BMC Genomics 2007, 8:363 http://www.biomedcentral.com/1471-2164/8/363 nium acetate and ethanol and then resuspended in 10 µl of TE. Lone linker LL1 (Fig. 1B) was prepared by annealing equimolar amounts of LL1L and LL1R (Fig. 1B) for 15 minutes at room temperature in TE. 1.2 µg of LL1 was then added to the cDNA and ligated overnight at 16°C (Methods-3). Ligated cDNAs were extracted with phe- nol:chloroform (1:1) and precipitated with sodium ace- tate and ethanol and resuspended in 50 µl TE. Fragments smaller than approximately 100 bp were removed by Sepharose CL-6B (Roche) gel filtration. The 250 µl flow- through peak was collected and the cDNAs precipitated with sodium acetate, 1 µl of glycogen carrier and ethanol and resuspended in 20 µl TE. This >100 bp cDNA popula- tion was amplified in a 10 cycle PCR reaction. Each 100 µl reaction contained 1 µg of the primer LL1P, 6 U and 0.6 U of Vent exo-, and exo+ thermostable DNA polymerases (New England Biolabs) respectively, 0.5 mM dNTPs, and 1 µl of α32P dCTP. An initial incubation at 72°C for one minute generated full-length 3' ends (Fig. 1B) (Methods- 4). The first 7 cycles were 95°C, 55°C, and 72°C for 1 min each. In the final three cycles, the 72°C extension steps were increased to 2, 4, and 8 min. respectively. Amplified cDNAs were purified by phenol/Chloroform extraction and ethanol precipitation. Self-subtraction 5 µg of amplified cDNA in 10 µl of annealing buffer (0.34 M NaCl, 0.1 M Na(PO4) pH 6.8, 1 mM EDTA) containing 300 ng of LL1F was overlayed with mineral oil, denatured by boiling for 5 min and then annealed at 60°C for one hour (Cot ~ 3 × 10-4 M·min). 100 µl of binding buffer (0.12 M Na(PO4), pH 6.8) was added to the bottom aque- ous phase which was then transferred to another tube. 100 µl of hydrated hydroxylapatite (Methods-5) sus- pended in 1.2 ml of binding buffer at 60°C was then added and the suspension incubated at 60°C for 10 min with frequent mixing. Bound dsDNA and hydroxylapatite were removed completely by discarding the pellets after two consecutive centrifugations (Methods-6). The 32P counts of sample aliquots taken before and after subtrac- tion, indicated that between 90 and 97% of the cDNA was bound to the hydroxylapatite and removed. The hydroxy- lapatite phosphate buffer was replaced with 10 mM Tris, 1 mM EDTA, pH8 (TE), by four consecutive exchanges in Centricon-100 filters (Amicon; Methods-7). The remain- ing single-stranded (ss) cDNAs were then subjected to a second round of amplification, and self-subtraction as described above, with the exception that the second rean- nealing time was 24 hours instead of one hour (Cot of ~ 7 × 10-3 M·min). Approximately 80% of the cDNA ampli- fied after the first subtraction was removed in the second self-subtraction. The phosphate buffer of the second self- subtraction reaction was exchanged for TE as described. The final double-stranded cDNA population for cloning was generated using the same PCR protocol used for the previous amplifications. Cloning Regenerated ds cDNAs were then digested with Bam HI and Asc I, and size-selected by agarose gel electrophoresis to obtain 200–300 bp cDNA fractions. These were then ligated into pKE-1 or pKE-2 vectors [19] and used to trans- form E. coli DH10B (Invitrogen) by electroporation. The three cDNA libraries – amphioxus, Drosophila and zebrafish – contained 6 × 106, 4 × 105, and 3 × 106 inde- pendent clones respectively. Analysis The cloning strategy was designed to preserve the orienta- tion of the cDNAs. The first strand directional random primer, DRP1, contained, from 5' to 3', 12 nt of defined buffer sequence, the 8 nt AscI restriction endonuclease sequence, 5 nt of random sequence and a 3' T nucleotide (Fig. 1A). The 5' buffer sequence was included to prevent destruction of the AscI site by the 5'->3' exonuclease activ- ity of E. coli PolI during second-strand synthesis. Using a primer which did not contain the buffer sequence resulted in the frequent appearance of cDNA clones that had lost the AscI site and therefore orientation (data not shown). T was added to the 3' end and As were excluded from the defined primer sequence at the cost of initiating cDNA synthesis at A (Fig. 1A-1, 2, 4) to eliminate an observed DRP1 self-priming artifact during first-strand synthesis. We tested the procedure by sequencing random clones picked from the different cDNA libraries. In an initial test, 43 clones from the Amphioxus library, 54 from the zebrafish library, and 15 from the Drosophila library were sequenced. Of the 112 clones, 9 clones contained no cDNAs and 4 clones yielded unreadable sequence data. The remaining sequences were compared to the NCBI non-redundant combined protein, combined DNA, and combined EST sequence databases, using the TBlastX search procedure [20]. In the self-subtracted libraries, 59 sequences matched translated RNAs, ESTs, or gene exons (e < 10-5) and 87% of these were in the forward reading frame. This fraction is within the 80–95% observed in directional cDNA libraries made by standard approaches using oligo dT primed cDNAs [21], indicating that the directional cloning strategy functions properly. that clon- ing was, as designed, directional. 16 matches to genomic DNA that were not part of any known functional or encoding RNA were identified. Since the first strand prim- ing technique was not completely random and the direc- tional primer included additional 5' GC-rich sequence (Fig. 1, parts 1,2), we were concerned that priming might be biased towards GC-rich regions. We examined this using clones which identified perfect matches in the sequence databases. From the 55 such clones we collected Page 3 of 7 (page number not for citation purposes)