Fragment Analysis provides size analysis, relative quantitation, and genotyping information for pathogens, variants, STR (Short Tandem Repeats) or SNPs (Single Nucleotide Polymorphisms). This technique is widely used in forensics (determining alleles that correspond to DNA fragments) and in animal species identification.
Fragment Analysis comprises a series of steps in which DNA fragments are fluorescent dye labeled, separated by capillary electrophoresis (CE), and compared to an internal DNA size standard. Typically, fluorescent dyes are attached to dedicated primers, and the selected fragments are amplified by PCR before electrophoresis. Frequently used dyes are FAM, TET, VIC, HEX, PET or NED. Prior to electrophoresis, each sample is mixed with the appropriate DNA size standard and formamide. DNA size standards are measured using separate dyes (ROX® or LIZ®).
Polymers, Buffers and Formamide Resuspension
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 is 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.
Seq-DI™ Formamide v2 is an ultra-pure highly deionized formamide used to resuspend sequencing samples, including DNA Size Standards, before electrokinetic injection on capillary electrophoresis systems. It is commonly used in fragment analysis. Seq-DI™ Formamide provides ready-for-use injection on 3130, 3500 and 3730 Series Genetic Analyzers and SeqStudio™ Flex, without any requirement for changes in protocol, conditions or spectral calibrations. The proven economical but high-quality alternative to Hi-Di™.
SeqStudio and Hi-Di are trademarks of Thermo Fisher Scientific.
Orange (LIZ® compatible) and Red (ROX®) DNA Size Standards
The Orange-500 and Red-500 DNA Size Standards are fluorescent labeled ultra-accurate internal size standards for the reproducible sizing of fragment analysis peaks during capillary electrophoresis and precise DNA fragment size comparisons between capillary electrophoresis runs. The 500 DNA Size Standard contains 16 Orange or Red dye-labeled, single-stranded fragments in the 35-500 nucleotides range with respective basepair lengths of 35, 50, 75, 100, 139, 150, 160, 200, 250, 300, 340, 350, 400, 450, 490 and 500 bp. Each of the DNA fragments is labeled with either LIZ® compatible or ROX® fluorophore resulting in sharp orange or red peaks under denaturing conditions. The Orange-500 and Red-500 DNA Size Standards are for use with five-dye or six-dye spectral calibration Genetic Analyzers, such as 3130, 3500 and 3730 and SeqStudio™.
LIZ, ROX and SeqStudio are trademarks of Thermo Fisher Scientific.
The Orange-600 and Red-500 DNA Size Standards contain 22 Orange dye-labeled, single-stranded fragments in the 45-595 nucleotides range with respective basepair lengths of 45, 72, 98, 128, 147, 167, 197, 227, 247, 267, 297, 327, 347, 367, 397, 427, 445, 465, 495, 525, 545, 565 and 595 bp. Each of the DNA fragments is labeled with either LIZ® compatible or ROX® fluorophore resulting in sharp orange or red peaks under denaturing conditions.
Please note: Orange-600 and Red-600 DNA Size Standards each contain 22 DNA size fragments (45 - 595 nucleotide range) versus 36 size fragments present in GeneScan™ 600 LIZ® and GeneScan™ 600 ROX®Dye Size Standards (20–600 nucleotide range).
GeneScan is a trademark of Thermo Fisher Scientific.
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 Kit for 3130/3730 Series Genetic Analyzers has 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 Kit 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 Fragment Analysis 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 Fragment Analysis 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.