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  • Analyzing (Re)Capping of mRNA Using Transcript Specific 5' End Sequencing.

Analyzing (Re)Capping of mRNA Using Transcript Specific 5' End Sequencing.

Bio-protocol (2021-03-05)
Daniel Del Valle Morales, Daniel R Schoenberg
ABSTRACT

The 5' cap is a ubiquitous feature of eukaryotic mRNAs. It is added in the nucleus onto newly synthesized pre-mRNA, and in the cytoplasm onto mRNAs after decapping or endonuclease cleavage. Cytoplasmic recapping can occur after loss of the cap at the native 5' end, or downstream within the body of the mRNA. The identification and location of recapping events is key to understanding the functional consequences of this process. Here we present an approach that addresses this problem, using the Lexogen TeloPrime® cDNA synthesis kit to tag recapped 5' ends. TeloPrime uses a proprietary DNA ligase to add a double stranded DNA oligonucleotide onto the 3' end of cDNA while it is base paired with mRNA. Specificity for capped ends is obtained by the oligonucleotide having an unpaired C residue that base pairs weakly with m7G on the mRNA 5' end. This is followed by PCR amplification of double-stranded cDNA using primers to the appended oligonucleotide and the mRNA of interest. The resulting products are gel purified and sequenced directly (if a single band) or cloned and sequenced. The sequence at the junction between the ligated oligonucleotide and the target mRNA provides the location of the cap on the corresponding transcript. This assay is applicable to all capped transcripts. It can be used with Sanger sequencing for small numbers of transcripts or adapted for use with Illumina library sequencing.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
LB Agar Ampicillin-100, Plates, pre-poured LB agar plates with 100 μg/ml ampicillin
Sigma-Aldrich
Ammonium persulfate, for molecular biology, suitable for electrophoresis, ≥98%