Twisted Intercalating Nucleic Acid
(TINA) – a novel group of molecules
with improved performance in PCR
and qPCR applications
Dr. Rainer Schubbert, Eurofins Medigenomix
19.03.2013
Twisted Intercalating Nucleic Acid (TINA) – a novel group of molecules with improved performance in PCR and qPCR applications
www.eurofins.de
Topics
Topics of this talk:
 What is TINA
 Applications: Analytical methods for species
determination
 Assay Setup and validation
 Results
Twisted Intercalating Nucleic Acid (TINA) – a novel group of molecules with improved performance in PCR and qPCR applications
2
Twisted Intercalating Nucleic Acid- TINA
3
Twisted Intercalating Nucleic Acid (TINA) – a novel group of molecules with improved performance in PCR and qPCR applications
TINA-PCR: PCR components
TINA intercalator
Forward primer
z
5´ ATAATCGACGTACGAGTC
3´
3´………TATTAGCTGCATGCTCAG………………………………CGCATTACGTGACTCATC……………5´
5´………ATAATCGACGTACGAGTC………………………………GCGTAATGCACTGAGTAG……………3´
Target
3´CGCATTACGTGACTCATC
5´
z
Reverse primer
Plus:
PCR buffer
TINA intercalator
Nucleotides
DNA polymerase
Twisted Intercalating Nucleic Acid (TINA) – a novel group of molecules with improved performance in PCR and qPCR applications
4
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.
Twisted Intercalating Nucleic Acid (TINA) – a novel group of molecules with improved performance in PCR and qPCR applications
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.
Twisted Intercalating Nucleic Acid (TINA) – a novel group of molecules with improved performance in PCR and qPCR applications
6
TINA: Introduction / Theory
This increase in specific primer annealing can
be utilized to increase the overall specificity
of a given assay.
Twisted Intercalating Nucleic Acid (TINA) – a novel group of molecules with improved performance in PCR and qPCR applications
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.
Twisted Intercalating Nucleic Acid (TINA) – a novel group of molecules with improved performance in PCR and qPCR applications
8
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.
Twisted Intercalating Nucleic Acid (TINA) – a novel group of molecules with improved performance in PCR and qPCR applications
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
Twisted Intercalating Nucleic Acid (TINA) – a novel group of molecules with improved performance in PCR and qPCR applications
10
9
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
Twisted Intercalating Nucleic Acid (TINA) – a novel group of molecules with improved performance in PCR and qPCR applications
11
Horse meat scandal
Twisted Intercalating Nucleic Acid (TINA) – a novel group of molecules with improved performance in PCR and qPCR applications
12
From sample to species determination
RST2010DNA
Twisted Intercalating Nucleic Acid (TINA) – a novel group of molecules with improved performance in PCR and qPCR applications
13
13
From sample to species determination
Biological sample
DNA extraction
PCR w/o other analysis
Species determination
RST2010DNA
Twisted Intercalating Nucleic Acid (TINA) – a novel group of molecules with improved performance in PCR and qPCR applications
14
14
From sample to species determination
Biological sample
- meat, meat products,
- fish and seafood
DNA extraction
- fat tissue, connective tissue
- organs like liver, kidney, heart
PCR w/o other analysis
- processed meat / fish
- all food containing meat / fish
Species determination
RST2010DNA
15
15
Twisted Intercalating Nucleic Acid (TINA) – a novel group of molecules with improved performance in PCR and qPCR applications
From sample to species determination
Biological sample
DNA extraction
- validated method in accredited
laboratory
PCR w/o other analysis
Species determination
RST2010DNA
Twisted Intercalating Nucleic Acid (TINA) – a novel group of molecules with improved performance in PCR and qPCR applications
16
16
From sample to species determination
Biological sample
DNA extraction
PCR w/o other analysis

RealTime PCR

PCR and Sequencing analysis
Species determination
RST2010DNA
17
17
Twisted Intercalating Nucleic Acid (TINA) – a novel group of molecules with improved performance in PCR and qPCR applications
From sample to species determination
Biological sample
DNA extraction
PCR w/o other analysis
Species determination

Specific amplification curves

Sequencing data
RST2010DNA
Twisted Intercalating Nucleic Acid (TINA) – a novel group of molecules with improved performance in PCR and qPCR applications
18
18
Mitochondrial DNA and nuclear DNA
Mitochondrion
Nucleus
Twisted Intercalating Nucleic Acid (TINA) – a novel group of molecules with improved performance in PCR and qPCR applications
19
Mitochondrial DNA and nuclear DNA
Mitochondrion with circular DNA
Nucleus with
Chromosomes (2x)
Twisted Intercalating Nucleic Acid (TINA) – a novel group of molecules with improved performance in PCR and qPCR applications
20
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
Twisted Intercalating Nucleic Acid (TINA) – a novel group of molecules with improved performance in PCR and qPCR applications
21
21
Mitochondrial DNA
RST2010DNA
Twisted Intercalating Nucleic Acid (TINA) – a novel group of molecules with improved performance in PCR and qPCR applications
22
22
Mitochondrial DNA
RST2010DNA
Twisted Intercalating Nucleic Acid (TINA) – a novel group of molecules with improved performance in PCR and qPCR applications
23
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
Twisted Intercalating Nucleic Acid (TINA) – a novel group of molecules with improved performance in PCR and qPCR applications
24
Analysis of mitochondrial DNA
Sequence of 16s rRNA from equine DNA
Twisted Intercalating Nucleic Acid (TINA) – a novel group of molecules with improved performance in PCR and qPCR applications
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
Twisted Intercalating Nucleic Acid (TINA) – a novel group of molecules with improved performance in PCR and qPCR applications
26
Analysis of mitochondrial DNA
RealTime PCR

specific method:
unique assay for every species
avoid cross - amplification
Twisted Intercalating Nucleic Acid (TINA) – a novel group of molecules with improved performance in PCR and qPCR applications
27
Analysis of mitochondrial DNA
RealTime PCR

specific method:
assay for cattle, pig, sheep, goat, horse….

