I'm attempting to amplify a region of unknown sequence, downstream of known sequence, starting from a genomic sample. For instance:
Ligation-mediated PCR is a technique that utilizes blunt-end ligation of a linker of known sequence to restricted genomic DNA. This "flanks" a chunk of unknown sequence, with two portions of known sequence, allow for PCR amplification and then sequencing of the amplicon. The process is as such:
1) Restrict the genomic DNA.
2) Creat a blunt end within your genomic DNA digest, and thermocycle using a primer specific to your known sequence. This is linear amplification, and utilizes one primer. This is where specificity arises - only your fragment of interest should generate a blunt end, and thus be the only eligible fragment for blunt-end ligation.
3) Ligate the linker to your restricted, now blunt-ended, genomic DNA. The linker is only blunt on one end, preventing linker-linker ligation.
4) Amplification, using two primers - one specific to the linker, one speific to the known sequence. Preferably, the primer to the known sequence will be different than that used in step 2, and closer to the unknown sequece. This increases specificity.
This protocol recommends using Vent polymerase. I'm not sure why this is preffered over a high-fidelity polymerase. Anyone have any insights?
I had a lot of background on my first try at this technique. I'm going to try to increase primer-specifity and annealing temperature. Any other ideas?