The Genom of Homo sapiens.pdf

MOUSE GENOME ENCYCLOPEDIA PROJECT 197presence of trehalose, the 5-kb FL cDNA product is visiblein lanes 2 and 3 without any partial products, showinglow electrophoretic mobility. In Figure 2b, naturalmRNA was used. Using the traditional protocol, thelongest size of the first-strand cDNA is around 9 kb.However, in the presence of trehalose, it is much improvedto 14–16 kb, covering almost all of the transcriptspresent.Selection Technology (CAP Trapper Technology)Figure 3a shows the structure of mRNA. The CAP siteshould be labeled to select FL cDNAs. We exploited the“diol” structure shown in this figure, which is a specificfeature of the CAP site. The CAP and poly(A) sites arethe only two sites where diol groups occur. The diolgroup can be oxidized by sodium periodine (NaIO 4 ) toproduce a dialdehyde group that could be connected to ahydrazide group. Biotin-hydrazide, which is commerciallyavailable, is very convenient for direct biotinylationin this reaction. This procedure is based on the chemicalreaction, and the efficiency is almost perfect(Carninci et al. 1996, 1997).Figure 3b shows the whole procedure of CAP trapping.After single-stranded cDNA synthesis, the CAP andpoly(A) sites are labeled using the protocol shown in Figure3a. RNase I is used to cleave remaining singlestrandedRNA, but does not act on RNA–DNA hybrids.Single-stranded RNA is cleaved by RNase I at a 5´ site.Cleavage also occurs at a poly(A) site (Fig. 3b). The nonpoly(T)complementary sequence is designed as a primerfor the first cDNA. Hybridization of mRNA with FLcDNA preserves the biotinylated sites, which would otherwisebe lost following RNase I digestion. Therefore,only mRNA hybridized with full-length single-strandedcDNA is trapped by avidin beads. The first-strand cDNAis then isolated and subjected to the subsequent cloningprocess.Normalization and Subtraction TechnologyA standard library reflecting the original mRNA populationis biased, causing unpermissible redundant clonesin the process of collecting FL cDNAs. Three populationsare defined within the collected cDNAs. These are “abundantcDNA,” “cDNAs already collected,” and “rare andnew cDNA.” We used the biotinylated RNAs of the startingmaterial, and biotinylated RNAs transcribed in vitrofrom cDNA already collected, as subtraction drivers(Carninci et al. 2000). The first-strand cDNA and thesedrivers are mixed, and hybrids are trapped using avidinbeads. Thus, the undesirable populations of first-strandcDNA are eliminated, and only rare and new cDNAs arecollected. In this protocol, we did not use PCR becausethis produces a large cloning bias. This resulted in a goodefficiency of new gene discovery in our library.New Cloning VectorWe developed cloning vectors λFLC1, λFLC2,λFLC3, and λFLC4, based on infection after packagingand in vivo circularization, using the Cre-lox recombinationsystem (Carninci et al. 2001). λFLC1 is the prototypeof our vector, which is based on the insertional λ vector.λFLC2 is the substitution vector for longer cDNAs, ofmore than 3.5 kb on average. The super-rare cutter sitesin λFLC3 are designed to transfer the cDNA to the expressionvector. Finally, λFLC4 was designed to clonesuperlong cDNA, with an average length of around 10 kb.λFLC4 carries the F-factor replication origin, which isvery stable even when carrying very long insert DNA; forexample, that used in BAC vectors for genomic DNAcloning.Summary of the Riken cDNA LibraryRiken cDNA technology created a FL cDNA library ofgood quality. The average length of cDNA is 2.0–2.5 kbin a standard library, and more than 3.5 kb in λFLC2 andλFLC3. Using λFLC4, cDNAs greater than 10 kb are easilycloned. The full-length coverage of the Riken libraryis such that 90–95% of clones in the standard library encodeat least initiation codon (ATG), and 60% of clonesin the accumulated cDNA bank cover whole coding sequences(CDS). In the accumulated bank, the full-lengthrate is lower than that of the single library, because wecompromised and incorporated partial cDNAs into thebank if the sequence was novel.Figure 3. (a) Structure of mRNA and the procedure of its biotinylation.Two diol groups are oxidized by NaIO 4 , and thencoupled with biotin hydrazide. (b) Scheme of CAP Trapper forFL cDNA selection.RISA SYSTEMAs explained above, a high-throughput sequence systemwith the capacity to finalize the collection of FLcDNA within a few years had not been developed whenwe started. The required capacity was 40,000 sequence