e103 Nucleic Acids Research, 2006, Vol. 34, No. 15 PAGE 2 OF 4 Table 1. Nested primer pairs used P53 Section A 50-UTR-AA249 P53 Section B AA156-30-UTR MLH1 section A 50-UTR-AA239 MLH1 section B AA206–AA496 MLH1 section C AA459–AA718 MSH2 section A AA6–AA311 MSH2 section B AA265–AA512 MSH2 section C AA441–AA742 MSH2 section D AA680-30-UTR 1st 2nd 1st 2nd 1st 2nd 1st 2nd 1st 2nd 1st 2nd 1st 2nd 1st 2nd 1st 2nd Forward GTCTAGAGCCACCGTCCAGGGAGCAGGT CGGGCTGGGAGCGTGCTTTCCACGA GACCTGCCCTGTGCAGCTGTGGGTTGA CGCGTCCGCGCCATGGCCATCTACA CTGAAGGCAC TTCCGTTGAG CAT CCTTGGCTCT TCTGGCGCCA AA AACAAGGAGA GACAGTAGCT GA CTACCCAATG CCTCAACCGT GGA GGGATACAAC AAAGGGGACT TCAGA GAGAAGAGAG GACCTACTTC CAGCA AGGTTTCGAC ATGGCGGTGC A CGAAGGAGAC GCTGCAGTTG GA CCTCAACCGG TTGTTGAAAG GCAA GTGCTGTATT GCCAGAAATG GAGA CAGGCTCTGG AAAAACATGA AGGAA CTGATCTTCG TTCTGACTTC TCCA GTTCCACATC ATTACTGGCC CCAA GACAAACTGG GGTGATAGTA CTCA Reverse GCCGGTCTCTCCCAGGACAGGCACA CTCCGGTTCATGCCGCCCATGCAGGA GTCCTACTCCCCATCCTCCTCCCCACAA GGGTGGGAGGCTGTCAGTGGGGAACA GCACTTCTTC ACTGAGTAGT TTGC AGGCTAGGGT TTTATCCTCA CATCCA CCTGGAGACT CAAAACACTA GTGA GGGGGGTACA AGCTGCAGTC A GTGTGAGCGC AAGGCTTTAT AGACA GTGTTCCACA GTCCACTTCC AGGA ACTTATTCAG CAAGGCAGCC AGA GTTAAGGGCT CTGACTGCTG CAA ATCCAAACTG TGCACTGGAA TCCA GTTTAATCTG TTTGCCAGGG TCCAA GTAGGTAGAA GTTCCTCTTC CCAA GAATCTTTGG TTGCAGACCT GA AACAGTCCTC AGTTACAGCT CTCA AAGTTGATAG CCCATGGGCA CTGA The forward and reverse, first and second round primers are shown for the gene sections indicated in the first column. The names of each section and the amino acid residue numbers covered are indicated. The nested PCR step was necessary as enrichment did not work on a PCR with a single pair of primers (data not shown). The reason is because although the simple PCR produced a similar amount of product, it is contaminated with a complex mixture of non-specific products that can cross hybridize with the main splice form of the target gene, thus attaching single-stranded DNA regions to it. RT–PCR Testis RNA was reversed transcribed with ‘Transcriptor’ reverse transcriptase (Roche) to avoid ‘false splicing’ arte- facts caused by some non thermostable polymerases [data not shown and (3)]. First round hot-start nested PCR followed by [(94 C 30 s, annealing temperature 30 s, 72 C 60 s) · 20 cycles, 72 C 5 min] was performed on testis cDNA with Expand High Fidelity T.Aq polymerase (Roche) in PCR buffer (Promega, 1· contains 50 mM KCl, 10 mM Tris–HCl pH 9, 0.1% Triton) also containing 0.25 mM dNTPs and 2 mM MgCl2. (Annealing temperatures were 69 C for TP53 and 59 C for MLH1 and MSH2, PCR volume 100 ml). A total of 0.1 ml of the ﬁrst reaction was used as template for the second reaction with the same conditions as before but with 35 cycles. The ﬁrst and second round primer pairs for ampli- ﬁcation of different sections of TP53, MLH1 and MSH2 are shown in Table 1. Splice enrichment protocol (1 h) PCRs were treated by addition of 100 ml of 1· L buffer [Roche 1· L buffer contains 10 mM Tris–HCl pH 7.5, 10 mM MgCl2, 1 mM dithioerythritol] containing 1 ml Exo- nuclease VII (USB) for 30 min at 37 C. No heat inactivation step is necessary and reactions can be stored at 20 C over- night either before or after this step. Fifty microlitres of the ssb-beads (which corresponds to 4 ml of the original slurry) were magnetically separated and liquid was removed, then 100 ml of exonuclease-treated PCR was added and left on the bench for 5 min. The beads were washed three times in EASI buffer (100 mM NaCl and 20 mM Tris, pH 7.5) then bound heteroduplexes were eluted for 5 min in 10 ml of EASI buffer containing 200 mM imidazole. Splice identification One microlitre of the eluate was used in a 25-cycle PCR to amplify the enriched material. The ﬁnal PCR (10 ml) was visualized on a 1.25% agarose TBE gel (Figure 1). Extra bands not annotated correspond to heteroduplexes of the different isoforms. For example in Figure 1b there are three strong un-annotated bands. These are the three possible heteroduplexes between the strong main band and isoforms 2 and 3. Each heteroduplex migrates about a quarter of the distance from the upper to the lower band that it is composed of, under our gel conditions. The remaining 90 ml of the enriched material was treated with T.Aq DNA polymerase (Promega) and 0.25 mM dATP at 72 C for 15 min, cleaned (PCR cleanup, Qiagen) and then cloned into pCR 4 TOPO (Invitrogen) TA cloning vector, and transformed into E.coli. To screen for alternative splices a ‘touch’ of bacterial colonies was then used as template in 6–24 PCRs for each enrichment [95 C 3 min, (94 C 30 s, 59 C 30 s, 72 C 60 s) · 35, 72 C 3 min with T.Aq DNA polymerase (Promega)] with the M13uni( 43) AGGGTTTTCCCAGTCACGACGTT and M13rev( 49) GAGCGGATAACAATTTCACACAGG primers. After a quick centrifugation to remove cell debris, PCR products were imaged by agarose electrophoresis and reactions of each size not conforming to the wild-type length were puriﬁed (Qiagen PCR cleanup) and sequenced with the M13 primers. RESULTS We ﬁrst developed the EASI technique using a known pair of alternative splice forms (from the HipK3 gene). A nested PCR from testis cDNA which contained both alternative splices in a >10:1 ratio (Figure 1a input lane) was bound (without any further treatment) to E.coli single-stranded DNA-binding protein (ssb) beads, T.Aq ssb beads or beads alone (Figure 1a). Bound material was eluted with imidazole and further ampliﬁed by PCR and visualized by agarose gel electrophoresis. Both ssbs caused enrichment of the rarer larger splice form but the T.Aq ssb beads enriched the isoform