Twisted Intercalating Nucleic Acid (TINA) a novel group of molecules with improved performance in PCR and qpcr applications

Transcription

1 Twisted Intercalating Nucleic Acid (TINA) a novel group of molecules with improved performance in PCR and qpcr applications Dr. Rainer Schubbert, Eurofins Medigenomix Topics Topics of this talk: What is TINA Applications: Analytical methods for species determination Assay Setup and validation Results 2

2 Twisted Intercalating Nucleic Acid- TINA 3 TINA-PCR: PCR components TINA intercalator z Forward primer 5 ATAATCGACGTACGAGTC 3 3 TATTAGCTGCATGCTCAG CGCATTACGTGACTCATC 5 5 ATAATCGACGTACGAGTC GCGTAATGCACTGAGTAG 3 Target 3 CGCATTACGTGACTCATC 5 z Reverse primer Plus: PCR buffer Nucleotides DNA polymerase TINA intercalator 4

3 TINA: Introduction / Theory The TINA molecule (Twisted Intercalating Nucleic Acid) from QuantiBact A/S enhances the thermal stability of a binding oligonucleotide duplex while leaving the ability to discriminate matching and mismatching oligonucleotides intact. 5 TINA: Introduction / Theory TINA labeling increases the primer annealing and melting temperature and therefore reduces the general probability of PCR primers to anneal unspecific. 6

4 TINA: Introduction / Theory This increase in specific primer annealing can be utilized to increase the overall specificity of a given assay. 7 TINA: Introduction / Theory Alternatively, by relaxing the stringency of the primer annealing, an increased sensitivity can be achieved without compromising specificity compared to an identical assay without TINA-modifications. 8

5 TINA: Introduction / Theory We present three examples from routine analyses (food authenticity and pathogen detection), where we have validated whether TINA modified oligos allows better discrimination in - allele specific PCR and RealTime PCR assays or - higher sensitivity in RealTime PCR assays compared to analyses with unmodified oligos. 9 Introduction DNA Campus Applied Genetics Laboratory specialised on DNA tests for food and agro market Eurofins Competence Centre for DNA analyses Accredited acc. ISO17025 for Food Authenticity Integrated into the Eurofins DNA campus Eurofins MWG Operon Preferred supplier for TINA oligos RST2010DNA 10

6 Introduction DNA Campus Food Authenticity testing by DNA analysis focus on Fish Species determination Animal species determination Rice / Plant variety / species testing Production strain determination / discrimination Food Forensics RST2010DNA 11 Horse meat scandal 12

7 From sample to species determination RST2010DNA 13 From sample to species determination Biological sample DNA extraction PCR w/o other analysis Species determination RST2010DNA 14

8 From sample to species determination Biological sample - meat, meat products, - fish and seafood DNA extraction PCR w/o other analysis - fat tissue, connective tissue - organs like liver, kidney, heart - processed meat / fish - all food containing meat / fish Species determination RST2010DNA 15 From sample to species determination Biological sample DNA extraction - validated method in accredited laboratory PCR w/o other analysis Species determination RST2010DNA 16

9 From sample to species determination Biological sample DNA extraction PCR w/o other analysis RealTime PCR PCR and Sequencing analysis Species determination RST2010DNA 17 From sample to species determination Biological sample DNA extraction PCR w/o other analysis Species determination Specific amplification curves Sequencing data RST2010DNA 18

10 Mitochondrial DNA and nuclear DNA Mitochondrion Nucleus 19 Mitochondrial DNA and nuclear DNA Mitochondrion with circular DNA Nucleus with Chromosomes (2x) 20

11 Species determination = = DNA Barcoding of mitochondrial DNA Mitochondrial DNA is partially highly variable between the species has conserved regions, which allow to design universal assays working for many species obtained sequences between are specific for the different species is abundantly in the cell is rather unsusceptible to degradation RST2010DNA 21 Mitochondrial DNA RST2010DNA 22

12 Mitochondrial DNA RST2010DNA 23 Analysis of mitochondrial DNA PCR and Sequencing analysis universal primers detect DNA from all species minor species detected between 5 and 10% admixture quantification difficult due to imbalanced amplification of different species with some universal primers, (could be avoided with specific assay design) direct detection method unexpected species would also be detected 24

13 Analysis of mitochondrial DNA Sequence of 16s rrna from equine DNA 25 Analysis of mitochondrial DNA RealTime PCR sensitive method (detection of 0.01 % of minor species) specific method (controlled by melt curve analysis) detection of PCR products in RealTime allows quantification wide linear range of amplification indirect detection method 26

14 Analysis of mitochondrial DNA RealTime PCR specific method: unique assay for every species avoid cross - amplification 27 Analysis of mitochondrial DNA RealTime PCR specific method: assay for cattle, pig, sheep, goat, horse. new request: assay for deer ( venisson burger ) 28

15 Specific detection of deer DNA Detection of deer (Cervus elaphus) DNA: with unmodified oligos parallel detection of roe deer (Capreolus capreolus) at similar Cp values target sequence at forward primer identical one SNP (C to T) 3 bases from 3 end (100 % homology to deer sequence both oligos were modified with TINA 29 Specific detection of deer DNA Dilution series of 2 ng, 200 pg, 20 pg, 2 pg total DNA from Cervus elaphus : Analysis in triplicates on Roche LC480 II, detection with SybrGreen; 2 pg corresponds to DNA from 1/3 cell (red arrow) 30

