An efficient strategy to isolate full-length cDNAs based on an mRNA cap retention procedure (CAPture).
The ability to generate cDNA libraries is one of the most fundamental procedures in contemporary molecular biology. One of the major drawbacks of current methods is that most cDNAs present in any given library are incomplete, rendering the characterization of genes an inefficient and time-consuming task. We have developed an affinity selection procedure using a fusion protein containing the murine cap-binding protein (eukaryotic initiation factor 4E), coupled to a solid support matrix, that allows for the purification of mRNAs via the 5' cap structure. When combined with a single-strand-specific RNase digestion step, specific retention of complete cDNA-RNA duplexes following first-strand synthesis is achieved. This method can be used to generate cDNA libraries in which polyadenylated and nonpolyadenylated mRNAs are equally represented and to enrich for full-length or 5'-end clones, thus facilitating cDNA cloning and promoter mapping.
Edery
I
I
Rutgers University, Piscataway, New Jersey 08854, USA.
Chu
L L
LL
Sonenberg
N
N
Pelletier
J
J
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D018076:DNA, Complementary / Q000302:isolation & purification*; D039561:Eukaryotic Initiation Factor-4E; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D010448:Peptide Initiation Factors / Q000235:genetics* / Q000302:isolation & purification; D012333:RNA, Messenger / Q000032:analysis; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification; D016415:Sequence Alignment
D018076:DNA, Complementary; D039561:Eukaryotic Initiation Factor-4E; D010448:Peptide Initiation Factors; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins