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The Fragment Analyzer™ Automated CE System

Product Description

The Fragment Analyzer™ Automated CE System is a fluorescence-based capillary electrophoresis instrument for both sizing and quantifying nucleic acids (DNA and RNA). By using a sensitive intercalating dye coupled with a powerful LED light source, Fragment Analyzer™ Automated CE System obviates the need for fluorescent labeled primers and can be used to separate dsDNA fragments and RNA (mRNA and total RNA). The Fragment Analyzer™ Automated CE System is the most flexible system on the market today, with the greatest sensitivity, the highest separation resolution over a wide range, the widest dynamic range and the fastest separation times.

This instrument comes available for customers who wish to run 12 samples at a time.

Currently kits are available to:

automate PCR gel electrophoresis
quantify and qualify RNA
quantify and qualify NGS fragments
quantify and qualify genomic DNA
do mutation detection, also known as TILLiNG, a reverse genetics technique
do microsatellite analysis

The application kits help laboratories to streamline workflow and avoid bottlenecks typically found with competitive lab-on-a-chip methods. Gels formulated to provide high resolution separation are also available for general fragment analysis, such as SSR/microsatellites, small and large PCR fragments (amplicons) and resolve fragments sized between 10bp and 40'000bp with resolution down to 2bp.

Parallel Capillary Electrophoresis : description of technology

Advanced Analytical's parallel capillary electrophoresis (CE) technology enables electro-driven analytical separations of DNA and RNA with on-line, simultaneous LED-based fluorescent detection.

Key features:

exchangeable capillary array assembly (33cm or 55cm for higher resolution)
fixed optical detection platform
high-voltage power supply
fluid pumping system
automatic tray positioning system

Customized software is tailored for specific applications and provides instrument control, data acquisition, and data analysis. End-users may view the CE separations as electropherograms or as digital gel images.

The instrument is designed to handle a 12 capillary array which can access all wells of a 96-well plate. Standard capillary dimensions are 50μm inner diameter (i.d.) and 200μm outer diameter (o.d.).

Anyone who was worked with agarose or acrylamide gel electrophoresis knows about the trade-off between speed vs. resolution. If you want high resolution, you use a long box. If you want answers quickly, you use a short box. Likewise with the Fragment Analyzer™ Automated CE System: various capillary lengths can be used, depending on the application, required resolution and desired speed of analysis. Longer arrays provide resolution down to 2bp for fragments under 300bp in length. Shorter arrays still provide good resolution with run times as fast as 15 minutes.

The inlet side of the capillary array is arranged in an 8x12 format with electrodes placed side-by-side with each capillary, allowing for direct injection via vacuum or voltage from standard 96-well plates. The capillaries are aligned in parallel in the detection window, which is positioned between the LED light source and the detector assembly. The outlets of the capillaries are bundled to a common reservoir where they are connected to a ground. The capillary arrays are modular in nature and a new capillary array can be exchanged in less than five minutes.

Room temperature air is circulated around the capillaries to modulate the temperature during the CE separation.

fs12diagram_v2c

Technology schematic (12 capillary version)

The optical platform consists of an LED light source (470nm excitation), camera lens and a CCD detector. Fluorescent light from the LED that passes through the windowed part of the capillary array, exciting the intercalating fluorescent dye present in the gel matrices, is focused on through the camera lens and spatially imaged onto the CCD detector. The capillary array is imaged continuously, providing real time, on-line detection of the CE separations.

The 20kV, 6mA total current high voltage power supply provides the appropriate voltage for electrokinetic sample injection and electrophoresis. The recommended operating limits are +/-16kV, 3mA total current (30μA per capillary).

A high-quality syringe pump provides controlled pressure or vacuum to the outlet reservoir of the capillary array. The ability to use vacuum is unique to the instrument. Vacuum injection can be used to inject samples without concern of salt interfering with the uptake of nucleic acids. Additionally, 2 modes of injection allow greater flexibility on the system and control of experiments when needed.

