Innovative Assembly Techniques for Large-Scale Gene Synthesis

The assembly technology for synthesizing large DNA fragments from scratch is the cornerstone of gene synthesis, encompassing methods such as BioBrick™, TAR, BglBrick, Golden Gate, Gibson assembly, CPEC, and Overlap PCR. Due to the limitations of current technology in accurately synthesizing DNA of gene length in a single step, it is necessary to combine in vitro and in vivo assembly techniques to assemble oligonucleotide fragments synthesized in segments into long DNA fragments.

In vitro assembly techniques

They are classified into various methods based on fragment size and sequence characteristics, with a common requirement for using tool enzymes. Overlap PCR, based on the DNA polymerase method, is simple and rapid in operation. The BioBrick and BglBrick methods, which rely on isoschizomers, enable standardized assembly, albeit with the BioBrick method being unsuitable for fusion proteins. The Golden Gate cloning method, based on type IIS restriction enzymes, allows efficient seamless ligation of multiple fragments. The Gibson assembly method, which utilizes a combination of multiple tool enzymes, enables seamless ligation with fragment sizes reaching several hundred kb. The standardized design of DNA components enhances the utilization rate of existing fragments.

In vivo assembly techniques

Although in vitro assembled fragments can reach several hundred kb in size, the yield is often insufficient, necessitating amplification using organisms such as Escherichia coli. In assembling fragments exceeding 20 kb, recombinant systems within organisms are often employed, including Bacillus subtilis, yeast, and engineered bacteria. Yeast, as a commonly used host, has been recognized for its efficient homologous recombination capability for many years. It can be used to assemble artificial chromosomes up to several megabases (Mb) in length. Additionally, Saccharomyces cerevisiae exhibits an extraordinary tolerance for chromosome length, providing a theoretical basis for utilizing yeast to construct ultra-long chromosomes in higher organisms.

Applications

Yaohai Bio-Pharma primarily employs two assembly methods for gene synthesis: ligase assembly and DNA polymerase assembly. The ligase assembly method connects overlapping and hybridized 5'-phosphorylated oligonucleotide fragments into double-stranded DNA using DNA ligase. The polymerase assembly method utilizes DNA polymerase to extend the overlapping oligonucleotide fragments through hybridization, obtaining a mixture of different lengths, and finally amplifying the successfully assembled full-length fragments using primers. Yaohai’s can help clients satisfy specific requirements in gene synthesis.

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