THE STANDARD FOR HIGH THROUGHPUT APPLICATIONS
High Throughput Sample Processing
for Real Time Analysis with Miniaturized
Reaction Volumes and Isothermal Chemistry
ABSTRACT
INTRODUCTION
The Nexar® Optimized for Real
Time Nucleic Acid Processing is
a fully automated solution for
low-volume, high-throughput
sample processing that
integrates liquid handling,
DNA amplification, and realtime fluorescence detection
in a single instrument. When
combined with Douglas
Scientific Array Tape™ and
EnviroLogix DNAble® chemistry,
it provides a fast, high-quality
alternative to real time PCR.
Douglas Scientific has developed a fast, cost effective, and robust solution
for high-throughput real time nucleic acid processing that simplifies sample
preparation, decreases cost per data point, and increases sample throughput.
When paired with DNAble® chemistry by EnviroLogix, the system can go from
sample to answer in 20 minutes or less.
This paper describes three proofof-concept experiments using
the Nexar Optimized for Real
Time Nucleic Acid Processing
and DNAble® isothermal DNA
amplification chemistry. These
experiments clearly show that
using the Nexar Optimized
for Real Time Nucleic Acid
Processing is a very powerful
and cost-effective method
for research and commercial
laboratories that want a fast
and accurate alternative to
traditional real time PCR.
Douglas Scientific Instrumentation
The Nexar® system used in the following experiments utilizes Array Tape™ in place
of standard microtiter plates, and is a fully automated, inline instrument designed
to handle low volume reactions.
• Nexar Optimized for Real Time Nucleic Acid Processing (Figure 1)
The Nexar Optimized for Real Time Nucleic Acid Processing provides real time
detection of amplification curves utilizing isothermal DNA amplification or
mainstream PCR chemistries. This system integrates liquid handling, assay
sealing, amplification and simultaneous detection, and real time data analysis
within one automated, inline instrument. Optimized for use with DNAble
isothermal chemistry, this instrument streamlines real time PCR detection
and allows laboratories to go from sample to answer in 20 minutes or less
with walk-away operation.
Figure 1. Nexar® Optimized for Real Time Nucleic Acid Processing
High Throughput Sample Processing
for Real Time Analysis
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• Array Tape (Figure 2)
Array Tape is a continuous polymer strip, serially embossed
with reaction wells in customizable volumes and formats,
including 96- and 384-well arrays. It is a flexible microplate
replacement.
samples without the gene of interest remain unchanged,
while samples containing the target DNA sequence amplify at
exponential rates.
MATERIALS AND METHODS
Materials
All reactions were completed using Array Tape™ and the
Nexar Optimized for Real Time Nucleic Acid Processing.
Figure 2. Array Tape™
• Intellics™ Software Analysis
The Douglas Scientific Intellics Software Suite was utilized
throughout these experiments. Array Builder™ was used to
design all experiment protocols and IntelliScore™ was used
to analyze the data.
ENVIROLOGIX DNAble CHEMISTRY
DNAble chemistry provides PCRquality results in 10 minutes or less at a
constant incubation temperature.
While traditional PCR chemistries rely
on multiple temperatures and one
enzyme, such as Taq polymerase, to
achieve DNA amplification, DNAble
utilizes two enzymes: a DNA
polymerase and a DNA-nicking
enzyme, which act together at one
temperature to achieve exponential
DNA amplification (figure 3). The
sequence-specific DNAble reaction
occurs at 56°C with the fluorescent
dye-labeled molecular beacon
generating a quantifiable signal. Both
PCR and DNAble chemistries produce
sigmoidal amplification curves when
monitored in real time. With purified
DNA templates, the duration of time at
56°C required for the molecular
beacon fluorescent signal to reach a
Figure 3.
detection threshold is proportional to
DNAble® Reactions
(Patents pending)
the amount of template DNA in the
reaction. This is referred to as
Threshold Time, and it is similar to the
Threshold Cycle used in mainstream quantitative PCR
reactions. In reactions utilizing DNAble chemistry, the
fluorescence readings of the no-template controls and DNA
High Throughput Sample Processing
for Real Time Analysis
A 2X reaction master mix was made by combining DNAble
isothermal DNA amplification reaction buffer, dNTPs, DNA
polymerase enzyme, DNA-nicking enzyme, oligonucleotide
primers, and molecular beacons labeled with FAM or ROX. One
stock of 2X master mix was used for all assays and reactions,
and sequence-specific oligonucleotide primers and molecular
beacons were added to each specific assay. The molecular
beacons for the human pathogen and E. coli Stx1 assays were
ROX-labeled, and the molecular beacon for the CMS assay was
FAM-labeled. Tris-EDTA buffer, pH 8.0 (TE buffer) was used as
the no-template control.
A gBlock Gene Fragment (purchased from IDT) containing the
DNA sequence of interest was used as the DNA template for the
human pathogen assay. A dilution series was generated from
seven consecutive 1:10 dilutions in TE buffer. The dilution series
started at 1x1010 and ended at 1x103 copies/µL. The E. coli Stx1
assay was completed with two different DNA templates:
purified bacterial genomic DNA from strains O157 and 3C were
diluted with TE buffer to a concentration of 1x102 copies/µL.
