High-Resolution Separation of Nucleic Acid Fragments

The invention of the polymerase chain reaction (PCR) in the early 1980s revolutionized molecular biology and molecular medicine. Since then, PCR has developed into a widely used technique for analyzing DNA in many common procedures such as the cloning of specific DNA fragments, genetic testing, and detection of infectious bacteria or viruses. PCR is used in many application areas—from cancer research, paternity testing, and veterinary diagnostics to food testing and molecular microbiology.

The most commonly used method for visualizing and separating PCR amplicons is gel electrophoresis using self-poured slab gels. While gel electrophoresis is relatively easy to adopt in any laboratory, it has a number of disadvantages, including highly labor-intensive preparation of slab gels, and user exposure to hazardous chemicals such as ethidium bromide as a staining agent. Moreover, separation times can be rather long, depending on the experimental settings, and slab gels usually do not provide the high resolution required to meet today’s research demands.

The QIAxcel Advanced System (QIAGEN GmbH, Hilden, Germany) overcomes these challenges by providing fully automated high-resolution capillary electrophoresis, thus increasing the efficiency and standardization of nucleic acid separation.

Fully automated multicapillary electrophoresis

The QIAxcel Advanced System is a benchtop multichannel capillary electrophoresis device that completely automates sensitive, high-resolution nucleic acid separation for a few samples up to 96 samples per run. These different throughput capabilities make the system suitable for laboratories with flexible throughput requirements.

Ready-to-run gel cartridges contain 12 separation microchannels, with a built-in gel matrix, enabling sample analysis with very little hands-on interaction, thus minimizing exposure to hazardous chemicals and reducing manual handling errors. The instrument includes a novel multiplexed fluorescence detection design, which includes an array of light-emitting diodes and micro-optical collectors that latch onto the capillaries within the gel cartridges. Fragments that are migrating through the gel matrix pass through excitation and detection windows, and signals are read using a computer and suitable software program (Figure 1).

Figure 1 – Sample separation using the QIAxcel Advanced System. Nucleic acid molecules are size-separated by applying an electrical current to a gel-filled capillary. A detector in the instrument detects the nucleic acid molecules as they migrate toward the positively charged terminus of the capillary. These data are passed through a photomultiplier before being converted to an electropherogram and gel image by QIAxcel ScreenGel software.

Easy data analysis and storage

The ScreenGel software provided with the QIAxcel Advanced System supports data collection and analysis. Interactive tools simplify analysis; facilitate rapid data interpretation; and provide the flexibility to offer different data views, including gel image and electropherogram formats. Results can be viewed individually, or overlay views can be created for sample and data comparison. All-in-one analysis for multiple data sets simplifies evaluation; a unique algorithm allows for automatic sizing of fragments.

Assay-specific process profiles can be set up and saved, including all steps of sample processing—from running samples to data analysis, generating reports, and exporting data to supporting standardization of routine applications.

Security features in the software include a password-protected user login to prevent unauthorized access and data manipulation, audit trail documentation for configuration files and system events, as well as automatic saving and archiving of write-protected raw data.

Applications

The QIAxcel Advanced System offers high detection sensitivity, enabling robust results with even low concentrations of nucleic acid. Preprogrammed methods, in combination with the corresponding gel cartridges (Table 1), permit the separation and analysis of a variety of nucleic acids, including single or multiple PCR fragments, DNA digested with restriction endonucleases, synthesized oligonucleotides, total RNA, and cRNA.

Table 1 – Available QIAxcel kits

High-resolution nucleic acid separation

Applications such as microsatellite instability testing, insertion/deletion studies, analysis of variable number of tandem repeats (VNTRs), short tandem repeats (STRs), and simple sequence repeats (SSRs) usually require separation of amplicons that differ by only a few base pairs in size, which is beyond the resolution capacity of standard agarose gels. With a resolution of 3–5 bp for fragments smaller than 0.5 kb, the QIAxcel Advanced System addresses these needs, providing higher accuracy than slab-gel methods and greater confidence in data interpretation.

Figure 2 – Detection of amplicons with small size differences. Alignment marker: 15/1000 bp. Method: OM500. Lanes—A01: pUC18/HaeIII in PCR buffer 10 ng/μL, A02: PCR fragment 96 bp, A03: PCR fragment 99 bp, A04: PCR fragments 96 and 99 bp, A05: PCR fragment 222 bp, A06: PCR fragment 225 bp, A07: PCR fragments 222 and 225 bp, A08: PCR fragment 332 bp, A09: PCR fragment 334 bp, A10: PCR fragments 332 and 334 bp, B01: pUC18/HaeIII in PCR buffer 10 ng/μL, B02: PCR fragment 441 bp, B03: PCR fragment 444 bp, B04: PCR fragments 441 and 444 bp.

Verification of best resolution was shown by generating PCR products of 96/99 bp, 222/225 bp, 332/334 bp, and 441/444 bp using the HotStarTaq® PCR kit (QIAGEN) according to the kit protocol. Amplicons were run on the QIAxcel with a DNA High Resolution gel cartridge individually and as a mixture, using the OM500 method and a 15/1000 bp alignment marker (Figure 2). The gel clearly shows two separated bands with size differences of 3 bp, indicating that the system is highly suited for the specific analysis of PCR products with small size differences. This characteristic is required when analyzing multiplex PCR reactions, e.g., for mutation detection (Figure 3).

Figure 3 – Analysis of 19-plex PCR. Multiplex PCR of 19 targets (99–955 bp) was performed using standard conditions for the QIAGEN Multiplex PCR Plus Kit, without further optimization, or using a variety of concentrations of a hot-start DNA polymerase from Supplier AII. a) Analysis using an agarose gel. b) Analysis using the QIAxcel Advanced System. M: GelPilot® 100 bp Plus Ladder (QIAGEN).

Summary

Easy-to-use, automated solutions that replace manual routine tasks are becoming more and more important, especially in laboratories seeking to streamline work flow and improve efficiency. The QIAxcel Advanced System fully automates nucleic acid separation and replaces tedious and time-consuming slab gel electrophoresis. With its fast run times, throughput flexibility, and high-resolution capabilities, it is an effective tool for the analysis of demanding multiplex PCR applications.

Dr. Carola Schade is Director, Head of Instruments Business Life Sciences; QIAGEN GmbH, Qiagen Strasse 1, 40724 Hilden, Germany; tel.: +49 2103 290; e-mail: carola.schade@qiagen.com.

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