Oligo-capping: a simple method to replace the cap structure of eukaryotic mRNAs with oligoribonucleotides.
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).
Maruyama
K
K
Institute of Medical Science, University of Tokyo, Japan.
Sugano
S
S
D000818:Animals; D017122:Bacteriophage T4 / Q000201:enzymology; D001483:Base Sequence; D017931:DNA Primers; D005821:Genetic Techniques; D005914:Globins / Q000096:biosynthesis*; D008969:Molecular Sequence Data; D009843:Oligoribonucleotides; D020648:Peptide Elongation Factor 1; D010445:Peptide Elongation Factors / Q000096:biosynthesis*; D010947:Plants, Toxic; D016133:Polymerase Chain Reaction; D011755:Pyrophosphatases / Q000378:metabolism; D012315:RNA Caps / Q000235:genetics* / Q000378:metabolism; D011130:RNA Ligase (ATP) / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics* / Q000378:metabolism; D011817:Rabbits; D014026:Nicotiana / Q000201:enzymology
D017931:DNA Primers; D009843:Oligoribonucleotides; D020648:Peptide Elongation Factor 1; D010445:Peptide Elongation Factors; D012315:RNA Caps; D012333:RNA, Messenger; D005914:Globins; D011755:Pyrophosphatases; D011130:RNA Ligase (ATP)