The CMS assay utilized purified CMS bacterial genomic DNA as
the DNA template. The DNA sample was diluted in TE buffer to
generate 1x104 and 1x102 copies/µL concentrations.
Methods
Samples and assays were dispensed into Array Tape using the
same method for all experiments, as described below.
1. 800 nL of each sample was simultaneously dispensed into
Array Tape using a 384-tip dispense pipette.
2. 800nL of 2X master mix was dispensed into each well using
the non-contact Dispense Jet module, giving a total
reaction volume of 1.6µL.
3. Amplification of all samples was completed in 10 minutes
at a constant temperature of 56°C. During that time, the
fluorescent signal of the molecular beacon was detected
every 30 seconds for real time analysis by the Nexar
Optimized for Real Time Nucleic Acid Processing.
RESULTS
The experiments described below utilize DNAble® chemistry
on the Nexar® Optimized for Real Time Nucleic Acid
Processing.
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Human Pathogen
The human pathogen assay demonstrated the dynamic range
of DNAble chemistry in Array Tape when analyzed using the
Nexar Optimized for Real Time Nucleic Acid Processing. This
reaction occurred very quickly, and the average threshold
time ranged from 42.1 seconds to 195.2 seconds for the
highest and lowest concentrations, respectively. The r2 value
of the linear regression was 0.961, indicating a good curve fit
over eight orders of magnitude. Real time amplification curves
of four dilutions (1x109, 1x107, 1x105, and 1x103) are shown in
figure 4.
The results show that there is no distinguishable difference
between the dilution samples, but there is a very large
difference in fluorescence intensity between the samples and
the no-template control.
Figure 6. Real time amplification curves of Stx1 with genomic.
Clavibacter michiganensis (CMS)
Figure 4. Amplification curves of human pathogen dilution series.
The regression analysis and curve fitting is shown in figure 5.
The CMS assay amplified purified bacterial genomic DNA in
a concentration-dependent manner, when analyzed using the
Nexar Optimized for Real Time Nucleic Acid Processing. These
results confirmed the quantification abilities of the DNAble
chemistry demonstrated above by the human pathogen
experiment. Two samples of purified CMS genomic DNA
(1x104 and 1x102 copies/µL) were analyzed with the DNAble
assay. The exponential amplification reaction was completed
quickly, but was also controlled by the concentration of target
DNA in the reaction. The average threshold times were
Figure 5. Regression analysis of the human pathogen dilution series.
These results indicate that even in a very fast acting DNAble
reaction such as this, the reaction is controlled by the
concentration of the input DNA and quantifiable results are
generated.
Escherichia coli (E. coli)
DNAble amplification of the Stx1 gene was completed with
DNA from two strains of E. coli, O157 and 3C, and the
reactions were analyzed in real time using the Nexar
Optimized for Real Time Nucleic Acid Processing. The
amplification curves are shown in figure 6.
High Throughput Sample Processing
for Real Time Analysis
Figure 7. Real time amplification curves of CMS from purified bacterial
genomic DNA.
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159 seconds for the sample containing 1x104 copies/µL and
179 seconds for the sample containing 1x102 copies/µL. These
results demonstrate that DNAble has quantification
capabilities with bacterial genomic DNA as well as purified
synthetic DNA templates. Real time amplification curves are
shown in figure 7.
CONCLUSIONS
The above experiments show that when DNAble chemistry is
combined with the Nexar Optimized for Real Time Nucleic
Acid Processing, amplified DNA is easily and clearly
distinguishable from the no-template controls, eliminating
the need for complex statistical analysis to separate out the
results. In addition, the amplification process is extremely
rapid and produces quantifiable results in 10 minutes or less.
Because the curves are linear (with r2 values in the region of
0.96), the amount of DNA present in the original sample can
be quantified with a very high degree of accuracy over a
very wide range of initial concentrations (from 103 to
1010 copies/µL).
When DNAble chemistry and the Nexar Optimized for Real
Time Nucleic Acid Processing is coupled with Douglas
Scientific Array Tape, another major advantage of the system
in comparison to mainstream PCR becomes evident, namely
extremely small reaction volumes (1.6μL). When sample
volumes are small, only small volumes of reagents are
required, significantly lowering reagent costs. In comparison
to mainstream PCR that typically requires sample volumes of
10-20μL, the DNAble/Nexar system requires just 1.6μL—a
roughly six to twelve-fold reduction.
The miniaturized reaction volumes and inline processing of
the Nexar® Optimized for Real Time Nucleic Acid Processing
enable laboratories to significantly decrease the cost of
chemistry for quantitative testing while increasing processing
capabilities. When the system is combined with DNAble®
isothermal DNA amplification chemistry, laboratories can go
from sample to answer in 20 minutes or less.
High Throughput Sample Processing
for Real Time Analysis
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