DNA Recombination

DNA Recombination

DNA recombination, also known as DNA shuffling, is a powerful technique developed to create a sizable library for directed evolution. Unlike random mutagenesis, this technology mimics the natural process of sexual reproduction to generate a library of offspring with variations. The process usually starts with several DNA sequences encoding proteins of similar function or a gene pool from random mutagenesis. A typical procedure of DNA recombination can be divided into four steps: i) The gene fragments are first created by DNase digestion. Each fragment may contain different mutations. ii) Full-length nucleic acids are generated by PCR. iii) After amplification, the DNA sequences are inserted into carrier plasmid. iv) All DNA variants are expressed to generate a library of protein mutants.

DNA recombination can be either homologous recombination or non-homologous recombination. In QuikChange Shuffling, a typical homologous recombination-based method, homologous genes are mixed and sonicated to produce short fragments, which are then assembled on the basis of sequence homology with primerless PCR. Non-homologous recombination, on the other hand, constructs hybrid proteins when two genes have no or little seuquence homology. A typical example is incremental truncation for creation of hybrid enzymes (ITCHY) described in 1999. Further improvement has been made on this method since then.

Creating a good mutant library is arguably the most critical component in all directed evolution exercises. Creative BioMart provides services of library construction by DNA recombination based on customer's need. With years of experience in this field, we offer mutant library construction with minimum existence of wildtype sequences, redundant sequences and truncated sequences:

• Homologous recombination.
• Non-homologous recombination.
• Sequence validation
• Transformation into desired host.

Reference:

1. Tee, K.L., Wong, T.S. (2013) Polishing the craft of genetic diversity creation in directed evolution. Biotechnology Advances. 31: 1707-1721.

Figure 1 Scheme of a typical example of DNA recombination.