Sanger Sequencing by capillary electrophoresis (CE) is used to determine the specific base sequence of a particular DNA fragment or gene segment. This method is based on the selective incorporation of four different fluorescent dye-tagged dideoxynucleotide chain terminators by DNA polymerase during in vitro DNA replication (cycle sequencing); the DNA polymerase adds the different nucleotides one by one onto a growing DNA template strand in a linear mode. This extension continues until a particular nucleotide is incorporated. Because of the nucleotide's dideoxy-configuration the polymerase cannot add any other base to this fragment and the extension is terminated. At the end of the selected number of cycles, numerous fragments with different lengths and one labelled nucleotide at the end are then generated.
Important aspects in the Sanger Sequencing are the purification of PCR products prior to cycle sequencing and the subsequent dyeterminator reaction cleanup step prior to loading for electrophoresis. The use of high-quality reagents during all phases of the workflow will eliminate any interference, and will optimize conditions to produce high-quality capillary electrophoresis peaks and sequencing data.
DyeTerminator Cycle Sequencing Kits, Polymers and Buffers
BrilliantDye™ Terminator Cycle Sequencing Kits provide all reagents (BrilliantDye™ Terminator, 5x sequencing buffer, pGEM control, M13 control primer) required for Sanger cycle sequencing reactions, in a ready for use pre-mixed format. The name brand drop-in replacement for BigDye® Terminator Cycle Sequencing Kits: no change to protocol, volume or setting. BrilliantDye™ can be used with other Sanger workflow products (e.g. NimaPOP™ or POP™ polymers) on 3130, 3500/SeqStudio™ Flex and 3730 Series Genetic Analyzers.
BigDye and POP are trademarks of Thermo Fisher Scientific.
NimaPOP™-4, 6 and 7 Polymers for 3130, 3500/SeqStudio™ Flex and 3730 Series Genetic Analyzers come in bottles or RFID-labeled pouches, compatible with all Data Collection Software versions. NimaPOP™ Polymers are widely used alternatives to POP™ Polymers and can be used without any change in run protocols, conditions, or spectral calibrations. NimaPOP™ Polymer combined with NimaPOP™ 10x Running Buffer us known for its excellent dynamic coating and separating capabilities. NimaPrime™ Conditioning Reagent is used to prime and wash the Genetic Analyzer polymer pump when switching between polymer types.
PCR Purification Kits
ExS-Pure™ Enzymatic PCR Purification Kit removes excess primers and unincorporated nucleotides from the PCR reaction prior to sequencing, as any leftover primers can interfere with the strand-specific cycle sequencing reactions that will follow. This kit provides a ready-for-use reagent containing a well-balanced mixture of two highly effective enzymes, recombinant hydrolytic and heat-labile enzymes, Exonuclease-I and Shrimp Alkaline Phosphatase. Prior to cycle sequencing, ExS-Pure™ is added directly to the PCR product, and cleanup is performed in a standard thermal cycler within 5 minutes. ExS-Pure™ is the widely adopted drop-in alternative to ExoSAP-IT™.
ExoSAP-IT is a trademark of Thermo Fisher Scientific.
The AmpliClean™ Cleanup Kit provides highly efficient and cost-effective magnetic bead-based technology for high-throughput purification of PCR products. AmpliClean™ removes, salts, primers, primer-dimers and dNTPs, leaving ultra-pure DNA of the desired length. It requires no centrifugation or vacuum filtration steps. Purified PCR products can be used directly for Sanger cycle sequencing. The AmpliClean™ protocol is highly flexible for manual operation and can be easily incorporated in any liquid handling system for automated processing. AmpliClean™ magnetic beads are the widely adopted alternative to AMPure XP beads for purification.
AMPure XP is a trademark of Beckman Coulter.
DyeTerminator Cleanup Kits
The iX-Pure™ DyeTerminator Cleanup Kit efficiently removes unincorporated dyeterminators and salt-ions from extension products cycle sequenced with BrilliantDye™ and BigDye™ chemistries. Resin-based clean-up is completed within 40 minutes. Purified dye-labeled extension products can be injected directly into Genetic Analyzers. The iX-Pure™ Dye Terminator Cleanup Kit is the proven drop-in alternative for BigDye XTerminator™.
BigDye and BigDye XTerminator are trademarks of Thermo Fisher Scientific.
The D-Pure™ DyeTerminator Cleanup Kit is the magnetic bead-based alternative to iX-Pure™ DyeTerminator or BigDye XTerminator™ and the high-quality alternative for CleanSEQ DyeTerminator Removal Kit. Magnetic cleanup is completed directly on the cycle sequencing plate, either manually or automated. Purified products can be loaded directly on Genetic Analyzers without further resuspension. Compatible with BigDye XTerminator™ run modules.
BigDye and BigDye XTerminator are trademarks of Thermo Fisher Scientific. CleanSEQ is a trademark of Beckman Coulter.
Pump and Capillary Array Maintainance
Capillary array as a carrier for polymers (e.g. POP™ and NimaPOP™ separation media) is critical for peak separation in Sanger Sequencing and Fragment Analysis. At the end of the array life, it will not generate well-separated peaks. ReCAP™ Capillary Array Regeneration Kits for 3130 and 3730 Series Genetic Analyzers have been designed for in-line cleaning of the capillary array, aimed at rejuvenating failing arrays to like new, maintaining optimal performance and improve operational life span. ReCAP™ Capillary Array Regeneration Kits contains 4 different buffer solutions, to be applied sequentially to the pump blocks and capillaries via the instrument’s inbuilt Water Wash Wizard protocol.
NimaPrime™ Conditioning Reagent is used to prime the polymer pump of Genetic Anayzers, as well as wash the polymer pump and channels when switching between polymer types or performing routine maintenance. The RFID labeled pouche is compatible with all Data Analysis Software versions of 3500 Series and SeqStudio™ Flex. NimaPrime™ Conditioning Reagent is a drop-in replacement for Applied Biosystems™ Conditioning Reagent, for 3500/SeqStudio™ Flex.
Applied Biosystems and SeqStudio are trademarks of Thermo Fisher Scientific.
Capillary Electrophoresis Instrument Compatibility
Automation of Sanger Sequencing based on capillary electrophoresis (CE) has been made possible with the development of a variety of DNA sequencing instruments and platforms, such as Applied Bioystems® Genetic Analyzers and Promega Spectrum Compact CE and the recent Superyears® Genetic Analyzer Classic and Honor Series.
Contact us to match the right NimaGen reagents and consumables with your CE instrument(s), supporting your whole Sanger Sequencing workflow.
Applied Bioystems® Genetic Analyzers comprise a range of 4-capillary (3130 Series), 8-capillary (3500/SeqStudio™ Flex), 16-capillary (3130 Series), 24-capillary (3500/SeqStudio™ Flex), 48-capillary or 96-capillary array (3730 Series) instruments.
Promega Spectrum Compact CE System is a 4-capillary array system, designed for use with existing sequencing chemistries using fluorescently labeled dideoxynucleotide triphosphate and 4-, 5-, 6- and next-generation 8-color STR kits commercially available.
Superyears® Genetic Analyzer Classic and Honor Series provide 6- and 8-color fluorescence technology.
Applied Biosystems and SeqStudio are trademarks of Thermo Fisher Scientific. Spectrum Compact is a trademark of Promega Corporation. Superyears is a trademark of Nanjing Superyears Genetechnology Co.