DESCRIPTION

Assembly of Sure-RACE cDNA 5 End Discovery Panels begins with the selection of high-quality mRNAs. The integrity of each poly A+ RNA is determined by Northern blot analysis using actin cDNA as the hybridization probe. Only mRNAs that fulfilled stringent criteria of intactness are used for cDNA synthesis.

It is well established that mRNAs have complex secondary structures that may act as strong stops during first-strand cDNA synthesis using standard reverse transcriptase (RT). As a consequence, the resulting single-stranded cDNAs may not be representative of the starting mRNAs as 5' ends may not be reverse transcribed. OriGene has successfully used a dual-cycling procedure to overcome this problem. Initial cDNA synthesis is performed using standard procedures with MMLV RT at 42C followed by a second round of synthesis with thermostable with DNA polymerase at 75C. Cycling of high-processivity/low-thermostability enzyme and low-processivity/ high-thermostability enzyme, which facilitates read-through of RNA secondary structures, is repeated a second time to ensure the efficiency of the reaction.

Conventional double-stranded cDNA synthesis involves the "blunting" of the cDNAs, which results in the loss of 5 end sequences of 20-200 nucleotides. To avoid sequence loss, first strand cDNAs are "tailed" at their 3' ends with dCTP (Fig. 1). The terminal transferase reaction is optimized to produce a tail of 12-15 dC residues. Second-strand cDNA synthesis is initiated by the annealing of an adaptor-oligo(dG) primer to the oligo(dC) tail at the 3' ends of the first-strand cDNA molecules. Escherichia coli DNA polymerase I is used for second-strand synthesis to reduce the nucleotide-incorporation error rate. Additionally, RNase H is added to nick the mRNA and to allow internal priming. Finally, T4 DNA ligase is added to facilitate joining of fragments primed by oligo(dG) and nicked mRNA.

SR Fig 1