new request:
assay for deer („venisson burger“)
Twisted Intercalating Nucleic Acid (TINA) – a novel group of molecules with improved performance in PCR and qPCR applications
28
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
Twisted Intercalating Nucleic Acid (TINA) – a novel group of molecules with improved performance in PCR and qPCR applications
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)
Twisted Intercalating Nucleic Acid (TINA) – a novel group of molecules with improved performance in PCR and qPCR applications
30
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
Twisted Intercalating Nucleic Acid (TINA) – a novel group of molecules with improved performance in PCR and qPCR applications
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;
Twisted Intercalating Nucleic Acid (TINA) – a novel group of molecules with improved performance in PCR and qPCR applications
32
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;
Twisted Intercalating Nucleic Acid (TINA) – a novel group of molecules with improved performance in PCR and qPCR applications
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)
Twisted Intercalating Nucleic Acid (TINA) – a novel group of molecules with improved performance in PCR and qPCR applications
34
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)
Twisted Intercalating Nucleic Acid (TINA) – a novel group of molecules with improved performance in PCR and qPCR applications
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
Twisted Intercalating Nucleic Acid (TINA) – a novel group of molecules with improved performance in PCR and qPCR applications
36
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
Twisted Intercalating Nucleic Acid (TINA) – a novel group of molecules with improved performance in PCR and qPCR applications
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;
Twisted Intercalating Nucleic Acid (TINA) – a novel group of molecules with improved performance in PCR and qPCR applications
38
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)
Twisted Intercalating Nucleic Acid (TINA) – a novel group of molecules with improved performance in PCR and qPCR applications
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
Twisted Intercalating Nucleic Acid (TINA) – a novel group of molecules with improved performance in PCR and qPCR applications
40
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
Twisted Intercalating Nucleic Acid (TINA) – a novel group of molecules with improved performance in PCR and qPCR applications
41
Pathogen detection
Twisted Intercalating Nucleic Acid (TINA) – a novel group of molecules with improved performance in PCR and qPCR applications
42
Pathogen detection
Twisted Intercalating Nucleic Acid (TINA) – a novel group of molecules with improved performance in PCR and qPCR applications
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
Twisted Intercalating Nucleic Acid (TINA) – a novel group of molecules with improved performance in PCR and qPCR applications
44
Pathogen detection
3,098
Amplification curves with non modified oligos 0,5 µm, detection with SybrGreen
500.000 to 50 target copies
Twisted Intercalating Nucleic Acid (TINA) – a novel group of molecules with improved performance in PCR and qPCR applications
45
Pathogen detection
17,174
Amplification curves with TINA modified oligos 0,5 µm, detection with SybrGreen
500.000 to 50 target copies
Twisted Intercalating Nucleic Acid (TINA) – a novel group of molecules with improved performance in PCR and qPCR applications
46
Pathogen detection
16,139
Amplification curves with TINA modified oligos 0,25 µm, detection with SybrGreen
500.000 to 50 target copies
Twisted Intercalating Nucleic Acid (TINA) – a novel group of molecules with improved performance in PCR and qPCR applications
47
Pathogen detection
Comparison of amplification curves with TINA modified oligos (high fluorescence)
and non modified oligos (low fluorescence), detection with SybrGreen
500.000 to 50 target copies
Twisted Intercalating Nucleic Acid (TINA) – a novel group of molecules with improved performance in PCR and qPCR applications
48
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
Twisted Intercalating Nucleic Acid (TINA) – a novel group of molecules with improved performance in PCR and qPCR applications
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
Twisted Intercalating Nucleic Acid (TINA) – a novel group of molecules with improved performance in PCR and qPCR applications
50
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
Twisted Intercalating Nucleic Acid (TINA) – a novel group of molecules with improved performance in PCR and qPCR applications
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
Twisted Intercalating Nucleic Acid (TINA) – a novel group of molecules with improved performance in PCR and qPCR applications
52
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
Twisted Intercalating Nucleic Acid (TINA) – a novel group of molecules with improved performance in PCR and qPCR applications
53
Pathogen detection
35
30
25
Non modified Oligo
20
TINA modified Oligo
15
TINA modified Oligo / non
modified Probe
10
5
0
500000
copies
50000
copies
5000 copies 500 copies
50 copies
Cp values at PCR with 50 to 500.000 target copies
Twisted Intercalating Nucleic Acid (TINA) – a novel group of molecules with improved performance in PCR and qPCR applications
54
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
Twisted Intercalating Nucleic Acid (TINA) – a novel group of molecules with improved performance in PCR and qPCR applications
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
Twisted Intercalating Nucleic Acid (TINA) – a novel group of molecules with improved performance in PCR and qPCR applications
56
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 )
Twisted Intercalating Nucleic Acid (TINA) – a novel group of molecules with improved performance in PCR and qPCR applications
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
Twisted Intercalating Nucleic Acid (TINA) – a novel group of molecules with improved performance in PCR and qPCR applications
58
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
Twisted Intercalating Nucleic Acid (TINA) – a novel group of molecules with improved performance in PCR and qPCR applications
59
TINA: Introduction / Theory
Thank you to Julia Mohrbacher in our lab
and
Thank you for your attention
[email protected]
Twisted Intercalating Nucleic Acid (TINA) – a novel group of molecules with improved performance in PCR and qPCR applications
60