Correctly cleaning up your sequencing reactions is an integral part of the Sanger Sequencing workflow. Without cleanup, you will get suboptimal sequence data. So how does sequencing cleanup work and what tools are available? Let’s find out.
To understand the importance of sequencing clean-up, you first have to understand the BigDye Terminator sequencing workflow and the basics of how the sequence information is captured. In short, the DNA that you want to sequence should be isolated and purified. This can be done by a variety of methods. But in general, the cleaner the product, the better the results.
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Once you have your purified DNA, you can now move on to the BigDye Terminator sequencing reaction. The BigDye Terminator Ready Reaction mix has all of the components necessary for sequencing. Just mix the Ready Reaction Mix, DNA Template, and a single primer and perform your cycle sequencing reaction. For best results, it’s usually better to set up 2 reactions per sample, one using a forward primer and one using a reverse primer. After the BigDye Terminator sequencing reaction, you will need to remove excess labelled nucleotides since these will interfere with the downstream sequence detection process. After the cleanup, the amplicons generated during the sequencing reaction are electrokinetically injected and separated by size, charge, and the dye, which is specific to each of the four nucleotides. The dyes are excited by the instrument laser and detected by the instrument’s camera.
If the Sequencing reaction cleanup step is missed or not performed properly, the residual dye in the reaction can out-compete the labelled amplicons for entry into the capillary and can cause reduced signal intensity which can interfere with the instruments ability to make clear base calls. As a result, the data generated will be of poor quality.
Several different methods have been developed over the years to cleanup sequencing reactions. Some methods, such as ethanol precipitation, can be labor intensive and time consuming. But there are other methods out there that makes sequencing cleanup easier and faster. The most efficient methods fall into two categories: column and bead based. Column based methods use centrifugal force to pass the solution through a substrate that will bind any unincorporated dyes. Bead based methods use particles in a solution that bind dyes that were not used up in the reaction. Removing the residual dyes from the sequencing reaction gives you much cleaner and more reliable results.
Thermo Fisher Scientific offers several different products for sequencing cleanup, such as BigDye Xterminator and Centri-Sep columns. For more information, please visit our website.
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But remember, when in doubt, just Seq It Out.