Hcp1 SELEX: Progress Report Two

Due principally to the inconclusiveness of the antecedent binding assay(s), the notion of persisting in selection via the RNA pool resulting from Round 16 was rejected. Thus, this succeeding progress report details the methodology, parameters, and therein ultimately the intentions of the first round wherein I began anew.

In a manner largely guided by the aforementioned failure, the initial round of selection exclusively involved beads. My justification for this is a purely theoretical one, as it, a seemingly pragmatic conclusion, seems readily available and yet has not appeared, to my knowledge, in relevant literature. It is such that, as the variability of the pool is greatest prior to the directed evolution within selection rounds, the most efficacious means of removing non-specific binding species (bead-binding species) from the RNA pool is within Round 1 via, in this case, a R0 N34 RNA pool. As the above gel depicts, W0 exhibited overamplification within each of the PCR cycles examined. Additionally, this gel denotes the degree to which the R0 N34 pool features affinity for the Ni-NTA beads.

As W0, the RNA population I will be taking into further, target-based, round of SELEX, was overamplified (as anticipated, yet stupidly not accounted for in ccPCR) following the cycle 6, obviously it was necessary, and likely integral to future successes, to reamplify the ssDNA derived from W0 reverse transcription in order to obtain a more favorable degree of resolution among lower cycles. The resultant gel, not shown, depicted an optimal amplification achieved following three cycles.


Nia_Fernandez said...

This is interesting. However, I believe that R0 pool selection is not necessarily used to "weed out" binders but more to test affinity. Maybe ask Brad or Gwen about this?

John Ostrominski said...

Definitely. Yet my general thought was such that, as the greatest variation exists within the R0 pool, it would be most efficient to try and eliminate latent bead-binding sequences from the pool in R1. Could this, in any way you know of, eliminate latent target-binding species from the pool? Thank you.