PCR Product Direct Sequencing Support
The information below provides detailed support on directly sequencing PCR products. If you are looking for trouble shooting information (e.g. because you are unhappy with your results), please go to our trouble shooting area. Should you not find the answer to your question/problem here, please contact us and we will do our best to help.
Direct PCR product sequencing means using the PCR product as a template for the sequencing reaction without first cloning it into a vector. It is important to realise that direct sequencing of PCR products has its benefits and its problems. The customer must decide whether s/he wants to try directly sequencing a PCR product or clone it into a vector and sequence that. There is no universal rule that will determine what is the "best" approach. However, below are some points to consider if you intend to try it:
- One really needs a very clean PCR product that contains a single amplified product.
- The product should be at least 200-300bp in size.
- The PCR reaction should give a high yield of product.
Should your PCR product not meet the above requirements (either before or after purification), then direct sequencing is not likely to be very successful.
One of the main advantages of sequencing a PCR product directly (as opposed to cloning it first) is that you will not see PCR-generated mutations. The reason for this is that you have a large population of templates in your initial PCR reaction and even a mutation introduced in one PCR product in the first round of amplification will only be present in that one product out of thousands produced by the reaction. This very low frequency means that this error is swamped out by the majority (correct) sequence and so is not seen. As the reaction proceeds, the error is propogated, but so is the proportion of correct sequence at the same place in other templates. Hence, the error is still only present at a very low frequency and is not seen.
Contrast the above scenario with what happens when you clone the PCR product. Each clone is obtained originally from a single PCR product being inserted into a single vector. Hence, if that product contains an error, all the cloned plasmids derived from it will contain the same error. For this reason, one must sequence at least three independent clones in order to be sure that you are seeing the actual sequence and not a PCR-generated error.
We can produce up to about 1kb of high quality sequence from a single primer. Hence, if your PCR product is less than or equal to 1kb, you should be able to sequence it all on both strands using the PCR primers (please note that both ends will only be sequenced on one strand due to the way sequencing works). If your product is bigger than 1kb, you will probably need internal primers to achieve full coverage. If your PCR primers are very long (over 30 bases), then it will probably be best to make shorter primers for performing the sequencing. This is because the very long primers might give a high background.
You may also wish to use "nested primers". These are primers that will bind slightly further into the PCR product than the primers used for PCR. This only needs to be a few bases. What this does is prevent any non-specific PCR products from taking part in the sequencing reaction and can, therefore, produce much cleaner sequence. Customers are strongly advised to consider the use of nested primers.
As noted above, it is important to have a substantial amount of product available. This does not mean that we need an enormous amount of material for the sequencing reaction, because we don't. What it means is that trying to sequence a vanishingly small amount of product, which constitutes the total amount you generated, is very unlikely to work well. It is much better to have a strong yield of product that needs to be diluted substantially before being sent. One reason why this is the case is that there are inevitably contaminants present in the PCR product nomatter how it has been purified. These may inhibit the sequencing reaction and so diluting them out is a good thing. Hence, there is actually no such thing as a "correct" volume for the PCR reaction that will be used for direct sequencing. What is important is the yield of PCR product.
PCR products must be cleaned up to remove unwanted PCR products (if present), excess PCR primers and remaining dNTPs from the PCR reaction. Failure to do this is very likely to result in a messy sequence for reasons that should be obvious. However, if they are not check out our trouble shooting section.
There are various options available for performing PCR product purification. What will be the best depends on various factors. If you have a very clean and strong single band, you might want to try EXO-SAP. This is a very simple method that removes residual primers and dNTPs (these are the things that cause problems for sequencing as noted above). The benefit is that you don't lose any product (it just gets diluted a little). However, this will not remove extraneous PCR products, so only works if you have a clean single band. It also will not remove primer dimers.
Any of the various column purification kits available will also work to remove primers and dNTPs. Yield is likely to be less than EXO-SAP, but it might remove primer dimers since these may not "stick" to the column. An alternative is to use a size cut-off membrane centrifugation approach to remove small molecules (dNTPs, primers, buffer components). Gel purification is also an option and needed if you do not have a single pure PCR product. The scalpel can be a very good purification tool! The trouble with this approach is that yield can be low and you run a real risk of introducing extra contaminants into the prep in the form of components from the agarose gel. It is vital to use a high quality agarose if doing gel purification.
The final option (maybe the first really?) is to simply dilute your PCR product in water with no further cleanup. It is certainly the quickest and cheapest option. If you have a well optimised PCR reaction, the residual primers and dNTPs will be low and diluting may be all that is necessary....
Once you have purified your PCR product, it is vitally important that you quantitate it by an appropriate method. This means NOT using a spec of any type. A lot of people think that they can just use a nanodrop or other type of spec. This is usually a disaster, because they forget that most of the absorbance can often be from free dNTPs and primers and not from their PCR product. This is especially important if you clean up your product by EXO-SAP or simply diluting it. Hence, people massively over-estimate the yield and consequently send far too little. What is needed is a ball-park concentration for the PCR product. It does not need to picogram accurate. For this reason, estimation on an agarose gel is best. Run a known volume of PCR product on a gel and also run a size marker (a couple of different loadings is good). Look at it and pick a marker band that is close in intensity to the PCR band. The marker product sheet will tell you that if you load Xµl, band Y will have Z nanograms of DNA in it. Hence, you can calculate your nanogram amount of PCR product and can work out the concentration. You can also use software to interpret the gel image and work this out for you, but that is not really needed. Some skill (and, more importantly, experience) is required for the estimation by eye, but not much. It is also imperative that any estimation of yield is done right at the end of the cleanup process. Do not just assume that you will recover all your product!
Information on the amount of sample to send for each of our contract research DNA sequencing options is given on the appropriate product page and so is not repeated here. However, please remember that we ask for enough template for two reactions supplied in 30µl total volume. This allows us to repeat things if disaster occurs.