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