LION PROBES

Information about LION PROBES

Published on September 14, 2007

Author: Naples

Source: authorstream.com

Content

Slide1:  LIONPROBESTM A new REAL TIME detection system 2007 Slide2:  WHY LIONPROBES? NEW DETECTION SYSTEM Tecnología patentada y protegida por Biotools The high potential of Real Time DNA Amplifications requires new detection systems The more systems that are on the market, the more open and accessible the technology will be Analysing the market situation and demand, BIOTOOLS opted for the design of a new detection technology for Real Time DNA Amplification: LIONPROBESTM Slide3:  LIONPROBESTM Tecnología patentada y protegida por Biotools The present detection systems applied for Real Time PCR require the 5’-3’exonuclease activity (or exo-) of the polymerase. The system designed by BIOTOOLS combines: Oligonucleotide with double labeling: a fluorophore and a quencher at each end DNA polimerasa with a 3´-5´exonucleasa proofreading activity (Pfu) Slide4:  Principles of LIONPROBESTM Technology patented and protected by Biotools When the probe is not paired (1-2 bp at the 3´ end) with the DNA template, Pfu repares the error, releasing the marker (fluorophore or quencher), which then allows the emission of fluorescence When the marker at the 3´ end of the probe is released, it acts as an amplification primer When the probe shows perfect hybridisation with the template DNA, the fluorophore is quenched and no fluorescence is emitted Slide5:  LIONPROBE system detects general SNPs AGTGCCAAGTC 5’ 3’ Pfu DNA polymerase TCACGGTTCAG ACACGGTTCAG Perfect LIONPROBE Hybridization with Template 3’ Mismatched LIONPROBE Hybridization with Template Thermal Cycle Step Denature Anneal Extension Step ACACGGTTCAG No release of fluophore GCACGGTTCAG or multiple mutation of codons CCACGGTTCAG TCACGGTTCAG Basic working scheme of LIONPROBESTM Slide6:  Principles of designing LIONPROBESTM Tecnología patentada y protegida por Biotools Design of the Primer-Probe Objective: determine the position of the fluorescent dye FAM and the quencher TAMRA in the oligo Method: primer-probes with identical sequences are used, which defer regarding the position of the fluorescent dye and the quencher. The primer-probes hybridise perfectly with the control plasmid (pMTB control), but show a mismatch at the 3´- end of one of the DNA mutants 1P, 1F and 2PF. Slide7:  LIONPROBESTM Technology Tecnología patentada y protegida por Biotools Figure 1: Resultas in Real Time using primer-probes LION (IS and IS-INV) and Pfu Fluorescence in the FAM channel B) Gel agarose (1.5 %) Primer-Probe LION IS-INV M 1F 1P 2PF pMTB 2PF + LION IS-INV 1P + LION IS-INV 1F + LION IS-INV pMTB-+LION IS-INV 2PF+ LION IS 1P+LION IS 1F+LION IS Primer-Probe LION IS M 1F 1P 2PF pMTB Slide8:  Tecnología patentada y protegida por Biotools Objective: confirme the absence of 3´-5´exonuclease activity of Pfu on labelled dyes Figure 2: Real Time Amplification of the sample LION IS-INV and an amplification mixture without templates and d-NTPs LIONPROBESTM Technology: confirming the absence of the probe degradation Slide9:  Tecnología patentada y protegida por Biotools Objective: confirme the absence of contaminating 5´-3´exonuclease activity in the preparation of the Pfu, which could interfere in the final results Method: amplification and detection system with a TaqMan probe that requires a 5´-3´ exonuclease activity for fluorescence emission. Three different DNA polymerases have been tested: Taq DNA polymerase 5´-3´exonuclease (exo+) Taq DNA polimerase (exo-) Pfu DNA polymerasa LIONPROBESTM Technology: confirming the absence of 5’-3’ exonuclease activity Slide10:  Tecnología patentada y protegida por Biotools Fluorescence is only observed when using a Taq polymerase exo+ Figure 3: Real Time amplification results using a TaqMan probe (5´FAM -3´TAMRA) with three different DNA polymerases (Taq exo+, Pfu and Taq exo-). LIONPROBESTM Technology: confirming the absence of 5’-3’ exonuclease activity (2) Slide11:  Tecnología patentada y protegida por Biotools Pfu does not show 5´-3´exonuclease activity and also no displacement activity along the chain Figure 4: Amplification results shown on an agarose gel LIONPROBESTM Technology: confirming the absence of 5’-3’ exonuclease activity (3) Slide12:  Advantages of LIONPROBESTM Tecnología patentada y protegida por Biotools The design of the LIONPROBESTM has shown to provide the system with a series of unique properties, in comparison with other Real Time detection systems: High amplification fidelity due to the proofreading activity of the Pfu As for the correct function of the system a displacement zone is required, it is possible to detect all the possible mutants caused by SNPs Slide13:  