In vitro Selection

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  randomized library of 1014 - 1016 DNA (or RNA) molecules that are subjected to successive rounds of screening. Nucleic acid sequences that express the desired function are enriched while inactive sequences are removed. The result of this test tube evolution experiment  will favour synthetic molecules that can either facilitate chemical reactions (termed DNAzymes) or function as molecular receptors (termed aptamers). Collectively, these molecules are known as Functional Nucleic Acids. 

The process

In vitro selection is an iterative process where every cycle aims to enrich sequences with a desired function. Each cycle can be broken down into three separate components: Selection (negative and positive), Enrichment, and Regeneration.

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, then the addition of your target ligand will force the DNA sequences to compete with each other to bind to the target. Sequences that fail to do this will be discarded while sequences that were successful are isolated and prepared for enrichment.

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.

The evolution

Since we are mimicking the evolutionary process, the condition for selection needs to be more stringent in the later cycles. After the first few cycles, the DNA library should contain many different sequences that can carry out the function of interest. We often categorize these sequences into classes, which are grouped by sequence similarity. Although each class now possess the desired function, their effectiveness will vary to a large degree. By increasing the selection condition, you will force the remaining classes to compete with one another. The end result will yield a functional DNA sequence that is highly efficient at performing the task of interest.

Overall, in vitro selection can be intimidating, however, we break it down into smaller sections to help you design the best in vitro selection experiment possible!