PCR-based approach for detection of novel Bacillus thuringiensis cry genes

Abstract

A two-step strategy, named exclusive PCR or E-PCR, has been developed to overcome the main limitation of PCR, which is the detection of already-known sequences only. This strategy allows the ability to detect and further clone and sequence genes for which no specific primers are available and in which a variable region exists between two conserved regions. This approach has been applied to Bacillus thuringiensis cryI genes by the use of mixtures of degenerate and specific primers recognizing well-known sequences. The first step allows the accurate identification of already-characterized cryI genes by the use of three primers. During the second step, the same sets of primers are used to exclude known sequences and to positively detect cryI genes unrecognized by any specific primer. The method, as well as its application to detect, clone, and sequence a novel cryIB gene, is described in this article.

PCR-based identification of Bacillus thuringiensis pesticidal crystal genes

“…Another strategy to expedite the screening is based on the use of a mixture of more than two primers (multiplex PCR) in the same reaction [14,24,25]. Usually, a single universal primer is combined with several speci¢c oligonucleotides that recognize individual genes.…”

“…The DNA used as template is obtained either by the typical lysis extraction and puri¢cation methods [14,18,29], or by simple time-saving lysis of a cell suspension using physical methods. In the latter case, DNA is usually released by boiling the suspension for 2^10 min [13,15,30], or by alternate freezing (370 ‡C) and boiling steps [17,24,26]. Template is added to the reaction mixture (¢nal volume 50^100 Wl) and the three-step ampli¢cation cycles are performed 25^35 times.…”

“…Kuo et al [59] reported the ¢rst primer pair able to recognize all the members of the cry1 gene subfamily known at that time, as well as some other genes coding for the actual cry4, cry3 and cry7. Jua ¤rez-Pe ¤rez et al [24], Jua ¤rez-Pe ¤rez [25], and Masson et al [22] also used the same concept of universal primers to amplify all the members of di¡erent subfamilies of the cry genes. Moreover, they introduced the use of degenerate primers in order to increase the probability of amplifying novel group members that did not exactly match the putative conserved region of the subfamilies from which the oligonucleotides were designed.…”

“…Another approach to detect new cry genes is based on the use of two sequential PCR reactions, using a multiplex PCR with speci¢c and universal primers [24]. This strategy, called exclusive-PCR (E-PCR), starts with the ampli-¢cation of already described cry1 genes, followed by a second conditional ampli¢cation that will occur only if a new putative cry1 gene(s) is present in the strain.…”

16S–23S rRNA internal transcribed spacers as molecular markers for the species of the 16S rRNA group I of the genus Bacillus

“…The biotechnological importance of some species, suc as B. subtilis, B. amyloliquefaciens, B. licheniformis and B. stearothermophilus, is due to the many applications related to the production of enzymes, antibiotics, solvents and other molecules [1]. Some species like B. thuringiensis and B. sphaericus are used for the control of insects for crop protection [2]. Bacilli (e.g.…”