AdvancedSeq LLC specializes in making high quality, reliable Sanger sequencing and PCR reagents and related products for Life Science research at competitive prices. Founded by scientists and product managers with extensive knowledge and experience in the life science industry, the company products are trusted and used by many companies and academic labs worldwide.
With the rapid development of next-generation sequencing (NGS) technology and its fast-growing applications, there is no doubt that Sanger sequencing will continue to serve as the gold standard with 99.99% sequencing accuracy. Sanger sequencing and NGS go hand in hand, both are required for the genomics community to serve the need in both research and clinical tests.
With much improvements being made for NGS, today NGS still needs Sanger sequencing, but not vice versa. Sanger sequencing plays an important role to identify the false-positive mutations occurred in NGS. In addition, Sanger sequencing is still used for large contiguous genomic fragment sequencing in an NGS genomic sequencing project.
For a mature technology, such as Sanger sequencing, lowering cost is essential for fast-developing research fields with constraint funding. With cost-effectiveness in mind, we have developed novel alternative products within the Sanger sequencing workflow that maintain high quality of sequencing. Some of these products, such as SupreDye™ Cycle Sequencing Kit, has been adopted worldwide in the community.
The sample (DNA template) purity and quantity are the most important factors affecting the success of sequencing results. As for purity, the template DNA should not be contaminated with proteins, polysaccharides, RNAs, or genomic DNA. As for the quantity, a proper DNA quantification method other than a mere spectrometer for 260 nm and 280 nm measurement should be used to measure the concentration of the template DNA. The right amount of template based on the length of the template should be used in the sequencing reaction. Most common Sanger sequencing templates could be a plasmid, PCR products or DNA made from Rolling Cycle Amplification (RCA).
Make sure plasmid of high quality is prepared for optimal performance. Plasmids that can be used as PCR products or digested with restriction enzymes may still not be good enough as Sanger sequencing templates. During plasmid preparation, it is critical to wash the purification columns thoroughly and elute DNA after removing traces of ethanol in the column.
PCR products are more favored as sequencing templates, but they should be of high purity and uniformity. Make sure high quality of single and specific PCR products are generated using good quality dNTPs, enzymes and primers. Although using gel purification of PCR products to achieve specific PCR products is a common practice, PCR condition optimization is a better solution to avoid generation of multiple PCR products and further gel purification. Purified PCR products from the agarose gels may get contaminated with sequencing inhibitors. Instead, magnetic beads, such as ADS™ PCR Cleaning Magnetic Beads, are often suggested for cost-effective clean-up of the PCR products.
Another common used method for cleaning up of the PCR products, especially for Sanger sequencing and NGS, is an enzymatic method to degrade left-over PCR primers and dephosphorylate dNTPs. ADS Exo-Alp PCR Cleanup Mix, alternative to ExoSAP-IT PCR Product Cleanup Reagent, can be directly added to the PCR tubes to remove the interference of PCR primers and dNTPs in 30 minutes and the reactions can be directly used for downstream applications without further purification.
For mutation screening purposes, plasmid template can be amplified directly from single colonies without growing the culture for plasmid isolation. This is especially popular when only a small fraction of colonies contains the plasmid inserts with desired mutations. The plasmid amplification can be achieved through rolling circle amplification (RCA) using Bacillus subtilis phi29 DNA polymerase, degenerate primers, and little cell lysate from single colonies. The amplification products can be directly used for Sanger sequencing without further purification.
| Template Products | |||
| ADS PCR and Purification Kit | ADS PCR and PCR Cleaning Beads | ADS Exo-Alp PCR Cleanup Mix | ADS phi29 DNA Polymerase and RCA DNA Amplification Kit |
| High quality for PCR success and cleaning | For efficient contaminant removal | For quick removal of PCR primers and dephosphorylation of dNTPs | For fast sequencing of DNA from single colonies |
Sequencing reaction is performed after the DNA template is properly prepared and quantified. Cycle sequencing or linear amplification method is used to prepare Sanger sequencing reactions. This method of preparation is different from traditional PCR methods that use two primers for exponential amplification of both DNA strands.
The sequencing reaction uses a single primer to base-pair one strand of the template and linearly amplifies the other strand of the DNA template in cycles of denaturation, annealing and amplification using Sanger’s dideoxy chain-termination method. In this process, a small fraction of the four types of fluorescently labeled dideoxynucleotides (ddATPs, ddTTPs, ddCTPs, and ddGTPs) is added as terminators to compete with their corresponding dNTPs for incorporation into synthesized DNA fragments. The elongation of DNA synthesis stops once the ddNTPs are incorporated instead of their corresponding regular dNTPs. The ladder of the synthesized DNA fragments with one nucleotide difference can then be separated on capillary electrophoresis on genetic analyzers or sequencers. Base-calling is made on the instrument software based on laser detection of fluorescent signals.
The sequencing reaction step is critical for achieving the best sequencing results. The following parameters influence sequencing quality:
The quality and the amount of DNA template and primers determine the sequencing quality. Higher than normal level or concentration of DNA templates generates strong signals at early peak readings but the signals get weak or faded at later peak readings. If the concentration of the DNA template or primer is too low, sequencing signals may be too weak for accurate reading.
Besides the combination of primers and templates, which differ from reaction to reaction, the rest of the sequencing components or sequencing reagents play an important role in achieving the best results. High-quality sequencing reagents, such as ADS® SupreDye™ Cycle Sequencing Kits, generate strong and uniform sequencing signals, allowing accurate sequence reading of >800 or even >900 base pairs.
