Increased specificity of reverse transcription priming by trehalose and oligo-blockers allows high-efficiency window separation of mRNA display

• DOI: 10.1093/nar/27.5.1345
• Publication Date: 2002-07-26T18:32:38Z
• Authors: Y Mizuno
• Keywords: Priming (agriculture)

We have developed a method for high-efficiency window separation of cDNA display by increasing the specificity priming in reverse transcription. In conventional method, two-base anchored oligo(dT) primers (5′dT16VN3′, where N is any base and V G, A or C) are used to make windows transcripts. However, transcriptase often extends misprimed oligonucleotides. To avoid mispriming from dT16VN primers, we two new technologies. One higher temperature with thermoactivated disaccharide trehalose. The other use competitive oligonucleotide blockers that hybridize non-selectively primed mRNAs, preventing VN site. These methods were combined improve restriction landmark scanning (RLCS), resulting elimination redundant signals appear different windows. This was achieved increased initiation trans-cription beginning poly(A) sites. paves way precise visualization transcripts allow expression profiles individual tissues at each developmental stage be understood.