Technology development for gene discovery and full-length sequencing Page: 3 of 14
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Q Further development and optimization of a vector designed to facilitate transposon-
mediated full-insert cDNA sequencing.
DESCRIPTION OF EXPERIMENTAL APPROACHES AND RESULTS
A. METHOD FOR PREFERENTIAL CLONING OF TISSUE-SPECIFIC AND OR RARE MRNAS
This method was developed to maximize the likelihood of representation of rare mRNAs in a
cDNA library. A number of factors contribute to making cloning of rare mRNAs challenging,
cloning efficiency being one of them. Typically, cloning efficiencies are not high enough to warrant
representation of rare mRNAs (1-5 copies per cell) in cDNA libraries. Reassociation-kinetics analysis
indicates that the mRNAs of a typical somatic cell are distributed in three frequency classes: (I) super
prevalent [consisting of about 10-15 mRNAs which altogether represent 10-20% of the total mRNA
mass), (II) intermediate [1-2,000 mRNAs; 40-45%] and (III) complex [15-20,000 mRNAs; 40-45%]
(Bishop et al. 1974). The probability that a given mRNA will be represented in a cDNA library can
be expressed by the equation P(x) = 1-(1-on, where f=frequency and n=number of recombinant
clones. Accordingly, the probability that an mRNA that is present at 1 copy per cell (1 in 500,000
total RNA molecules) will not be represented in a cDNA library of 1 million recombinants is 14%.
To minimize this problem we devised a strategy based on the idea that cloning of rare mRNAs
could be facilitated by depletion of all previously identified mRNAs from the mRNA population
prior to cDNA synthesis. We reasoned that if the representation of rare mRNAs in a total cellular
RNA preparation could be increased by depletion of all previously identified mRNAs, their cloning
would become less challenging. This can be accomplished in a two-step process involving
hybridization of total cellular poly(A)+ mRNA with a driver comprising a comprehensive non-
redundant collection of cDNAs representing thousands of transcripts previously identified in that
mRNA population, followed by selection and isolation of the mRNAs that remain single strand. The
latter can then be amplified according to established methodologies and the amplified RNA utilized
for synthesis and cloning of cDNAs, thus generating a library enriched for rare mRNAs. Selection
and isolation of unhybridized mRNAs can be accomplished in different ways, e.g. RNase H
digestion of the RNAs in RNA:DNA heteroduplexes followed by isolation of the intact poly(A)+
single-stranded mRNA using streptavidin-coated oligo-[dT]-beads, or alternatively upon binding of
the heteroduplexes to streptavidin-coated magnetic beads if a biotinylated driver is utilized.3
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Soares, Marcelo Bento. Technology development for gene discovery and full-length sequencing, report, July 19, 2004; United States. (https://digital.library.unt.edu/ark:/67531/metadc779591/m1/3/: accessed April 18, 2024), University of North Texas Libraries, UNT Digital Library, https://digital.library.unt.edu; crediting UNT Libraries Government Documents Department.