Overview
Functional genomics, the development and application of experimental approaches
to assess gene function, begins with a full-length copy of the gene of
interest. The availability of such clones allows researchers to pursue the
functional dissection of genes, many of which code for hypothetical proteins
with no known functions.
We have utilized high-throughput cloning and expression profiling to isolate
and catalog the TrueCloneTM Collection - a searchable gene bank of
over 20,000 full-length human cDNA clones suitable for transfection and direct
in vitro expression. This collection includes over 75% of the NM sequences in
the NCBI RefSeq database, covering 65-80% of the predicted human cDNA
repertoire. All clones match annotated mRNA reference sequences found in the
public domains.
Quality Control
Each cDNA clone in the TrueClone Collection is sequenced at its 5' end and the
resulting sequence matched to a corresponding known reference at the start-site
of the longest ORF (Open Reading Frame). When a transcript has more than one
putative ORF, or the ORF is located far downstream of the 5?end, transcript
matching occurs at the extreme 5?end of the reference. That match is confirmed
through 3?end sequencing and re-matched to the same reference. If the
reference lacks 3?UTR sequences (because its ORF was generated by prediction)
or contains a truncated 3?UTR, attempts are made to either 1) demonstrate that
the 3?read is derived from genomic sequences located downstream of the
putative ORF or 2) sequence the end of the putative ORF using a primer located
immediately upstream of the termination codon. When 5?and 3?reads overlap,
clones are sequenced in entirety. Lengths of the complete cDNA inserts are
confirmed similar (variation <15%) to their expected size by release from the
cloning vector using an appropriate restriction enzyme.
It has to be noted that many predicted reference sequences are only partially
correct. The availability of physical clones to these predicted sequences
serves to confirm existence and either validate or disprove authenticity.
Search for a Gene
The TrueClone Collection may be searched by following methods.
Nucleotide Sequence: Enter the mRNA sequence and perform a BLAST search.
Accession Number: Enter the NM or XM NCBI RefSeq accession number to
search the database. The NM/XM accession number can be obtained easily on
NCBI抯 Web site by entering the accession number with which you are familiar
and using the "Links" function to return "Related Sequences". Approximately 10
percent of TrueClone reference sequences have not yet been accorded a RefSeq
NM/XM number. For such these clones, a BLAST search via nucleotide sequence is
recommended.
Protein Domain(s): The TrueClone database can be search for genes that
encode a specific protein domain or a combination of up to three protein
domains.
Keyword(s): Keyword(s) from the NCBI gene description may be used to
search the TrueClone database. Only keywords from the NCBI gene description
should be used for this search to avoid missed location or the return of
incorrect or irrelevant entries.
Product Application
High-throughput screening options are highly productive means of finding useful
drug targets. All the genes in TrueClone collection are housed in a series of
nonproprietary CMV vectors, allowing for uniformed screening methods. The
full-length cDNA fragment is inserted with the open reading frame located
downstream from a eukaryotic transcriptional promoter capable of driving
heterologous gene expression in a variety of mammalian cell lines in culture
and tissues in transgenic mice. This feature facilitates the investigation of
gene functions and the development of transfected or transgenic cells for drug
screens. The expression vector also contains a prokaryotic transcriptional
promoter which supports coupled transcription-translation of the cDNA sequence
in an appropriate cell-free system. This approach can be utilized to generate
recombinant proteins for target identification and testing activities in vitro.
TrueClones may also be used to generate hybridization probes, for DNA
immunization to generate antibodies, and to search for gene polymorphisms and
alternatively spliced forms.
Ordering Information
Product Delivery & Storage
Plasmid DNA containing an insert of the appropriate cDNA fragment is provided
in a complex environment* on a dry filter, ready for elution and transformation
in a competent bacterial host. In some cases the transformed bacteria is
supplied as an agar stab. Clones are shipped at ambient temperature and should
be stored at room temperature upon arrival. Transformed bacteria should be
grown in LB / Amp medium and stored in 10% glycerol at -80癈.
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