Sanger sequencing is still a workhorse of most molecular biology labs. Even with the advent of next-generation sequencing we still need to sequence our clones and PCR products. In this article I have listed some of the tips and tricks we used in our Sanger services.
(1)Dilution of BigDye:
I’d expect this to be a tip everyone knows, but BigDye can be massively diluted and still give good results. There are many publications describing ways to do this and there are commercial providers of BigDye dilution buffers.
(2)Colony PCR instead of plasmid prep:
You don’t need to plasmid prep your clones for Sanger sequencing. After picking clones for culture, simply swirl your cocktail and stick into a PCR master mix with primers for your multiple cloning site (probably M13 F&R). Submit the PCR for Sanger sequencing and you can have results back the next day. You then only need to midi-prep the clones that you really need to continue your project.
(3)Tailed primers make life easier with almost all PCR amplicon sequencing:
PCR is an easy way to capture a specific region of DNA and you don’t need to do any cloning to get Sanger sequence data back. There are some simple tips you can follow which make PCR amplicon sequencing easier and quicker;
M13 tailed PCR primers: M13 is the most commonly used multiple-cloning priming site. Nearly all Sanger sequencing providers carry M13 forward and reverse primers ready for you to use free of charge. Using a pair of ready-made sequencing primers means you don’t need to worry about how your primers will work in a Sanger sequencing reaction where the kinetics are quite different from a standard PCR. To make use of these easy and free primers you’ll need to include them in the design of your locus specific PCR primers, simply add the M13 sequences to the 5′ ends. Order these from your oligo provider and your PCR is (almost) ready to sequence.
PCR clean-up: You need to get rid of excess primer before starting your Sanger sequencing reaction. Most Sanger sequencing service providers will ask you to send gel- or column-purified PCR products, if you don’t then there is a risk that you will end up with very poor or noisy sequence data. This is because the PCR primers produce secondary reactions in the tube. However, Sanger sequencing quality requirements are different for different people. If you are simply screening clones to find a specific mutant, or checking your PCR for heterozygous variation, then a little noise is tolerable. The methods below all require a little tweaking but this is well worth the effort as it saves a lot of clean-up in the long run. Especially if you are sequencing 10 exons in 100 patients!
- Dilute the final PCR: You can simply dilute your PCR products 1:10 with low-EDTA TE buffer or water. This will reduce the amount of oligo that can act as a primer in your final Sanger reactions. The amount of dilution needed will vary depending on your reaction kinetics.
- Reduced primer input: By reducing the amount of primer used in your PCR you can make sure the reaction plateaus because it becomes primer-limiting. Very little oligo is left that can act as a primer in your Sanger sequencing reactions.
- Asymmetric PCR: You can get away without any dilution using an asymmetric PCR where the pair of oligos are not used in equimolar amounts. Again this can require some tweaking to get the conditions right, but if you are working on a large project it is worth the effort upfront as no final dilution or clean-up is required.
(4)High GC content:
Life Technologies sell dGTP chemistry to sequence template with high GC content. If you know your templates are likely to have secondary structure issues, then it can save you time and money to spend a little extra on the first round of sequencing and request it is done with a dGTP mix. Most providers should offer this as an option. An alternative if you are doing this yourself is to use a mix of BigDye and dGTP chemistry which means 95% of templates should work first time round with little or no optimization.
Are you already using any of the above tips in you sequencing? Perhaps you know other you would like to share with fellow Bitesizers?!