Oligo-capping: a simple method to replace the cap structure of eukaryotic mRNAs with oligoribonucleotides

doi:10.1016/0378-1119(94)90802-8

Gene

Volume 138, Issues 1–2, 28 January 1994, Pages 171-174

https://doi.org/10.1016/0378-1119(94)90802-8 Get rights and content

Abstract

We have devised a method to replace the cap structure of a mRNA with an oligoribonucleotide (r-oligo) to label the 5′ end of eukaryotic mRNAs. The method consists of removing the cap with tobacco acid pyrophosphatase (TAP) and ligating r-oligos to decapped mRNAs with T4 RNA ligase. This reaction was made cap-specific by removing 5′-phosphates of non-capped RNAs with alkaline phosphatase prior to TAP treatment. Unlike the conventional methods that label the 5′ end of cDNAs, this method specifically labels the capped end of the mRNAs with a synthetic r-oligo prior to first-strand cDNA synthesis. The 5′ end of the mRNA was identified quite simply by reverse transcription-polymerase chain reaction (RT-PCR).

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^∗: Present address: Department of Hygiene and Oncology, Tokyo Medical and Dental University, School of Medicine, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113, Japan. Tel. (81-3) 3813-6111, ext. 3172; Fax (81-3) 3813-0852

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Gene

Abstract

References (14)

Isolation and characterization of the human chromosomal gene for polypeptide chain elongation factor la

J. Biol. Chem.

Two-step binding of eukaryotic ribosomes to brome mosaic virus RNA 3

Nature

End labeling of enzymatically decapped mRNA

Nucleic Acids Res.

Rapid production of full-length cDNAs from rare transcripts: amplification using a single gene-specific oligonucleotide primer

A highly sensitive method for mapping the 5′ termini of mRNAs

Nucleic Acids Res.

A blocked structure at the 5′ terminus of mRNA from cytoplasmic polyhedrosis virus

Nature

Transcription of the human adenovirus E1a gene in Succharomyces cerevisiae

Gene

Cited by (536)

Global approaches for profiling transcription initiation

The human chd8 gene is transcribed from two distant upstream promoters

A 5′ UTR-Overlapping LncRNA Activates the Male-Determining Gene doublesex1 in the Crustacean Daphnia magna

Genome-Wide Mapping of 5′ Isoforms with 5′-Seq

RNA LIGASE-MEDIATED RACE: AN EFFECTIVE METHOD FOR THE CLONING OF FULL-LENGTH cDNAENDS

FUNCTIONAL ANALYSIS OF HUMAN GENES

Short communication Oligo-capping: a simple method to replace the cap structure of eukaryotic mRNAs with oligoribonucleotides

Abstract

J. Biol. Chem.

Two-step binding of eukaryotic ribosomes to brome mosaic virus RNA 3

Nature

End labeling of enzymatically decapped mRNA

Nucleic Acids Res.

Rapid production of full-length cDNAs from rare transcripts: amplification using a single gene-specific oligonucleotide primer

A highly sensitive method for mapping the 5′ termini of mRNAs

Nucleic Acids Res.

A blocked structure at the 5′ terminus of mRNA from cytoplasmic polyhedrosis virus

Nature

Transcription of the human adenovirus E1a gene in Succharomyces cerevisiae

Gene

Short communication
Oligo-capping: a simple method to replace the cap structure of eukaryotic mRNAs with oligoribonucleotides