Academic World: Introduction to Common Library Construction Methods in Directed Evolution

Common library construction methods include error-prone PCR, whole plasmid large primer PCR, saturation mutation, DNA shuffling, in vitro random recombination, and cross-extension, etc.

Error-Prone PCR is the most commonly used method for creating random mutation libraries. This method takes advantage of the low fidelity feature of Taq enzyme, supplemented by high concentrations of Mg2+ and Mn2+, to increase the probability of base mismatch in the amplification system. Theoretically, it can cover all mutants. However, false positives may exist in mutant libraries and are greatly affected by template plasmids. Effectively removing template plasmids is the key to this method.

Megaprimer PCR of Whole Plasmid (MEGAWHOP) overcomes the shortcomings of error-prone PCR product digestion and ligation, namely the influence of template plasmids. The whole plasmid large primer PCR method mainly consists of four steps, namely, preparing DNA large primers, PCR amplification of whole plasmids, DpnI treatment of PCR products and transformation.

Saturation Mutagenesis refers to a mutation method in which the amino acid at the target site replaces 19 other amino acids. According to the basic principles, it can be classified into oligonucleotide directed mutation, box mutagenesis, overlapping extension PCR and other methods. Different methods have their own advantages and disadvantages, and the choice can be made based on the actual situation.

Table 1 Advantages and Disadvantages of Different Saturation Mutation Methods

DNA Shuffling is a process that uses DNASE I to enzymatize the mutated target gene, causing it to form many random fragments of different sizes. These fragments are then amplified without a template. The random fragments serve as primers and templates for each other and recombine during the amplification process to obtain the mutant.

The target gene can be obtained through error-prone PCR, but when shuffling, primer-free PCR assembly may all come from the same parent, reducing the mutation efficiency. Therefore, the use of DNA Family Shuffling based on ssDNA can reduce the influence from parents. The target gene of the parent was phosphorylated by 5 ', and then the double-stranded DNA was degraded by λ exonuclease to obtain ssDNA. The ssDNA was mixed in equal molar ratios and then subjected to enzymatic digestion and PCR amplification by DNASE I.

Figure 3 Schematic diagram of the principle of DNA Shuffling

Random-Priming in Vitro Recombination (RPR) uses single-stranded DNA as a template, supplemented by merging primers, to generate short DNA fragments complementary to different sites of the template. There will be a small number of point mutations in the short DNA fragments, and in the subsequent PCR reaction, They recombine with each other as primers to form complete gene lengths.

Figure 4 Schematic diagram of the principle of in vitro random recombination

The interleaved Extension (Staggered Extension Process, StEP) is actually two PCR processes. In the first PCR, only annealing and extension were set, with the annealing and extension times only set at 5 to 15 seconds to obtain short fragments. In the second PCR, standard PCR was set to amplify the full-length gene.

Figure 5 Schematic diagram of interlaced extension

The above is an introduction to some commonly used methods for constructing mutant libraries. Besides, there are also methods such as RACHII and ITCHY, which need to be combined with efficient screening methods to achieve the purpose of high-throughput screening.