The ADS™ SupreDye™ Cycle Sequencing Kits are a powerful and demonstrated alternative to BigDye™ Terminator v1.1 or v3.1 Cycling Sequencing Kits. They include kits for regular templates and high-GC templates. These kits are formulated to deliver increased robustness, even peak heights, and long read lengths.
| Cycle Sequencing Products | |
| SupreDye Cycle Sequencing Kits | ADS 5x Sequencing Buffer |
| High-performance alternative for BigDye Terminator v.3.1 or 1.1 Cycling Sequencing Kits | Cost-effective choice for sequencing reactions |
Although every single step in the Sanger sequencing workflow plays an important role in generating good sequencing results, two major criteria are critical for high-quality results from Sanger sequencing: high template quality and quantity and thorough clean-up of the sequencing reactions. Variations in these criteria may contribute to uncertainty in sequencing success.
Clean-up of sequencing reactions is performed to remove unincorporated dyes (fluorescent labeled ddNTPs), dNTPs, primers, and enzymes from the completed reaction mixture. Presence of these contaminants can generate poor quality of the sequence readings.
Different methods can be used for clean-up of the cycling sequencing reactions, including size exclusion, affinity purification, ethanol precipitation, binding with magnetic beads (such as ADS™ Sequencing Reaction Cleaning Beads) and proprietary methods (such as in ADS™ BD XT purification kit). Although ethanol precipitation is the most cost-effective method, sequencing quality obtained is often poorer the first 40 nucleotides close to the primer compared to above methods.
We developed two clean-up products, ADS™ BD-XT Purification Kit and ADS™ Sequencing Reaction Cleaning Beads for different clean-up preferences. The ADS™ BD-XT kit uses specialized resin for contaminant binding and can reduce sequencing signal loss and prevent any potential contamination during the wash and elution steps, without repeating washing steps.Therefore, the sequencing signal strength is often better with the ADS™ BD-XT kit compared to magnetic beads. However, use of magnetic beads is cost-effective and is best for large-scale sequencing to save sequencing cost while still achieving high-quality sequencing results.
| Clean-Up Products | |
| ADS BD-XT Purification Kit | ADS Sequencing Reaction Cleaning Beads |
| Fast, simple and effective purification for DNA sequencing reactions. | Rapid purification for clean electropherogram with low dye blob frequency |
Once the cycle sequencing reactions are cleaned up, the sample reactions are ready to load for capillary electrophoresis (CE), for instance, via electrokinetic injection. After the reaction passes through the capillary array, fluorescent sequencing signals are detected. Conversion of these signals to DNA sequences by base-calling are performed by the analysis software.
Resuspending the sample in highly deionized formamide, such as our deionized high-purity TruPure™ formamide, before injection helps stabilize the DNA fragments and maintain consistent signals for both long and short fragments. Formamide is especially used for resuspension of sequencing samples after ethanol precipitation and commonly used in fragment analysis.
The sequencing fragments are separated on CE using polymers as separation matrices. High-quality polymers such as PwrPOP™ Polymers allow robust separation with appropriate peak space for long read length.
We provide PwrPOP™ P4, P6, and P7 polymers to accommodate different analyzers:
| Sample Loading and Capillary Electrophoresis Products | ||
| TruPure Formamide | PwrPOP Polymers | ADS 10x Sequencing Running Buffer |
| For High performance sequencing sample resuspension | For better signal intensity and stability | For cost-effective sequencing results. |
After multiple sequencing runs, polymer deposit and protein contaminants from the sequencing samples may accumulate on the capillary array surface and compromise the capillary array performance and jeopardize the sequencing quality. Without proper regeneration to remove the adhesion of the contaminants, the array may have to be replaced early, which significantly increases the preventable cost for a sequencing facility.
By monitoring the sequencing read length and peak spacing and comparing their value differences at normal runs, a sequencing operator may be able to judge if the capillary needs regeneration. Other factors, such as the age of the running buffer and the polymer, may also affect these changes. It is important to perform sequencing using reagents according to the manufacturer’s instructions and to consider all these influencing factors before regenerating the capillary array.
The ADS™ Capillary Regeneration Kit cleans the capillary array and restores its performance for generating long sequencing read length and for lowering noise and background.
| Regeneration Products |
| ADS Capillary Regeneration Kit |
| For sustaining high-quality sequencing |
Fragment analysis refers to a genetic analysis that identifies the PCR fragment pattern difference by size among individual samples. It uses specifically designed and labeled PCR primers to generate PCR products from sample DNA. Then, specifically labeled PCR products mixed with fluorescent size standard are denatured and run on the capillary electrophoresis (CE) instrument to separate fragments by size. Different from Sanger sequencing in reaction mechanism, fragment analysis does not involve cycle sequencing reactions that generates nested extension products with one nucleotide difference; However, similar to sequencing extension products, fluorescently labeled fragments are separated and detected through CE by size difference.
Fragment analysis can be used in multiple areas including:
The fragment workflow include DNA sample preparation, PCR fluorescent sample preparation, sample mixing and denaturing, and capillary electrophoresis and data analysis. We offer cost-effective products at different workflow steps for high-quality fragment analysis.
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DNA Sample Prep
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Labeled PCR Prep
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Sample Mixing & Denaturing
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Capillary Electrophoresis
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Data Analysis |
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ADS™ PCR and purification Kit
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TruPure™
Formamide |
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PwrPOP™ P4/P7 Polymers
ADS™ 10x Running Buffer |
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