Selection rounds

The second item in the menu bar is (Data), which lists the selections (Round, Column Width), respectively (Choose Vectors). 

The raw data table is displayed with the numbers on a white background and the headers and index on a gray one a yellow How-to-Procede panel gives instructions, respectively confirms choices two white panels display the file size and the presently selected rounding option. 

The selection efficiency is evaluated by the ratio of the output over input titers. After each selection, the phages are amplified and can be subjected to anew selection round. If some clones of the library are effectively selected, the ratio should increase from around 10-5 (background level) to around 10-2. Typically, the ratio reaches a plateau after about 4 to 8 selection rounds - depending on the starting diversity and on the power of the selection itself. 

It can be interesting to increase the selection pressure from one selection round to the next by doubling the number of washes, by decreasing the suicide substrate concentration, or the time of incubation by a factor of 10. This could lead to the selection of the most efficient catalysts. 

The out/iiC Ratio Does Not Increase with Selection Rounds... 

If sufficient target is available, new matrix can be used in every selection round. Otherwise, the matrix must be treated so that no denaturation or degradation of the target molecules occurs. Correspondingly, elution of bound RNA and regeneration of the matrix have to be carried out. 

The selected RNA must be reverse-transcribed into cDNA for further amplification by PCR. Therefore, the RNA is precipitated and subjected to a reverse transcription reaction according to the protocol of the reverse transcriptase supplier. If the RNA concentration is very low (as in the first selection rounds) a coprecipitant like glycogen has to be added to recover the RNA quantitatively. 

After the ssDNA pool has been established, it must be de- and renatured as described for RNA aptamers (see Section 7.3.1.5). Then preselection and selection are done, and the selected ssDNA is used in a PCR to create the new dsDNA pool of the next selection round. 

Figure 8. A sketch of the SELEX technique used to select for molecules with optimal binding constants to predefined target molecules. The SELEX procedure selects for molecules with sufficiently high binding constants, so called aptamers, in two steps. Target molecules are attached to a chromatographic column which allows for selective retention of sufficiently strong binders. A different solvent is applied to release the binders and canalize them to the next selection round. Commonly some tens of selection cycles (Figure 7) are sufficient to isolate optimal binding RNA molecules.

The theory of molecular evolution and the in vitro evolution experiments suggest practical applications to the design of biopolymer molecules as they were proposed already in the 1980s [4], The basic principles of the so-called irrational design of biomolecules are indeed identical with Darwin s natural law of variation and selection. Molecular properties are improved iteratively in selection cycles in order to achieve an optimal match with the predefined target function. The process is sketched in Fig. 5. Every selection cycle consists of three phases amplification, diversification, and selection. In these experiments, the fitness of genotypes is tantamount to their probability to enter the next selection round. 

Sequential protocols in which one starts with a range of multivalent banks, and works towards higher affinities with monovalent libraries, mutational libraries or extension libraries, derived from the enriched clones, have been shown to be highly effective (e g. Refs. 33, 85 and 94). This and the evolutionary approach in highly mutagenic E. coli hosts over many selection rounds (e.g. Ref. 41) will surely influence further experimental design. 

Increased thermal stability of proteins used for biosensors is desirable to allow for robust devices that can withstand a variety of storage, assay, and regeneration conditions. In addition, some evidence suggests that starting from the most stable version of a bioreceptor by yeast surface display will aid in later affinity maturation efforts (38-40). In addition, thermally stable mutants can enable higher expression levels as soluble proteins from yeast or E. coli (19, 20, 38). Thermal stability selection rounds have been carried out on many of the proteins that were later mutated for high affinity (17,19-21, 38 2). 

The recently described method of ribosome display has been proposed as a method to affinity-mature antibodies. This approach involves the translation of proteins in vitro and their selection while attached to ribosomes [88,89]. This can occur if the mRNA lacks a stop codon. In this case, the ribosome is not able to detach, and mRNA and encoding protein remain attached together and can be selected on a relevant ligand. After selection, the mRNA from selected polysomes is converted into cDNA, amplified by PCR, and used for the next transcription, translation, and selection round. Ribosome display has been used for the display [89,90] and evolution of a scFv antibody in vitro [89], but no improvement in affinity was reported. However, the combination of this display technique (which potentially allows access to unlimited genetic diversity, unlike phage display) with methods such as DNA shuffling will see greater affinity improvements... 

Whereas oligonucleotide libraries were traditionally partitioned by filtration or capture on beads or on affinity columns, capillary electrophoresis has been validated as a promising alternative. Aptamers displaying a high affinity were obtained in a very limited number of selection rounds. Nonequilibrium capillary electrophoresis of equilibrium mixtures led to the identification of DNA aptamers... 

---