21 TINA-modified Primers Impact on Multiplex PCR Complex sample material

22 TINA Multiplex PCR crude NC crude NC 1000 bp rrs elt estah Unmodified primers 5 -TINA modified primers Octaplex end-point PCR by unmodified primers and 5 -TINA modified primers. The red box highlights the lack of amplicons for the estah gene with unmodified primers. 10-fold target dilution series for an Escherichia coli strain entailing three of eight target genes. Assay with unmodified primers published by Brandal et al, J Microbiol Meth (2007) 68:

23 Multiplex PCR complex test material Soil Feces

24 TINA Multiplex PCR Degenerate Primers 27.0 DNA primers 25.5 TINA primers TINA improves: multiplex PCR efficiency - from 82.6% to 95.6% multiplex PCR sensitivity - from Cq=27.0 to Cq=25.5 (10 3 copies)

25 Direct detection of DEC in feces with Multiplex TINA-PCR Assay Patient Turn-around Time Sample prep. Simple lysis at 95C Standard sampling MagPix detc. PCR prep. Multiplex TINA-PCR with MagPlex beads Transport to laboratory PCR run Laboratory workflow: Total turn-around time: 2.5 hrs

26 Cliffhanger vs. Current Gold Standard PCR No. of patients stx1 eae estah estap rrs (internal control) ipah elt stx2 Cliffhanger Standard PCR All Standard PCR positive samples were also tested positive by Cliffhanger stx1 eae estah estap rrs (internal control) ipah elt stx2 Standard PCR Cliffhanger

27 TINA PCR - Conclusion Improved PCR efficiency No need for special primer design, just add TINA to the 5 terminal position of the priming sequence Works with end-point as well as Real-time PCR Increases Ta and decreases C primer Increased freedom of primer design Improves PCR efficiency whenever primers are limiting Improves sensitivity Faster cycling parameters Improved PCR robustness Protects primers against exonuclease activity Improved multiplexing Improved efficiency and sensitivity of multiplex PCR

28 When to use When you need a more efficient PCR - the primers are limiting the reaction - you need design freedom (e.g. shorter primer) - you want to shorten the PCR cycle parameters - you want to use more stringent buffers (low salt buffer) When you are working with complex test material - you are testing impure sample material (you need robustness) - you are designing degenerated primers When you are designing multiplexed PCR assays - you are designing a multiplex real-time PCR - you are designing a multiplex end-point PCR TINA-modified primers

29 Thank you for your attention! We are looking forward to your questions: Dr. Jutta Huber Managing Director Eurofins MWG GmbH Gorm Lisby, MD PhD Senior Vice President, Founder QuantiBact, Inc. Ursula Doer Product Manager Eurofins MWG GmbH

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