High Throughput DNA Sample Processing and

THE STANDARD FOR HIGH THROUGHPUT APPLICATIONS
High Throughput DNA Sample Processing
and Target Detection for Endpoint and
Real Time Isothermal Analysis
ABSTRACT
INTRODUCTION
Douglas Scientific has developed
automated, inline instruments
for high throughput endpoint
and real time isothermal DNA
amplification.These instruments
have been validated for isothermal
chemistry provided by EnviroLogix
to create a pair of novel and very
powerful analytical tools. This
paper describes a number of
proof-of-concept experiments
using the lectin gene amplified
from soybean DNA on these
instruments.
Douglas Scientific has developed a fast, cost effective, and robust solution for
high-throughput genetic analysis that simplifies sample preparation, decreases cost
per sample, and increases sample throughput. When paired with the EnviroLogix’
proprietary DNAble® chemistry, the system can generate genetic answers in 20
minutes or less.
• The Nexar® system enables
end-point and real-time
isothermal reactions in Array
Tape™.
• Inline liquid handling, sealing,
incubation/amplification,
detection, and data analysis
with walk away time.
Douglas Scientific Instrumentation
Two Nexar systems were used in the soybean lectin experiments described in detail
below. Both instruments utilize Array Tape in place of a standard microtiter plate,
and both are fully automated, in-line instruments that are designed to handle low
reaction volumes.
• Nexar Optimized for Endpoint Isothermal DNA Amplification (Figure 1)
This Nexar couples endpoint analysis of isothermal DNA amplification with high
sample throughput and minimal operator interaction. The amplification occurs
in a heated chamber (with temperature control from ambient to 70⁰C) for time
periods from 4–60 minutes. The system integrates liquid handling sealing, amplification/incubation, detection, and data analysis in an inline system. Fluorescence
detection occurs after the amplification process with each 384-well plate being
read in less than 40 seconds. This system enables laboratories to go from sample
to answer in less than 15 minutes resulting in hundreds of thousands of data
points in a day.
• DNAble® chemistry works with
both purified and crude sample
matrices.
• The solution enables small
volume reactions (1.6 µL) and
high-throughput sample
processing.
• With 10 minute isothermal
amplification time, labs go from
sample to answer in 20 minutes
or less.
Figure 1. Nexar® Optimized for Endpoint Isothermal DNA Amplification
Endpoint and Real Time
Isothermal Analysis
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• Nexar Optimized for Real Time Nucleic Acid Processing
(Figure 2)
This Nexar provides real time detection. Go from sample
to result in just 20 minutes. This system integrates liquid
handling, sealing, amplification with simultaneous
detection, and data analysis in an inline system. Optimized
for DNAble isothermal chemistry, this instrument tames
the cumbersome process for real time PCR detection in a
fully automated, inline instrument with high specificity,
repeatable results, and walk-away operation.
Figure 2. Nexar® Optimized for Real Time Nucleic Acid Processing
• Array Tape™ (Figure 3)
Array Tape™ is a continuous polymer strip, serially embossed
with reaction wells in customized volumes and formats
including 96- and 384-well arrays. It is a flexible microplate
replacement for applications that enables a streamlined
lab process.
ENVIROLOGIX DNAble CHEMISTRY
DNAble assays utilize two sequence-specific oligonucleotide
primers to amplify a genetic region of interest, as well as a
third, sequence-specific, fluorescent
dye-labeled molecular beacon that
facilitates detection of the amplified
product. The DNAble reactions are
shown in figure 4 at right.
Unlike PCR, which relies on multiple
temperatures and one enzyme (such as
Taq polymerase) to achieve exponential
DNA amplification, DNAble employs
two enzymes—a DNA polymerase and
a DNA-nicking enzyme. These enzymes
work together at one temperature in
the presence of EnviroLogix’ proprietary
chemistry* to achieve exponential DNA
amplification, and like qPCR, produce
amplification curves in real time that
can be employed in the construction
of a standard curve for nucleic acid
quantification.
Figure 4.
DNAble® Reactions
(Patents pending)
MATERIALS AND METHODS
Materials
DNAble amplification reaction buffer, lectin-specific
oligonucleotide primers, a FAM-labeled molecular beacon,
dNTPs, DNA polymerase enzyme, and a DNA-nicking enzyme
were provided by EnviroLogix. A 2X reaction master mix was
created with these components and kept on ice until time
of use.
Two samples of soybean genomic DNA (purified, and unpurified
in crude sample extracts) were used for these experiments.
Figure 3. Array Tape™
• Intellics™ Software Analysis
The Douglas Scientific Software package was used to
analyze the data. IntelliScore™ provided scoring for
endpoint and real time data.
