Generation of random mutant libraries with multiple primers in a single reaction

Abstract

Characterization of multiple sites in a single gene that are important in biological phenotypes is challenging due to the difficulty to generate many mutants representing all or a majority of combinations of mutations in the gene. Using the HIV-1 env and pol genes as templates, four random libraries were generated representing different combinations of mutations introduced by up to 36 mutagenesis primers in a single assay. Over 86% of the clones contained mutations and the mutants tended to have single or fewer mutations in the libraries. When protein size was used as a screening marker, all identified clones contained at least 2 mutations and up to 12 mutations were detected in a single clone. Nearly all mutant clones in each library contained unique mutations, indicating that mutants in the library were generated at random. Closely related mutations which were overlapped by neighboring mutagenesis primers were often introduced in this system. Analysis of the env library showed that some potential N-linked glycosylation sites did not increase the Env molecular mass significantly, suggesting they were not used for glycosylation or only limited carbohydrate moieties were added at these sites. This novel method can serve as a powerful tool to study the biological phenotypes of genes whose functions are determined by multiple sites.

Figure 1

Identification of clones containing introduced mutations by Western blot and sequencing analysis. The same amount (0.5 μg) of each plasmid DNA was transfected into 293T cells. Equal volume (12 μl) of culture supernatant was loaded into NuPAGE® Novex 4–12% Bis-Tris gels. Anti-HIV-1 Env mAb 13D5 and Alexa Fluor® 680 conjugated goat anti-mouse IgG were used to detect Env proteins. The image was acquired using the Odyssey Infrared Imaging System (Li Cor Bioscience, Lincoln, NE). The clones with added PNLGs showed increased molecular weight of the expressed Env Proteins. The mutation sites introduced in individual clones were confirmed by sequence analysis.

Figure 2

Generation of clones with mutations close to each other by overlapping mutagenesis primers. Sequences of overlapping primers, parent clone MCon3 and resulting mutants are aligned. The codon in which the mutation was introduced is underlined. The mutation positions are indicated with black dots. (A) Mutagenesis primers overlap each other but do not overlap the actual mutation bases. (B) Mutagenesis primers overlap each other and the mutations.