PCR Product Sequencing
How To Sequence Your PCR Products
Once you amplify your DNA sequence of interest, you usually need to check the amplification quality and accuracy using DNA sequencing services. There are several methods to do this and each method has its advantages and disadvantages.
Direct Sequencing of PCR Products
Direct sequencing of PCR products is obviously the quickest method although it is not always the most successful method. It is possible to directly sequence a PCR product without having first cloned the fragment into a sub-cloning vector such as a TOPO plasmid. Obviously there are many advantages to the direct sequencing approach. However, there are several disadvantages and steps which many people bypass only to have failed DNA sequencing results.
Direct sequencing of PCR fragments is hardly successful unless you learn how to make it successful. We have generated the following tips to make sure you don't fail in your direct sequencing attempts.
1) Make sure that you have the right PCR product before you send your PCR product for sequencing.
With sub-cloning PCR products into a vector, people usually restriction enzyme digest the bands out of the vector to check if it is the right sequence. Make sure you check the size of your PCR product carefully on the gel before you send it for sequencing. Also, if there are known restriction sites in the PCR fragment, make sure you use these to check that your product is the right one.
2) Make sure you don't have many non-specific products (bands on agarose gel)!
PCR reactions rarely produce only a single (one) band. Usually you will have many bands (non-specific products). Even if you can't see them, your PCR reaction may have many bands (non-specific products) that will cause your DNA sequencing to have many signals at each nucleotide (you get a jumbled sequence i.e N (all 4 nucleotides) instead of 1 nucleotide).
Using nested PCR primers can reduce this problem. It is also less likely that non-specific product will co-amplify through a second round of PCR with internally-
3) Remove all residual PCR primers and unincorporated nucleotides before sequencing.
The good thing with sub-cloning is that you clone in the PCR fragment into the vector, amplify it, and purify it. This way you have a very clean batch of DNA. After PCR amplification however, you have a lot of unincorporated nucleotides, pcr primers, salts and proteins that can interfere with DNA sequencing. Make sure you at least use a PCR clean-up step to ensure your DNA sequencing doesn't fail.
4) Ensure you don't send a very concentrated DNA sample!
Make sure that you double-check the template DNA concentrations before you send your PCR for sequencing. This is because PCR fragments are smaller than plasmids, and thus you get more DNA per sample. This makes PCR products more effective sequencing templates than the usual plasmids. Therefore you need to have lower concentrations for PCR products.
For example, a 200 bp fragments needs to be just 2 ng/ul! If your samples are at too high a concentration, not only will they NOT sequence any better but may cause your DNA sequencing to fail. Estimate your PCR product concentration by loading them with standards on an agarose gel. Usually laboratory spectrophotometers cannot with accurately measure the small amount of DNA that a PCR reaction generates (unless you use the newer micro-specs i.e. "Nanodrop"). Based on your analysis, make sure you dilute them.
Subcloning PCR Products and Sequencing them
The most effective way to sequence PCR products is by subcloning them into vectors or plasmids. Usually this was done with primers that contained restriction enzyme sites. There were many problems with this including the fact that restriction enzymes have difficulties cutting at the ends of a PCR fragment.
After cutting the PCR fragment, one would have to ligate the PCR fragment into a pre-cut vector that has compatible ends. After amplification and purification of the plasmid, one would simply send the fragment for DNA sequencing. Most vectors have T7, T3, M13, and other primer sites which allows easy DNA sequencing using universal primers (such as the commonly available T7 primer, T3 primer, and M13 primers).
TOPO Vectors for PCR sub-cloning
The latest greatest innovations allowed many to bypass the difficulties or subcloning PCR products into regular vectors. TOPO cloning allowed the RAPID and easy cloning of PCR products into vectors. TOPO cloning is very quick (5 minutes for ligation) and very effective. You can easily clone your PCR fragment into the vector without any restriction sites. This is due to the fact that many DNA polymerases add an extra A to the ends of the DNA fragment. This allowed the cloning of the PCR fragments into TOPO vectors (or TA vectors) which contained free T ends. TOPO vectors contain Topoisomerase enzyme which enhances the cloning of PCR fragments into the vector.
TOPO vectors are available at Invitrogen
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