The instrument provides space for up to six 96-well plates. The positions of the stages are typically assigned to a (from the top) inlet/run buffer tray, waste tray, marker tray and three sample trays, respectively. When marker fragments are added directly to the samples (as designed in the NGS Fragment Analysis Kit, RNA Kits, and genomic DNA Kits), 4 sample trays (384 samples) can be loaded onto the instrument at one time. For automated sample addition and robotic access to the tray positions, modified (open faced) drawers are available.

12_capillary_version

Operational software provides complete automation for performing capillary conditioning, sample injection, electrophoresis separation and data processing while the analytical software, PROSize™ allows user to quickly review and assess data in a digital format.

Instrument capabilities

A fluorescence based system for both sizing and quantifying dsDNA fragments and RNA samples. The system was especially designed for separating nucleic acids. With the highest resolution and fastest run times of any CE system for dsDNA, the Fragment Analyzer™ Automated CE System is the ideal system to improve laboratory throughput and reduce analysis time.

System features

The Fragment Analyzer™ Automated CE System is the ideal system for dsDNA fragment and RNA analysis.

Features	Comment	Benefits
Capillary array cartridge	A long array (80cm total length) and a short array (55cm total Capillary Array Cartridge length) can be used to tailor separation times and resolution.	Run 12 samples in one experiment. Direct, parallel injection and separation of an entire plate at once, limits sample evaporation and handling.
Fast run times	Separations can be as short as 15 minutes or up to 90 minutes, depending on resolution requirements and application	Get more done in less time. The fastest separation time of any CE system available. Individual samples can be viewed in real-time as they separate.
High sensitivity	Detection limits as low as 5pg/µl for single fragments, and 100pg/µl for smears.	Allows direct injection of PCR samples without desalting or clean up. Better sensitivity means less sample is needed for analysis.
Accurate sizing	Better resolution equals better sizing.	More definitive interpretation of data.
Wide dynamic range	Dynamic range over 3 orders of magnitude (5pg/µl to 5ng/µl)	Confidence to visualize fragments without knowing sample concentration.
High separation resolution	Four gel formulas are available for various sizes of DNA fragments, each designed to achieve the highest resolution possible.	Resolve a wide range of dsDNA fragments, up to 40'000bp. Achieve as low as 2bp resolution for fragments <300bp. Custom gels can be developed.
Reagent kits	Kits are available for NGS fragment analysis, RNA analysis (total RNA and mRNA), genomic DNA and mutation detection (TILLiNG)	Premeasured, performance tested for ease of use and accurate results generation
Multiple injection modes	Premeasured, performance tested for ease of use and accurate results generation	Vacuum injection mode allows sample injection even in the presence of high salt (500mM).
Robotics interface	Only truly automated system for dsDNA and RNA analysis	Allows unlimited unattended sample analysis. No chips to fill, remove or clean.
User friendly software	Calculate fragment size and quantity, display data in multiple formats, color code results for quick reference and analysis.	Fast analysis of results for quick and accurate decision making. Reduce time to results

Applications

Gel electrophoresis automation

The fastest and easiest way to read your PCR products. Take the plate out of the PCR instrument, insert it onto the Fragment Analyzer and start the run. No more pipetting step, no more ethidium bromide !!

RNA Analysis

Assessment of RNA quality and RNA quantitation is critical for many molecular biology techniques. Manual assessment methods for both quantity and quality can use large amounts of precious materials and take a considerable amount of time to complete.

Several analytical instruments exist for the evaluation of RNA. However, these instruments either lack acceptable detection sensitivity or require significant hands-on time to complete chip loading and preparation. The Fragment Analyzer™ Automated CE System process for RNA – either for total RNA and/or messenger RNA – is completely automated, requiring minimal hands on time.

Total RNA sheet

NGS Fragment Analysis

Accurately quantifying and qualifying Next Gen Sequencing (NGS) fragments in a high throughput automated fashion is becoming critically important for more laboratories. The labs are seeing increased demand from researchers who want to sequence smaller amounts of information, but from larger pools of organisms.