LIONPROBESTM has broad applications Technology patented and protected by Biotools Bandamp;M Labs,S.A. Following applications have been tested with LIONPROBES™: Primer-Probe: acts as an identification probe and as an amplification primer at the same time Liquid hybridisation: as an identification probe for (geno-)typing of previously amplified products Probe: as a sequence that is not directly involved in the amplification Slide14:  Applications of LIONPROBESTM Tecnología patentada y protegida por Biotools Figure 5: Fluorescence signal that is obtained using the primer-probe LION IS-INV for the detection of the amplified samples (control and mutants) Assay: the reaction mixture contains the primer-probe LION IS-INV and all the required reagents for an amplification except the dNTP, so that no polymerase reaction can take place Slide15:  Developed products: BIOCMV QT Kit Tecnología patentada y protegida por Biotools Kit for the quantitative detection of Cytomegalovirus in clinical samples. The detection methodology uses LIONPROBESTM: CMV LION labelled primer-probe (5´TAMRA - FAM3´) that in combination with the reverse primer CMV-REV amplifies a highly conserved region of CMV (human) Pfu releases the fluorescent dye FAM INT LION labelled primer-probe (5´CAL FLUOR 610 - BHQ-23) that in combination with the reverse primer INT-REV amplifies the internal control Pfu releases the quencher BHQ-2 Slide16:  Figure 6: Analysis of fluorescence curves obtained in the FAM-channel using positive controls (5x 101- 5x 105 copies/reaction) provided with the BIOCMV QT kit Tecnología patentada y protegida por Biotools Developed products: BIOCMV QT Kit (2) Slide17:  Tecnología patentada y protegida por Biotools Figure 7: Analysis of fluorescence curves using the detection channel for the internal control included in the samples used in figure 6 Developed products: BIOCMV QT Kit (3) Slide18:  Tecnología patentada y protegida por Biotools Kit for the quantitative detection of Mycobacterium tuberculosis in clinical samples. The detection tehcnology used is based upon LIONPROBESTM: Figure 8. Analysis of the obtained fluorescence curves. A) FAM channel using positive controls (5x 101- 5x 105 copies/reaction) provided with the BIOTUB QT Kit. B) Internal control channel Developed products: BIOTUB QT Kit Slide19:  Randamp;D Outlook: analysing the Pfu efficiency Tecnología patentada y protegida por Biotools Objective: Studying the cutting efficiency of Pfu with different targets Assay: a liquid hybridization assay is performed confronting the LION probe with different targets that present at the 3´ end a single non-paired base Slide20:  Tecnología patentada y protegida por Biotools Figure 9: Fluorescence curves obtained in the liquid hybridisation assay with LIONPROBESTM G Negative fluorescence values are observed in case of the negative control (sample without substrate) as well as with the substrate C without non-paired bases Randamp;D Outlook: analysing the Pfu efficiency (2) Slide21:  Tecnología patentada y protegida por Biotools Figure 10: Fluorescence curves obtained for the liquid hybridisation assay using LIOPROBESTM A Negative fluorescence curves are only obtained with the negative control (sample without substrate) Randamp;D Outlook: analysing the Pfu efficiency (3) Slide22:  Tecnología patentada y protegida por Biotools Screening the cutting efficiency of the Pfu Protecting sequences with phosphorthioate the modified bases with phosphorthioate are resistant to the exonuclease activity of the Pfu Protection of the cutting site using degenerated bases The use of degenerated bases would allow at a certain point the detection of the incorporation of any other base without the requirement of a label of this Randamp;D Outlook Slide23:  Outlook of LIONPROBESTM TECHNOLOGY Technology patented and protected by Biotools LIONPROBESTM offer a highly sensitive technology for the detection and quantitative determination of DNA targets The design and use of LIONPROBESTM is extremely simple LIONPROBESTM offer an attractive alternative to the present methods LIONPROBESTM is the first primer detection system with a probe that uses a proofreading polymerase, providing in this sense a high amplification fidelity (error rates using the Taq polymerase are very high, which decreases to an important way the sensitivity of the assays using this system) The versatility of the system allows the detection of SNPs and the protection of sequences at critical targets Slide24:  Biotools, Bandamp;M Labs, S.A THE REAL TIME COMPANY C/Valle de Tobalina, 52, nave 43 Madrid 28021 Spain Tfn:(+34) 917100074 Fax:(+34) 915053118 E-Mail:[email protected] www.biotools.net

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