Why is a clean template for your Sanger sequencing reaction important? And what are the ways to get it without losing too much precious samples? Let’s find out today.
The sequencing workflow usually starts with PCR also known as Polymerase Chain Reaction, where the target fragment is replicated into thousands of copies under a controlled condition using primers, DNA polymerases and deoxynucleotides. Before the PCR product is ready for Sanger sequencing, you must do one more thing, a PCR clean up step. Because Sanger sequencing is a highly accurate technique for you to read DNA sequence base by base, it is very important to clean up your reaction mixtures so that those unincorporated primers and dNTPs won’t interfere with your results.
So how do you perform this PCR clean up step and what are your options?
There are several methods for PCR cleanup such as ethanol precipitation, bead or column based purification, and Enzymatic approaches.
Ethanol precipitation is most cost effective but is also the most labor intensive. Bead or column based purification can be an effective method for many applications. However, this route tends to be a bit pricier compared to other options. This method of purification also requires transferring samples in and out of the column which can often result in the loss of some of your precious samples so this may not be the best method for those with limited starting materials.
An enzymatic cleanup approach has a really simple and straightforward workflow. In fact, it’s only one single pipetting step! It is also relatively affordable compared to the bead or column based purification methods. Basically, you add an enzyme mix to your finished PCR reaction and let it sit at a certain temperature.
But to understand this method better, let’s take a look at our lab book.
One of the enzymes, exonuclease I(one), digests excess primers. At the same time the other enzyme in the mix, alkaline phosphatase, dephosphorylates nucleotides in the reaction. Dephosphorylation means that the enzyme renders the dNTPs from your reaction useless, so that they don’t disturb the sequencing reaction. This is important because the sequencing reaction has a very specific ratio for its nucleotides– and you don’t want to change that ratio.
Now the enzymes have done their work and you don’t need them anymore. You can simply heat up the reaction mix to 80 degrees and this will deactivate the enzymes. The result is a very clean amplicon that is now ready to go into your sequencing reaction.
You don’t need to perform any other steps. And you didn’t lose any of your template either. You are done! But I am sure you’ll have more questions on PCR cleanup. Submit your question at thermofisher.com/ask and subscribe to our channel to see more videos like this.
But remember, when in doubt, just Seq It Out.