Advice for Sanger Sequencing for DNA Barcoding

How to find a Sanger sequencing service and send your PCR products for sequencing

An overview of Sanger Sequencing for DNA barcoding

During Sanger sequencing, a cleaned PCR product containing a single amplicon is used in a cycle sequencing PCR similar to a conventional PCR, but differing in:

• Using a single primer (forward or reverse)
• Being a linear amplification (due to the single primer)
• Incorporating fluorescently labeled dideoxy terminator nucleotides that stop extension and further amplification when incorporated into a new copy of DNA

When using appropriate concentrations of PCR product, primer, and fluorescently labeled dideoxy terminator nucleotides, a cycle sequencing reaction produces a range of partial copies of the PCR amplicon of different lengths (from very small to full-length fragments), all starting at the primer binding site and ending with a fluorescently tagged nucleotide, with a different colour for each nucleotide (A, C, T, G).

These fragments are then run on a capillary electrophoresis machine that separates the product of the cycle sequencing reaction by size (since small fragments can migrate through the capillaries faster than larger fragments). The machine then detects the colour of the fluorescently tagged nucleotides at each size of fragment (from near the start of the original amplicon to the full length amplicon) as that size range passes through the machine.

Finding a Sanger Sequencing service

The availability of sequencing services in your area will vary depending on where you are located; and whether you are requesting sequencing as part of an institution or company, or as a private individual. Please note that some sequencing companies are reluctant to allow private individuals to sign up for their services.

If you are part of an educational institution or company you may have in-house sequencing capacity. If not, you should be able to sign up with a commercial sequencing company, for example GeneWiz and Macrogen.

In some countries, some universities provide a sequencing service to their staff, students, and others, and may be willing to extend a sequencing service to private individuals.

We recommend you ensure that you can access a Sanger sequencing service before planning any DNA barcoding work.

Preparing PCR amplicons for sequencing

Requirements for Sanger sequencing will vary depending on your sequencing service, but usually involve something resembling the following procedure:

Cleaning your PCR product: Primers, small fragments of DNA, and nucleotides need to be removed from your PCR product before they can be sequenced successfully, otherwise they will interfere with the sequencing process. The easiest option is to request cleaning as part of your sequencing service at an extra cost. Alternatively you could clean the PCR product yourself using a spin-column cleanup kit or a magnetic bead cleanup kit.

Diluting your PCR product: The PCR product needs to be diluted to an appropriate concentration for cycle sequencing. The requested final volumes are often 10 µL or 20 µL, and the requested concentrations will vary depending on the service. Usually only a few µL of PCR product in a total volume of 10 µL is required for sequencing (for example 2 µL of PCR product and 8 µL of water, or 4 µL in 20 µL. Often weaker PCR products sequence better than stronger ones. This is because excessive amounts of DNA template cause a large amount of the fluorescently tagged terminator nucleotides to be incorporated in the early stages of the cycle sequencing PCR, producing many small fragments with a strong signal, and the terminator nucleotides are then depleted later on in the cycle sequencing process, resulting in a very weak signal for larger fragments. Conversely, a very weak PCR product may produce a good sequence because little of the terminator nucleotides are used up at the start, leaving them available for incorporation throughout the cycle sequencing PCR.

PCR products can be supplied to sequencing facilities with a sequencing primer mixed in (for guidance on this consult your sequencing service guidelines), for example by adding 1 µL of a 10 µM primer to the mix. This may be worth doing if you are cleaning your PCR products yourself.

Alternatively, other sequencing services allow the option of sending the diluted PCR product on its own, with an accompanying primer in a different tube at a given concentration (usually 10 µM), in a volume sufficient for a given number of samples.

Posting PCR products for Sequencing

DNA is stable at room temperature for multiple days in PCR grade water if it contains no microbial contaminants or DNA-degrading enzymes, but it is best to coordinate postage so your samples spend as little time travelling as possible. Whenever possible, post your envelope just before the post is to be collected to minimise transit time.