NGS analysis sheet

Microsatellites/SSR

Microsatellite (SSR) analysis by PCR is a powerful molecular tool for screening organisms and determining polymorphic loci and genetic profiles. The Fragment Analyzer™ Automated CE System is an equally powerful and flexible fluorescence-based capillary electrophoresis instrument.

It provides size and quantification information for microsatellites and DNA fragments generated using unlabeled PCR primers specific to polymorphic microsatellite regions. With the highest resolution and fastest run times of any dsDNA fragment analysis capillary electrophoresis system, the Fragment Analyzer™ Automated CE System improves laboratory throughput and reduces analysis time of microsatellites.

Genomic DNA Analysis

Genomic DNA (gDNA) extraction from tissues, cells or organisms is a critical first step for all subsequent DNA analyses. Until now, assessing the quantity and quality of genomic DNA has typically required the use of two separate instruments: one for quantity, one for quality.

With the Fragment Analyzer™ Automated CE System platform, both genomic DNA quality and genomic DNA quantity can be assessed at the same time, using the same aliquot of sample and in less time than to do both by traditional methods.

Plasmid DNA

Separating the 3 forms of Plasmid DNA can be challenging. Each form -- nicked, supercoiled or linear -- can run differently depending on its' size and dye intercalation. The Fragment Analyzer™ Automated CE System is the only multichannel capillary electrophoresis instrument that can successfully separate the 3 plasmid DNA forms.

Large DNA Fragments

Would you undertake different research projects if you had an automated CE instrument that could separate large fragments ?

PCR fragments are not always small in size and sometimes larger amplicons (>1'500bp) need to be analyzed. Some competitive DNA analysis instruments are based on single-strand technology, and are very limited in the fragment sizes they can analyze. The Fragment Analyzer™ Automated CE System platform has several gels for larger fragments, including a gel for efficient separation of fragments <12'000bp and another gel for <40'000bp fragments.

Mutation Detection / TILLiNG

TILLiNG (Targeting Induced Local Lesions in Genomes) is a reverse genetics technique designed to detect unknown mutations in a gene by forming DNA heteroduplex as a result of mixing wild-type and chemically-induced mutated genes.

In order to increase experimental throughput, the genes are pooled first, then PCR amplified. This is followed by heteroduplex formation, then the samples are enzymatically digested. Next, digested cleaved DNA fragments are analyzed for scoring. The enzyme specifically recognizes the mismatched base pairs and cleaves the DNA at the site of mismatch.

Until now, detecting unknown mutations was both a challenging and a time intensive process. Advanced Analytical has developed a streamlined TILLiNG process that requires NO fluorescently labeled primers and NO sample clean-up. Detecting unknown mutations has never been easier, especially with this process developed for the Fragment Analyzer™ Automated CE System platform (see below). In comparison to the traditional method, the Fragment Analyzer™ Automated CE System method requires less than ½ the hands on time and analyses can be completed twice as fast (total time) compared to competitive methods.

Currently, Fragment Analyzer™ Automated CE System customers are using their instruments to do reverse genetics TILLiNG of rice, corn, wheat, lettuce, tomato and soybean.

Regardless of whether your lab wants to do TILLiNG (mutation detection) with diploids, tetraploids or hexaploids, the Fragment Analyzer™ Automated CE System has excellent sensitivity for analyzing medium and large-sized TILLiNG pools.

Mutation detection sheet

Software

Prior to using our system, many customers told us that data analysis with manual slab gels or poured vertical acrylamide gels was often a tedious process. We designed our system with their experiences in mind.

With the PROSize™ software, fragment analysis actually becomes enjoyable because it's quick, easy and completely automated. The easy-to-use software analyzes results within minutes of finishing a separation. Just import the data, align the upper/lower sizing marker and let the software do the rest. Instantly, both size and fragment quantity is at your fingertips. With a few simple manipulations, your 'print quality' results are ready.

Because readings are collected in digital format as electropherograms, the data can be viewed in multiple ways:

a representative gel view, similar to what you would normally see with agarose gels
as electropherograms
or in table format, depending on what is needed

Fragments are sized and quantified using ladders and marker fragments. While marker fragments are run in every lane, the ladder is run in one capillary only. After alignment of the upper/lower markers to each other to normalize the capillaries and to the ladder, the sample fragments can be accurately quantified and sized. Additionally, the data is exportable and can be hooked into a LIM system (additional programming required).

