G-less Cassette In Vitro Transcription Using HeLa Cell Nuclear Extracts

INTRODUCTION

The G-less cassette is a 365-nucleotide (nt) segment of DNA lacking guanine (G) residues on the nontemplate strand. In principle, a full-length transcript can be generated in an in vitro reaction lacking GTP, an omission that leads to suppression of most random, nonspecific transcription throughout the plasmid. This method, like runoff transcription, generates radiolabeled RNA products, directly bypassing the necessity and extra time required to perform primer extension or other indirect mRNA product measurements. Unlike runoff transcription, which requires a cleaved end, the G-less assay can be performed on a circular, supercoiled plasmid, which in many systems is a more efficient template. In practice, most crude systems, such as HeLa nuclear extracts, contain low amounts of contaminating GTP that lead to small amounts of background transcription and occasionally can cause random upstream transcription to read through the G-less cassette. To minimize these artifacts, the reaction generally contains 3′-O-Me-GTP, a chain-terminating analog of GTP that causes transcription to cease when it is incorporated into a growing transcript, much like the dideoxy analogs used in DNA sequencing. The reaction products are cleaved with T1 RNase, which cleaves RNAs at G residues, further reducing background transcription; the G-less mRNA remains intact, whereas small random RNAs are digested.