Unique Library Format

Each FirstDraft ™ cDNA Library is provided as 2 Master-pools corresponding to the entire complexity of the two libraries obtained after different sizing procedures and 48 Sub-pools corresponding to a lower density (~12,000 clones per pool). Each one of these pools is provided as glycerol stock and super-coiled DNA. The DNA template corresponding to the small pools of cDNA clones can be used to synthesize large quantities of RNA in vitro for gene expression studies or proteomic applications. The DNA (or the corresponding RNA) prepared from these primary clones can also be used to transfer the cDNA molecules into many other systems (e.g. two-hybrid, phage display, ribosome display), currently being developed and used in drug discovery programs.


Schematic representation of the library construction for a group of cell lines or tissues:
The first strand cDNA molecules synthesized by oligo d(T) priming were size-fractionated by electrophoresis in low melting point agarose gel. One-fourth of the total cDNA was used to select molecules longer than 800 bp, while the remaining three-fourths was used to select molecules longer than 2 kb. The two fractions were cloned separately and the two resulting libraries were directly grown on agar plates. At this step, 48 pools of approximately 12,000 clones per plate were created (24 pools for each of the 2 sized libraries) and kept separately while the remaining clones were plated at a higher density and pooled together to represent the entire complexity of each library.


Each FirstDraft ™ cDNA Library is represented by two different sets of clones obtained from the same mRNA source after different size selection procedures to ensure representation of the longest transcripts without losing representation of smaller full-length molecules. These two fractions of cDNA molecules complement each other to more completely represent the entire set of expressed genes, independently of their size or abundance. The libraries have between 3x10⁶ and 10⁷ authentic primary clones with an extensive coverage of insert size, typically from 1 to 8 kb, that maximizes the probability of good representation of even the rarest and longest sequences.

The libraries were directly grown on agar plates at the initial transformation step to accumulate the biological material corresponding to the real primary clones. At this step, we made a special effort to separate the primary clones into groups of 12,000 (± 2,000) clones, which offers crucial advantages for downstream applications (see figure). For example even a rare transcript represented at 1 in 500,000 molecules in the entire library is now represented at 1 in 12,000 clones if present in a specific pool, so any assay capable of detecting signal, binding or activity in this range of complexity is now applicable. Depending on the type of experiment, multiple genes might also compete with each other such that only the most abundant or the most reactive molecules are selected. When the same experiment is carried out at a lower complexity, the probability of having the different genes represented without a competitor in one of the pools is increased. This is also true for PCR cloning applications in which different isoforms may be successfully isolated from small pools of clones but not from the entire library when only one of the isoforms may take over during the amplification process.