Other CE manufacturers often charge exorbitant seat licensing fees for software. Instead, we offer an affordable pricing structure for software seat license fees, so that researchers can use PRO Size™ software on their own PC or laptop computer. They aren't tied down to using only the computer that comes with the instrument. The extra user seat licenses aren't free, but they are reasonable.

Global configuration settings

For data files being processed for the first time, the Global Configuration Settings box opens allowing the user to set the parameters for the analysis. Settings for picking peaks, markers and the ladder calibration are adjustable. Other criteria for calculating quantity and size of fragments can be set when the file opens are set after the data is initially processed. Advanced settings allow the user to specify marker concentration, dilution factor and calculation mode (RNA, DNA, gDNA, NGS and Mutation Analysis):

fragment_analyzer_global_configuration

Gel view

View all 96 capillaries at the same time. After normalization of all lanes to the upper/lower marker fragments, all of the samples can be compared simultaneously. Each electropherogram separation can be viewed separately, in this view, by moving through the gel view image or clicking through the 96 well plate image. Fragment size can be easily determined using the pull down ruler, showing fragment size on both the electropherogram and on the gel view. A table, which can be exported, shows both individual peak sizes and concentrations of the selected separation:

fragment_analyzer_gel_view

Sample overlay view

For a close up view of any sample, the sample overlay view is used. Gel order can be arranged as needed and the gel view image is right clickable for easy pasting into another electronic media. This view shows both a gel view image and the selected electropherograms. Further editing capabilities include a sizing ruler, fragment color and background selection, copy/paste function and annotation:

fragment_analyzer_sample_overlay_large

Flagging feature

This feature was designed to reduce back end analysis time. Analytical parameters entered into the 'Flag' criteria windows can be saved and recalled for quick review of sample plates. Combining Boolean logic and limitation for bp units range or concentration allows for maximum flexibility. Results are provided in a table represented as a '1' or a '0' showing if the criteria were met:

fragment_analyzer_flagging_feature

Smear analysis

NGS fragments, RNA and genomic DNA can be quickly analyzed after separation to size and quantify smears of nucleic acids. Smear concentration can be calculated several ways, either by the software or through user defined smear boundaries. The software calculates the concentration in both ng/ul, nM, average size and %CV. Sample information can be exported to Excel©:

fragment_analyzer_smear_analysis

Report generation

Data can be downloaded as a .pdf or .xls file extension for all or part of the samples. The report contents can be tailored to output specific and critical information and easily modified to include more or less information:

fragment_analyzer_report_generation

Technical Specifications

Max sample throughput (12 capillary)	12 samples in 30 minutes for NGS fragments 12 samples in 45 minutes for RNA 12 samples in 12 minutes for DNA fragments
Max sample throughput (96 capillary)	96 samples in 60 minutes for NGS fragments 96 samples in 70 minutes for RNA 96 samples in 20 minutes for DNA fragments
Sample per run	Up to 96
Max unattended sample capacity	288 samples
Minimum sample volume	20µl; typically 2µl of sample is required for dilution
Gel sizing ranges	NGS: 35 - 6'000bp Total RNA and mRNA: 19 - 6'000 base Genomic DNA
Resolution	Gel dependent, as low as 2bp
Sizing accuracy	Typically 5% or better
Sensitivity	<100pg/µl for smears
Light source power	One 1'000mW LED
LED wavelength	470nm
Emission collection device	CCD detector
Software	Fragment Analyzer™ system software and PROSize™ data analysis software
Data export format	Microsoft© Excel spreadsheet
Environmental conditions	Indoor use, normal laboratory environment 20 - 23°C
Relative humidity range	<80% (non-condensing)
Electrical	100-200VAC; 50-60Hz; 15A
Instrument dimensions (LxWxH)	51.0 x 35.0 x 61.0cm
Instrument weight	38.5kg