BMC Genomics 2006, 7:73 http://www.biomedcentral.com/1471-2164/7/73 FMiegcuhraeni3sm of recombination between mutant loxP spacers Mechanism of recombination between mutant loxP spacers. Input lox71 left inverted repeat mutant (LE) and lox66 right inverted repeat mutant (RE) oligonucleotide sequences with scissile phosphodiester bonds highlighted with blue arrows. Cre-mediated recombination between the LE mutant and RE mutant resulted in one loxP site containing both mutants and another with wild-type inverted repeats. The input LE/RE spacer mutants of the reacting oligonucle- otides were inferred by working backwards from the loca- tion of the scissile bonds in the final sequenced recombinant. oligonucleotides was ambiguous because two different pairs of oligonucleotides could produce the same PCR products and sequence reads. Due to this ambiguity, this category was excluded from the majority of analyses. A Perl script (see Additional file 3) was run on these sequences to transform them to their inferred original for- mat and all intermediates based on the reaction mecha- nisms (Figure 3) (see Additional file 4). There were a total of 47 Type I reactions, 303 Type II reactions, 284 Type III reactions and 2,490 Type IV reactions with an average number of mismatches/spacer of 0, 1.5, 2.05 and 3.56 respectively (Table 3). The minimum number of mis- matches varied by type: zero in Type I or perfect matches, 1–2 in Type II, 1–4 in Type III, and 2–6 in the Type IV. Type II reactions had an approximately equal number of single and double mismatches (150 and 153 respec- tively). Type III reactions had 94 single mismatches, 102 double mismatches, 60 triple and 19 quadruple mis- matches. Lastly, Type IV reactions had 379, 878, 785, 368, and 80 of two, three, four, five and six bp mismatches, respectively. The observation of 80 Type IV reactions with six mismatches reflects the ability of the loxP spacer to tol- erate numerous mutations and continue to recombine. Sequence characteristics of the Cre-loxP spacer region Cre-loxP recombination is mediated by the cooperative binding to the inverted repeat of two Cre proteins to each loxP site [1,14]. We used the suite of successful input spacer sequences to build a binding profile, in the same manner that sequences from a SELEX scan of a transcrip- tion factor are used to determine the consensus binding site [23]. We decided to use all types of spacers in our data for this analysis including Type IV. It is reasonable to include Type IV reactions in this analysis because the nucleotide composition at each position can be unambig- uously determined and is all that is necessary for this anal- ysis (Figure 4D). The 6,248 spacers (from 2 × 3,124 successful input LE and RE oligonucleotides) that success- fully recombined showed no base preference at position 8, but a trend towards guanine at each of the remaining five degenerate positions (Table 4). A sequence logo [24] representing the consensus Cre-loxP spacer sequence is shown in Figure 5. Of all sites, the one most active in recombination was GGGTAGGC, which recombined with itself 6 times and also reacted with 26 other distinct spacer sequences in a total of 44 reactions. Further, we evaluated the effect that deviation from the most promiscuous spacer by 1 bp at each position had on recombination rate (Figure 6). As supported by the binding profile (Figure 5), position 8 was most impervious to substitutions with transversions from cytosine to guanine having the least effect. Transition to thymine and transversion to adenine had roughly the same effect dropping the recombination rate to around 65% of the maximum. Recombination rates dropped precipitously upon transitions of G to A or transversions to A at all positions with a slightly less pro- nounced effect at position 6. On the other hand, transver- sions to thymine were the most tolerated at each position save for position 3 where it mirrored the effect of C and A substitutions. Positions 3 and 7 appear to be the most sen- sitive overall to changes from the most promiscuous sequence. Novel recombining LE/RE-spacer mutants and their promiscuity profile Each sequence read had two, usually non-identical, loxP spacers representing a distinct recombination reaction. Page 6 of 13 (page number not for citation purposes)