CAPswitch based PCR technology for full-length cDNA library construction from small amount of RNA

We describe a CAPswitch based PCR cDNA library construction method. In this method, a modified oligo(dT) primer is used to prime the first-strand reaction, and a CAPswitch oligonucleotide serves as a short, extended template at the 5' mRNA end for reverse transcriptase (RT). When the RT reaches the 5' mRNA, the enzyme switches templates and continues replicating to the end of the oligonucleotide. Then LD-PCR is performed using combination of CAPswitch and oligo(dT) primers to generate high yield of full-length ds-cDNA. By using this CAPswitch technique, we successfully constructed 10 cDNA libraries each with an average of 1.90 x 10independent clones. More than 90% of clones contained inserts as detected by B/W screening and insert PCR analysis. In comparison with conventional cDNA construction methods, CAPswitch method has the following benefits: (1) High yields of cDNA can be synthesized from small quantities of RNA (minimum amount for total RNA is SO ng, for mRNA is 25 ng). (2) Plaque hybridization by 5'- and 3'- probes show that CAPswitch cDNA libraries contain more full-length cDNA clones. In addition, sequencing ten identified CAPswitch cDNA clones indicated that they had an extra 1-54 nucleotides on the 5' end, compared with the longest corresponding cDNA sequences deposited in GenBank. (3) A database search of 50 random CAPswitch cDNA clone sequences showed that the libraries had less mitochondria! and ribosomal RNA clones. (4) The method is fast, simple and does not require any radioactive materials. The method will be useful to generate cDNA libraries from biopsy tissues or rare cell lines, and has a potential to identify transcription start sites.

Duke Scholars

Author Luda Diatchenko Anesthesiology