In vitro selection, also referred to as "test-tube evolution"
In vitro selection is a test tube-based experiment that mimics the evolutionary process found in Nature. A population, organism or even molecule that can adapt to their environmental pressure will thrive while those that fail to derive key traits to survive will wither away. The purpose of an in vitro selection experiment is to identify DNA or RNA molecules that have unique molecular functions. Every in vitro selection begins with a large
In vitro selection is an iterative process where every cycle aims to enrich sequences
Negative Selection: This is a crucial yet often overlooked step of in vitro selection. To obtain functional sequences of high specificity, DNA species that can cross-react or have the capabilities to bind multiple targets are removed. This is a fine-tuning that allows the user to determine what kind of binding properties is desired.
Positive Selection: This is where the magic happens. The DNA sequences within the library must be able to carry out the function of interest or it will be eliminated. For example, if you are identifying aptamers,
Enrichment: Sequences that survived the selection step will be allowed to multiply. This is done using polymerase chain reaction (PCR). However, something special also happens during PCR. As the DNA is being synthesized by the polymerase, minor mutations are introduced and may provide the sequence with a fantastic advantage in the upcoming cycle!
Regeneration: The end result from PCR will produce a double stranded library that needs to be separated to regenerate the single stranded DNA pool. At InnovoGENE Biosciences, we use a specially designed primer that will allow differentiation between the sense and anti-sense strand during denaturing PAGE. In addition, selections of many DNAzymes or Ribozymes require further modification prior to positive selection.