My primers work fine in PCR - why not in sequencing??
PCR primers CAN work very well in sequencing, but there are limitations:
Discussion below centers both on the use of PCR primers to sequence non-PCR
templates, and on using your PCR primers to sequence products they generated.
There's also a separate page with information on
performing direct PCR sequencing.
- PCR is exponential, sequencing is not.
With two primers replicating opposite strands, a PCR reaction is exponential. That has some HUGE effects
on the results. Primers that are inefficient can still work for PCR. Most people amplify beyond the linear
part of the reaction, meaning inefficient primers will produce as much product as efficient ones. For
sequencing, this is NOT the case. Inefficient primers will give only weak bands. Also - and this is often
very important - PCR will amplify even if the target DNA is only a small proportion of the DNA present.
99% of your plasmid prep may be some junk DNA, but the remaining 1% will amplify just fine. It will not
- PCR reactions can be tailored to the primer; sequencing reactions cannot.
You probably 'tweak' your PCR conditions to improve yield and specificity. The annealing temp and Mg++
concentration are usually critical, but adjustments in extension and denaturation temps and times can be
helpful as well. Unfortunately, we simply cannot do that in a Sequencing Core. Samples are processed 48-96
at a time, and we have to use consensus conditions throughout. Please study our guidelines for the design
of sequencing primers; they were written with these facts in mind.
- PCR reactions actually can use a mismatched priming site; sequencing rarely can.
A primer may start out mismatched against the template, but if even ONE primer manages to anneal, even
briefly, the extension product will now have a perfect match and will amplify extremely well during
subsequent cycles. Sequencing reactions have no such benefit. With only one primer copying one strand, the
reaction will ALWAYS be less efficient for a mismatched primer.
- Sequencing PCR products with the PCR primers: One (apparent) band doesn't guarantee good sequence.
Most people analyze their PCR products using gels with limited resolution. You may see only one band, but
there could easily be multiple bands present, but at very similar sizes. We saw a reaction like this quite
recently - a single band on a PCR gel gave mixed peaks that ultimately proved to be multiple PCR products.
Also, that dim background smear or those faint bands near the bottom of the gel may look insignificant, but
they may be more DNA than you think, adding background bands.
- You can use your PCR primers to sequence PCR reactions, BUT there are a few caveats:
- You MUST remove residual PCR primers from the reaction before you submit it for sequencing!
- PCR reactions generally can NOT be quantitated by spectrophotometer. Please use an analytical
gel to estimate the concentration (unless you have an unusually sensitive spectrophotometer).
- Your primers must be designed to be compatible with our sequencing conditions.
Here's the bottom line:
- Just because your primers work in PCR reactions does NOT mean they'll work for sequencing.
- Your PCR reaction may look good on your gel, but a sequencing reaction can reveal much
that you cannot see. Your primers may not be working as well as you had thought.
- Removal of PCR primers from your reactions is critical.
If you have any questions of comments, please contact Dr. Robert Lyons,
Director of the DNA Sequencing Core.