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Multi-line split DNA synthesis: a novel combinatorial method to make high quality peptide libraries - PubMed

  • ️Thu Jan 01 2004

Comparative Study

Multi-line split DNA synthesis: a novel combinatorial method to make high quality peptide libraries

Ichiro Tabuchi et al. BMC Biotechnol. 2004.

Abstract

Background: We developed a method to make a various high quality random peptide libraries for evolutionary protein engineering based on a combinatorial DNA synthesis.

Results: A split synthesis in codon units was performed with mixtures of bases optimally designed by using a Genetic Algorithm program. It required only standard DNA synthetic reagents and standard DNA synthesizers in three lines. This multi-line split DNA synthesis (MLSDS) is simply realized by adding a mix-and-split process to normal DNA synthesis protocol. Superiority of MLSDS method over other methods was shown. We demonstrated the synthesis of oligonucleotide libraries with 1016 diversity, and the construction of a library with random sequence coding 120 amino acids containing few stop codons.

Conclusions: Owing to the flexibility of the MLSDS method, it will be able to design various "rational" libraries by using bioinformatics databases.

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Figures

Figure 1
Figure 1

Scheme of the MLSDS method. The case of three-lines is shown. Uppermost three boxes indicate the state in the DNA synthesizers after the first three synthetic cycles, that is, partial mixtures of triplet codons attached to the CPGs. 1, 2 and 3 denote 1st, 2nd and 3rd letter mixture in the line-1 DNA synthesizer, respectively. Their A:T:G:C mixing ratios are designed with the GA program so that 123 gives a partial mixture of triplet codons without any stop codons. In the same way, 456 and 789 denote corresponding partial mixtures of triplet codons in the line-2 and the line-3 DNA synthesizers, respectively. The equimolar mixture of 123, 456 and 789, which is obtained after split-and-mix procedure, can be designed to give approximately the target amino acid composition without stop codons. Examples of 1,2,.....,9 are shown in Table 1.

Figure 2
Figure 2

Dependence of the final fitness on the number of DNA synthesizers for various libraries. Ordinate: final fitness (=correlation coefficient). Abscissa: line number of DNA synthesizers. Libraries are "Uniform", "Natural", "No Met" and "No Cys".

Figure 3
Figure 3

Production of 2×, 4×, and 8× length library. Lane-M: Size marker. 10 base ladder (left) and 100 base ladder (right). Lane-S: Initial synthesized library (87 mer). Lane-1U: Initial single-unit library (94 mer) made by PCR amplification. Lane-2U: Double-unit library (138 mer) made by ligation product of digested 1U. Lane-4U: quadruple-unit library (240 mer) Lane-8U: octuple-unit library (444 mer). PAGE was done with 5 % polyacrylamide, TAE buffer (pH 8.0), 8 M urea, 65°C and stained with SybrGreen I and visualized with a fluorescence imager (Bio-Rad FX).

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