Combination of error-prone PCR (epPCR) and Circular Polymerase Extension Cloning (CPEC) for improving the coverage of random mutagenesis libraries.

Random mutagenesis, such as error-prone PCR (epPCR), is a technique capable of generating a wide variety of a single gene. However, epPCR can produce a large number of mutated gene variants, posing a challenge in ligating these mutated PCR products into plasmid vectors. Typically, the primers for mutagenic PCRs incorporate artificial restriction enzyme sites compatible with chosen plasmids. Products are cleaved and ligated to linearized plasmids, then recircularized by DNA ligase. However, this cut-and-paste method known as ligation-dependent process cloning (LDCP), has limited efficiency, as the loss of potential mutants is inevitable leading to a significant reduction in the library's breadth. An alternative to LDCP is the circular polymerase extension cloning (CPEC) method. This technique involves a reaction where a high-fidelity DNA polymerase extends the overlapping regions between the insert and vector, forming a circular molecule. In this study, our objective was to compare the traditional cut-and-paste enzymatic method with CPEC in producing a variant library from the gene encoding the red fluorescent protein (DsRed2) obtained by epPCR. Our findings suggest that CPEC can accelerate the cloning process in gene library generation, enabling the acquisition of a greater number of gene variants compared to methods reliant on restriction enzymes.

Mutagenesis in Rice: The Basis for Breeding a New Super Plant.

The high selection pressure applied in rice breeding since its domestication thousands of years ago has caused a narrowing in its genetic variability. Obtaining new rice cultivars therefore becomes a major challenge for breeders and developing strategies to increase the genetic variability has demanded the attention of several research groups. Understanding mutations and their applications have paved the way for advances in the elucidation of a genetic, physiological, and biochemical basis of rice traits. Creating variability through mutations has therefore grown to be among the most important tools to improve rice. The small genome size of rice has enabled a faster release of higher quality sequence drafts as compared to other crops. The move from structural to functional genomics is possible due to an array of mutant databases, highlighting mutagenesis as an important player in this progress. Furthermore, due to the synteny among the Poaceae, other grasses can also benefit from these findings. Successful gene modifications have been obtained by random and targeted mutations. Furthermore, following mutation induction pathways, techniques have been applied to identify mutations and the molecular control of DNA damage repair mechanisms in the rice genome. This review highlights findings in generating rice genome resources showing strategies applied for variability increasing, detection and genetic mechanisms of DNA damage repair.