In vitro template-change PCR to create single crossover libraries: a case study with B. thuringiensis Cry2A toxins

Abstract

During evolution the creation of single crossover chimeras between duplicated paralogous genes is a known process for increasing diversity. Comparing the properties of homologously recombined chimeras with one or two crossovers is also an efficient strategy for analyzing relationships between sequence variation and function. However, no well-developed in vitro method has been established to create single-crossover libraries. Here we present an in vitro template-change polymerase change reaction that has been developed to enable the production of such libraries. We applied the method to two closely related toxin genes from B. thuringiensis and created chimeras with differing properties that can help us understand how these toxins are able to differentiate between insect species.

Figure 1. Single-crossover recombination process.

Only the forward recombination process is shown. The small triangles represent ddATP molecules, and the gray regions in primers or DNA fragments represent flanking sequences used for selective amplification. (A) Generation of reverse ssDNA templates from cry2Aa (using primers F and R) and cry2Ad (using primers F’ and LR) by asymmetric PCR. (B) Generation of randomly terminated cry2Aa forward chains using primer LF. (C) Re-annealing and extension of cry2Aa forward chains using the cry2Ad ssDNA template to generate chimeric double-stranded DNA amplicons. (D) Use of the flanking sequence primers (SF and SR) for selective amplification and resolution of the chimeras.

Figure 2. Crossover regions of the generated chimeras relative to a cry2Aa-cry2Ad gene alignment.

The crossover occurred in a region containing identical DNA sequences between cry2Aa and cry2Ad, and these are indicated by open rectangles. 2Aa and 2Ad refer to the cry2Aa and cry2Ad genes respectively. R01 to R37 are the crossover regions of chimeras, the numbers in parentheses means the number of sequenced chimeras with crossovers within that region. Regions of sequence identity are indicated by gray shading and the vertical lines represent the boundaries between the three structural domains.

Figure 3. Biological activity of the chimeras.

2Aa and 2Ad refer to the Cry2Aa and Cry2Ad toxins respectively, and R01 through R37 represent the individual chimeras. The y-axis indicates the toxin’s relative toxicity following exposure of the test insect to 50 ppm toxin. The relative activity of each toxin was normalized to the activity of Cry2Aa. The original mortality data are shown in Supplementary Table 2.