16 Specific detection of deer DNA MeltCurve analyis after PCR with DNA from dilution series of 2 ng, 200 pg, 20 pg, 2 pg total DNA from Cervus elaphus : Analysis in triplicates on Roche LC480 II, detection with SybrGreen; Identical melt curves, no unspecific amplicon 31 Specific detection of deer DNA PCR with 2ng total DNA from Cervus elaphus (red arrow)/ Capreolus capreolus (blue arrow) and Equus caballus (green arrow) Analysis on Roche LC480 II, detection with SybrGreen; 32

17 Specific detection of deer DNA 5 Cp PCR with 2ng total DNA from Cervus elaphus (red arrow) / Capreolus capreolus (blue arrow) and Equus caballus (green arrow) Analysis on Roche LC480 II, detection with SybrGreen; 33 Specific detection of deer DNA MeltCurve analyis after PCR with 2ng total DNA from Cervus elaphus (red arrow) / Capreolus capreolus (blue arrow) and Equus caballus (green arrow) 34

18 Specific detection of deer DNA Detection of deer (Cervus elaphus) DNA: with TINA oligos we have a sensitive and specific assay for deer, one SNP (C to T) 3 bases from 3 end leads to 5 Cp difference Next validation: Avoiding cross amplication of roe deer DNA by shift of the reverse primer of one or two bases (locate SNP nearer / directly to the 3 end) 35 Specific detection of pig DNA Detection of pig (Sus scrofa) DNA: development of an new assay with very short amplicons for detection of highly degraded DNA: no amplification with unmodified oligos both oligos were modified with TINA 36

19 Specific detection of pig DNA Dilution series of 2 ng, 200 pg, 20 pg, 2 pg total DNA from Sus scrofa: Analysis in triplicates on Roche LC480 II, detection with SybrGreen; 2 pg corresponds to DNA from 1/3 cell 37 Specific detection of pig DNA PCR with 200 pg genomic DNA from Sus scrofa, Bos taurus, Ovis aries and Equus caballus : Analysis on Roche LC480 II, detection with SybrGreen; 38

20 Specific detection of pig DNA MeltCurve analyis after PCR with 200 pg genomic DNA from Sus scrofa (red), Bos taurus (blue), Ovis aries (green) and Equus caballus (black) 39 Specific detection of pig DNA Detection of pig (Sus scrofa) DNA: In this case also oligos modified with TINA does not lead to a sensitive and specific assay 40

21 Pathogen detection Detection of pathogens in buildings: Red-flag organisms should not be present (causing health problems) Detection by RealTime PCR with DNA extracted from filter swabs 41 Pathogen detection 42

22 Pathogen detection 43 Pathogen detection Detection of Streptomyces spp.: Comparison of performance of an assay with non modified oligos and TINA modified oligos with identical oligos sequences under identical PCR conditions 44

23 Pathogen detection 3,098 Amplification curves with non modified oligos 0,5 µm, detection with SybrGreen to 50 target copies 45 Pathogen detection 17,174 Amplification curves with TINA modified oligos 0,5 µm, detection with SybrGreen to 50 target copies 46

24 Pathogen detection 16,139 Amplification curves with TINA modified oligos 0,25 µm, detection with SybrGreen to 50 target copies 47 Pathogen detection Comparison of amplification curves with TINA modified oligos (high fluorescence) and non modified oligos (low fluorescence), detection with SybrGreen to 50 target copies 48

25 Pathogen detection MeltCurve analyis after PCR with 500 copies template DNA with non modified oligos (green) and TINA modified oligos (red): sharper peak and higher fluorescence values with modified oligos 49 Pathogen detection MeltCurve analyis after PCR with 500 copies template DNA with non modified oligos (green) and TINA modified oligos (red): slight increase of temperature maximum 50

26 Pathogen detection MeltCurve analyis after PCR with 500 copies template DNA with non modified Oligos, 0,5µm (green) and TINA modified oligos (0,5 µm, 0,375 µm and 0,25 µm (red): sharper peak and higher fluorescence values with modified oligos 51 Pathogen detection MeltCurve analyis after PCR with 500 copies template DNA with non modified oligos (green) and TINA modified oligos (red): slight increase of temperature maximum 52

27 Pathogen detection Detection of Streptomyces spp.: comparison of performance of - SybrGreen detection - TINA modified primer / non modified probes with identical oligos sequences under identical PCR conditions 53 Pathogen detection Non modified Oligo TINA modified Oligo TINA modified Oligo / non modified Probe copies copies 5000 copies 500 copies 50 copies Cp values at PCR with 50 to target copies 54

28 TINA: Summary Influence of TINA molecule at three assays: 1. Deer versus Roe deer and other species: The assay is very sensitive and specific for Deer; SNP located 3 bases from 3 end of primer leads to 5 Cp/Ct difference 55 TINA: Summary Influence of TINA molecule at three assays: 2. Detection of porcine DNA: This assay is only slightly better compared to non modified oligos but still not sensitive and cross-amplify with other species 56

29 TINA: Summary Influence of TINA molecule at three assays: 3. Detection of Streptomyces spp. DNA: The assay is more sensitive: about 3 Cp/Ct difference to assay with non modified Oligos Melt curve peaks are higher and sharper (please note that melt max switches ) 57 TINA: Summary The TINA molecule can help to increase sensitivity and specificity of difficult assays The increase in melt temperature of the modified oligos can allow to design new assays in AT rich sequences SNP in primer binding site can have bigger influences compared to non modified oligos 58

30 TINA: Introduction / Theory TINA is no universal solution to make your assay work but if your primers or primer design are the limiting factor, TINA may be the modification of choice to make it work Therefore: You must try it by yourself 59 TINA: Introduction / Theory Thank you to Julia Mohrbacher in our lab and Thank you for your attention RainerSchubbert@Eurofins.com 60