BMC Genomics 2006, 7:73 http://www.biomedcentral.com/1471-2164/7/73 FReigcuormeb7ination profile of self-recombining loxP sites Recombination profile of self-recombining loxP sites. For each self-recombining spacer, the light blue portion of the stacked bar represents the count of self-recombination reactions and maroon the count of recombination reactions with other spacers. tion and not once did this sequence undergo self recom- bination (see Additional file 4). Our data show that GGGTAGGN is the sequence most favoured by Cre recombinase (Figure 5). Third, Lee and Saito argued that domains exist in the spacer region which cannot tolerate mutations due the requirements of strand exchange and resolution of the Holiday junction in the loxP intermedi- ate [7]. Lee and Saito described three spacer domains, two in which homology was required for exclusive self-recom- bination. We did not observe this in our data as all possi- ble positions in the spacer sequence could tolerate mismatches and proceed to recombine in our in vitro assay. The observation of 80 reactions in our data that had all six possible degenerate spacers mismatched suggests there are few absolute spacer homology requirements required for Cre-mediated recombination (at least in an in vitro setting). Finally, it has been assumed that directed, non-promiscuous recombination is best mediated by identical spacer mutants. In fact, none of our self-recom- bining spacer mutants that reacted more than once were non-promiscuous. Further, we identified 16 pairs of non- self recombining spacers; some of which recombined at levels similar to the top self-recombiners (Table 5). For example, the non-self recombining spacers TGGTAGGC and TGGTAGGT reacted together four times which is identical to the number of GGGTAGGG self-recombina- tions (Table 5, Figure 7). All spacers, save for GGG- TAGGG, reacted with equal probability with a suite of spacer sequences however in most cases there was one spacer that had a higher number of reactions. These results question the necessity, or preference, of the Cre recombi- nase to react with homologous identical sequences. In future Cre-loxP applications it may prove reasonable to select any two sites that show specificity for each other, regardless of whether or not they share identical spacer sequence. Several published spacer mutants have been described in the literature as self-recombining and are non-promiscu- ous (at least with the wild-type loxP spacer sequence). Some sites are extremely non-reactive with other loxP sites, namely lox2272 [7], m3, and m7 [8]. We screened our data set for published sequences (Table 1) to see if these trends were recapitulated. The wild-type spacer was detected in one reaction but no other published spacers were detected at all. We did detect a number of the spacers in the Lee and Saito data [7] but only one of their spacers, AGGTATGC or lox23, was detected in our self-recombining data. These results do not challenge the utility of published spacer mutants but indicate that published spacer sequences are potentially not kinetically favoured by Cre recombinase relative to other spacers. Additionally, there has been speculation in the literature that spacers at positions 2 and 7 are essential for exclusive recombination, at least between loxP mutants and the wild-type spacer [7,8]. In our set of self-recombining sites we detected recombina- tion at both positions 2 and 7 indicating that mismatches at these positions resulted in spacer promiscuity (see Additional file 4). In fact, all position, but not all nucle- otide, mismatch combinations were detected in the com- plete dataset. However, our results cannot rule out the possibility that certain combinations of specific nucle- otide mismatches can block promiscuous recombination of self-recombining spacers. When using inverted repeat mutants the recombination reaction yields two different tandem loxP sites; one site with double (LE and RE) inverted repeat mutants and the other with wild-type sequence. These products are assumed to be resistant to subsequent Cre-mediated recombination. We screened all of our sequences for evi- dence of failure of the inverted repeat mutants to block further rounds of recombination. We found only three Page 10 of 13 (page number not for citation purposes)