ExactStart™ Full-Length cDNA Library Cloning Kit

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Last modified: 2008-07-15 14:57:15

The ExactStart™ Full-Length cDNA Library Cloning Kit facilitates the precise identification of the 5' and 3' ends of coding and some noncoding RNAs. It complements functional genomic studies using DNA microarrays and serial analysis of gene expression (SAGE), which typically cannot identify these important transcript features. Unlike other full-length cDNA cloning methods, the ExactStart approach does not add template-independent nucleotides to the cDNA, simplifying the identification of the true transcription initiation site.

The cloning method used by the ExactStart Kit allows a wider range of RNAs to be cloned than other cDNA library kits. Many of these transcripts are novel, noncoding RNAs similar to those recently discovered by the ENCODE project.

The ExactStart Kit uses a simplified TAP/T4 RNA oligo adapter ligation to selectively tag the 5' end of capped or 5'-monophosphate-containing RNA transcripts. cDNA synthesis is primed with an oligo-d(T) primer that places a second tag on the 5' end of the cDNA. These tags are then used to amplify the cDNA via PCR. The double-stranded DNA products can then be cloned using the included vector.

Two strategies are incorporated in the cDNA synthesis method used in the ExactStart Kit to ensure that the cloned transcripts are derived only from full-length RNA:

1. The cap structure or the 5' phosphate(s) of the RNA is replaced with a RNA oligo adapter that serves as a common priming sequence. This oligo adapter ligation event prevents most RNA degradation products from having the necessary 5' tag sequence.
2. Oligo(dT) priming of first-strand cDNA synthesis, which selects for RNA molecules that have the characteristic poly(A) tail of eukaryotic mRNA, mitochondrial RNA (human) and potentially other novel coding and noncoding RNAs.

The ExactStart Kit contains most of the reagents needed for the creation of a directionally cloned cDNA library from as little as 1 µg of total or up to 250 ng of poly(A) RNA. The researcher will need to supply a PCR system and two restriction enzymes to complete the cloned library.

Last modified: 2008-05-14 16:46:46

Applications

• Creating a cDNA library of full-length transcripts from total RNA or enriched mRNA
• Studying alternate transcriptional start sites
• Discovering and cloning novel noncoding RNAs similar to those discovered by the ENCODE project

Benefits

• cDNA is produced only from full-length RNA
• The novel cDNA synthesis method generates clones of coding and noncoding RNAs that other kits cannot

Last modified: 2008-05-14 16:46:46

Protocols for: ExactStart™ Full-Length cDNA Library Cloning Kit

(catalogue number ES0907)

Please note: all protocols off site are the responsibility of the products supplier

Last modified: 2008-05-14 16:46:46