Direct Sequencing of PCR Products
Sounds a bit dismal, right? Not necessarily. There are some very good reasons you might want to go ahead and sequence directly from a PCR product. Here are some:
Direct sequencing is much quicker. If you're screening hundreds of patient samples for mutations in a gene, you do NOT want to be gel-purifying all those PCR reactions, and you CERTAINLY don't want to clone them all before sequencing.
Direct sequencing doesn't show any PCR mutations. Common PCR protocols (Taq polymerase under standard cycling conditions) generate mis-incorporations occasionally (about once per 3 kb, in my hands). If you clone those PCR products and sequence several of them, you will see point mutations in some of the clones.
If you directly sequence the PCR product, though, what you'll see is the consensus base at each position. Although many of the individual products have mutated nucleotides, these mutations are scattered randomly, and are different for each individual product fragment. Consequently, at any one nucleotide, most of the clones will be correct, and you'll be seeing the original sequence with no mutations.
We have many clients who have successfully sequenced thousands - even tens of thousands - of PCR products, with outstanding results. Be critical of the quality of your PCR product and, if necessary, optimize the PCR conditions or gel-purify the desired fragment. Prove you have the right fragment before you invest in large-scale sequencing, and you'll be pleased with the results.