Purified DNA
Purified soybean genomic DNA was purchased from ZYAGEN
and diluted to 5ng/µL in Tris-EDTA (TE) buffer, pH 8.0. The
TE buffer was used as a no-template control. For dispensing
consistency and simplicity, 24 replicates of each sample type
(purified DNA, unpurified DNA, and TE buffer) were hand
pipetted into one 384-well microtiter plate. This sample plate
was used for all reactions.
Crude DNA
Unpurified genomic DNA was prepared using a simple crude
preparation protocol. Ground soybeans were added to room
temperature TE buffer, pH 8.0 at a 1:10 weight-per-volume
ratio (100mg into 1mL of buffer in a 2mL screw-top tube) and
heated in a dry heat block at 98C for six minutes. Samples
Endpoint and Real Time
Isothermal Analysis
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were cooled to room temperature, mixed, and centrifuged at
14,000 RPM in a micro-centrifuge for five minutes to collect
the spent ground soybeans. The supernatant was collected
and used without further dilution as the unpurified DNA
sample for the DNAble reaction.
Methods
The soybean lectin gene was amplified at a single temperature,
56⁰C for 10 minutes, using a DNAble isothermal DNA
amplification chemistry. The soybean lectin gene was chosen
as the genetic target for this experiment because it is wellstudied and highly regulated during the soybean life cycle. It
is commonly used as a housekeeping gene for genetic analysis
in soybeans. In this experiment, reactions were dispensed,
heated, and analyzed in low volume reactions (1.6µL) in Array
Tape on the Douglas Scientific Nexar Optimized for Endpoint
Isothermal DNA Amplification and the Nexar Optimized for
Real Time Nucleic Acid Processing systems.
Nexar Optimized for
Real Time Nucleic Acid Processing
The real time detection of lectin amplification in purified
and crude DNA samples demonstrated exponential curves
for all samples. As expected, wells or reactions containing
purified DNA (figure 6) increased in fluorescence in a much
more uniform way than wells containing unpurified or crude
preparation DNA (figure 7), but all samples achieved similar
endpoint fluorescence values. Most importantly, each reaction
curve contains an early exponential phase that can be used
to measure the point at which it crosses an experimentally
determined threshold just as that observed with qPCR systems. These threshold values, when collected from dilution series of
three or more dilutions, can be used to create a standard curve
for quantitative analysis of nucleic acids. These results indicate
that the sample preparation method, purified or crude, and the
detection method, endpoint or real time, can be specifically
tailored to fit the requirements and goals of each experiment.
RESULTS
The experiments described below utilize DNAble chemistry
on the Nexar Optimized for Endpoint Isothermal DNA
Amplification and the Nexar Optimized for Real Time Nucleic
Acid Processing systems.
Nexar Optimized for
Endpoint Isothermal DNA Amplification
Figure 5 shows the results of the DNAble reactions completed
in the Nexar Optimized for Endpoint Isothermal DNA
Amplification using purified and crude sample preparation
lectin DNA from soybeans.
Figure 6. Real Time Amplification Curves of Purified Soy DNA with DNAble
Lectin Assay
Figure 5. Endpoint Results of Soy DNA Amplification with DNAble Lectin Assay
The lectin gene was efficiently amplified in both purified
and unpurified soybean DNA samples. All samples showed
successful amplification—with an average fluorescence value
of 3,657 RFU for unpurified DNA and 4,526 RFU for purified
DNA. Both purified and unpurified samples were easily
distinguished from the no-template controls, which had an
average fluorescence value of 253 RFU.
Endpoint and Real Time
Isothermal Analysis
Figure 7. Real Time Amplification Curves of Crude Soy DNA with DNAble
Lectin Assay
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CONCLUSIONS
The Nexar system is a very powerful new analytical tool
for endpoint and real time isothermal data generation and
analysis. These experiments show that the Nexar system
specifically and exponentially amplifies genetic targets in 10
minutes or less at a constant temperature of 56⁰C when paired
with DNAble chemistry. In some cases, the amplification is
as fast as four-five minutes. They also show that the soybean
lectin gene was amplified in both purified and crude soybean
DNA samples, created no false-positive signals in the notemplate controls, and used very low reaction volumes of just
1.6µL. The real time data gives rise to its use in quantitative
analysis. The main advantages of the Nexar Optimized for
Endpoint Isothermal DNA Amplification and the Nexar
Optimized for Real Time Nucleic Acid Processing instruments
are very rapid results, significantly reduced chemistry costs
as a direct result of lower reaction volumes, significantly
increased throughput and complete in-line automation of the
entire process.
Endpoint and Real Time
Isothermal Analysis
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