Multiple types of templates can be used for Sanger sequencing. Both double-stranded and single stranded DNA can be sequenced. The most commonly used DNA templates include: plasmid, PCR products, and rolling cycle amplification products. In addition, bacterial artificial chromosome, genomic DNA, and cosmid DNA have also served as templates.
It is necessary to purify PCR product templates because after a PCR reaction, unincorporated dNTPS, primers and DNA polymerases remain in the reaction. Without purification or cleanup, these contaminants may change or interfere with the cycle sequencing reactions, resulting in suboptimal or undesired extension products.
There are different methods for purification of PCR products, including enzyme treatment, binding to spin columns or magnetic beads or ethanol precipitation. Our ADS™ PCR cleanup beads are cost-effective for PCR product purification.
SupreDye Cycle Sequencing Kits are our proprietary Sanger sequencing kits that are powerful alternatives to BigDye® Terminator Cycle Sequencing kits with similar performance. The SupreDye Cycle Sequencing Kits can be successfully used on ABI 310, 3130 series, and 3730 series of Genetic Analyzers without changing the BigDye’s experimental protocols or other reagents.
Four types of SupreDye Cycle Sequencing Kits are available. The Table below lists these kits and their uses.
|SupreDye Cycle Sequencing Kits||BigDye Alternatives||Applications|
|BD1||BigDye Terminator v1.1||Optimized for short read or near primer reading; for regular template|
|BD3||BigDye Terminator v3.1||Long read for de novo sequencing or resequencing, regular andlarger template|
|BD1 dGTP||dGTP BigDye Terminator v.1.0||Optimized for short read or near primer reading; for high G content (GC or GT) templates|
|BD3 dGTP||dGTP BigDye Terminator v.3.0||Long read for de novo sequencing or resequencing, high G content (GC or GT) templates|
Use SupreDye Cycle Sequencing Kits and 5X sequencing reaction buffer as alternative to BigDye Terminator Cycle Sequencing Kits and its 5X sequencing Buffer. Keep everything else (reagents and protocols) the same. SupreDye Cycle Sequencing Kits can also be used with all the other ADS™ reagents for sequencing.
The SupreDye Kits are shipped with dry ice and should be stored at -20°C after arrival.
The completed sequencing reactions still contain unincorporated dNTPs and ddNTPs, primer and enzyme components. Loading these components, especially the fluorescently labeled ddNTPs, to the capillary electrophoresis can interfere with migration of extension products and signal detection or basecalling (such as dye blob).
There are different ways to purify the sequencing reactions, such as size exclusion chromatography, affinity purification, magnetic beads cleanup, and ethanol precipitation. Any of these can be chosen for purification. We provide ADS™ BD-XT Purification Kit as an alternative to XTerminator Purification Kit or ADS™ Sequencing Reaction Cleaning Beads for effective removal of contaminants from sequencing reactions.
Although both products can remove contaminants for optimal sequencing results, the two products have different mechanisms and end results. The BD-XT Purification Kit binds to the contaminants while the magnetic beads bind to extension products. Since there is no wash step in the purification protocol for BD-XT Purification Kit, the recovery of extension products is better; after the mixing step, samples are directly loaded to the sequencer. The Sequencing Reaction Cleaning Beads are optimized for dye-blob removal with minimal wash and thus yield good extension product recovery. If there are budget constraints, use of magnetic beads is more cost-effective.
Sequencing reaction purification methods determine how the samples are loaded. If BD-XT Purification Kit is used, the sample will be directly loaded to the sequencer. If the products are purified by ethanol, the products are often resuspended into highly deionized formamide, such as TruPure™ formamide. C peak tailing may occur in samples resuspended in water; sample resuspension in formamide can overcome the problem.
TruPure Formamide is highly deionized and purified. It increases sequencing signal intensity and stabilityand has been successfully used as an alternative to Hi-Di™ formamide.
Different polymers are chosen for different sequencing needs (long or short reads) and also based on compatibility with sequencers. Our POP-7™ is the only polymer that can be used on 3730 series for optimal long reads.
PwrPOP P7, P6, and P4 polymers are high-performance alternatives to POP-7, POP-6™, and POP-4™, respectively, using the same protocols and without making any operational changes. These polymers provide high-quality extension product separation with even peak space and long read length.
After hundreds of runs, polymer debris or proteins may accumulate on the capillary array. This negatively affects the separation performance of the polymers, such as in peak space and read length. Therefore, regeneration of capillary is necessary to restore the optimal performance of the array without replacing the array itself.
ADS™ Capillary Array Regeneration Kit is designed to restore capillary array performance for all the ABI genetic analyzers. Just follow the protocol provided with the product to achieve optimal regeneration.
In a capillary array with optimal performance, read length and peak space are uniform, although the values will vary with different arrays. After hundreds of uses, accumulation of contaminants on the array leads to shorter read length, wider peak space, and tailing peaks in all four colors, even after careful cleaning. This is the sign that the array needs to be regenerated. We recommend the capillary array be regenerated with our Capillary Array Regeneration Kit every 500 runs.
In the ADS Capillary Array Regeneration Kit, we provide a detailed protocol for cleaning and regeneration of the array.
It is more cost-effective to regenerate the capillary array. The regeneration cost is only a fraction of the replacement cost.
After regeneration, the array will last for hundreds of runs depending on how the sequencers are maintained. Our experience indicates that a regenerated array can run another 500 times before the next regeneration. An array can be generated multiple times before replacement becomes necessary.