RE TGGTAGGT GGGTATGG GTGTAGTG GTGTATGG TGGTAGTG GTGTACGC GGGTAGGA GTGTATAC GGGTATGT GGGTAGTC GTGTAAGG GGGTAGGA GGCTAGGC GAGTAGGG GTGTATGT CTGTAGGG BMC Genomics 2006, 7:73 Table 5: Non-self recombining spacers LE TGGTAGGC GGGTATGC GTGTAGTT GGGTATGG TGGTAGTC GTGTACGG TGGTAGGA GGGTATAC GGGTAAGT TGGTAGTC GTGTAAGA GGGTATGA GGGTATAC GAGTAGGA TTGTATGC TTGTAGGC examples of additional recombination within our set of 3,124 successful reactions. The fact that even the most prolific spacer sequences such as GGGTAGGC did not react with the double mutants suggested that products were stable post-recombination. There is a need for functional genomics tools that can expand the number of transgenic DNA segments that can be specifically incorporated into a target sequence. For example, synthetic biology applications require a mecha- nism to stitch together disparate DNA molecules from var- ious sources in an ordered, controlled manner. Serializing RMCE or insertional recombination via inverted repeats has been limited by the small number of stable, non-pro- miscuous loxP sites identified to date. Furthermore, previ- ous attempts to discover novel loxP spacers have focused on mutants slightly different than, and non-reactive with, the wild-type sequence. In this work we deploy a high- throughput pipeline that rapidly identifies novel loxP spacers, regardless of sequence composition, with stability post-recombination and limited promiscuity. The set of 12 spacer sequences detected with these properties have an increased probability of successfully tolerating multi- ple loxP recombinations in vivo and, therefore, these sites have potential utility. Conclusion In this study we introduce a novel, high-throughput assay to rapidly screen for successful recombination between loxP spacers using standard molecular biology techniques and high throughput sequencing. Using the results from 3,124 successful reactions, we provide the first profile and comprehensive analysis of sequence spacers favouring Cre-loxP recombination. Increasing the guanine content at all spacer positions save for position 8 resulted in http://www.biomedcentral.com/1471-2164/7/73 Reactions 4 3 3 2 2 2 2 2 2 2 2 2 2 2 2 2 increased recombination reactions. Interestingly, self- recombination is not preferred over non-self recombina- tion and the collection of known loxP spacer mutants do not appear to be favoured by Cre recombinase. We have identified novel self and non-self recombining loxP spacer sites that expand the repertoire of sites available for fur- ther study and potential in vivo recombination applica- tions. Further, given that these sites have been shown to be functional in combination with inverted repeat muta- tions, they should be useful for mediating direct serial insertions into a given target construct, chromosome or genome. Methods Generating two pools of LE/RE-spacer mutants Two oligonucleotides were designed that contained loxP sites with six degenerate spacer nucleotides (positions 1,2,3,6,7,8) and two central fixed spacer nucleotides (4,5) (Table 2). One of the two oligonucleotide pools (LE) con- tained the lox71 left arm mutant sequence and the other (RE) contained the lox66 right arm mutant sequence [12]. These oligonucleotides were synthesized (Invitrogen), purified by polyacrylamide gel electorophoresis, and dou- ble-stranded product was generated by PCR. Generating a library of circularized LE-spacer mutants The amplified, double stranded LE-spacer fragment was double digested with EcoRI and HindIII, agarose gel puri- fied, and ligated to EcoRI/HindIII linearized and agarose gel purified pUC19 vector to create a library of LE mutant sequences (Figure 1). Ligation products were transformed into DH10B cells (Invitrogen) and amplified on 2xYT solid media. Supercoiled plasmid DNA was isolated by alkaline lysis and then subsequently agarose gel purified. Page 11 of 13 (page number not for citation purposes)