Troubleshooting

Primer related problems

Problems with primers can be divided into four areas based on the results seen in the data:

• No readable sequence
• Very weak sequence
• Two or more sequences
• Primer-dimers

No readable sequence

If there is no priming site for the primer in the template DNA, no sequence data will be obtained. The data will show a flat line except for excess dye terminator peaks. A result like this can be obtained if, for example, the incorrect primer is used for a particular vector or if there is a mutation in the primer binding site in the the vector.

Very weak sequence

A weak signal strength can be produced if the primer anneals poorly because of a low melting temperature (Tm). Primers should have a Tm greater than 45°C.

Two or more sequences

More than one sequence can be present in the data if there are multiple primers present in the sequencing reaction. This occurs when the unincorporated primer from PCR products have not been completely removed and thus can be carried over into the sequencing reaction.
Another cause of multiple sequences is when there is a secondary hybridisation site for the primer in the template, resulting in an electropherogram showing peaks under peaks where the multiple sequences overlapp. Hybridisation at a secondary site with an imperfect match for the primer usually occurs when the recommended primer concentration is exceeded.

Primer-dimers

Primer-dimers are caused when two primers with the same or different sequences hybridise. Usually this is found in PCR reactions where the forward and reverse PCR primers hybridise but can also be found in sequencing reactions where primer-dimers between two primers with the same sequence are generated. This appears as a 25-60 base pair long product.