Determination of 5' ends of specific mRNAs by DNA ligase-dependent amplification.
We established a novel way to clone 5' ends of unknown length and sequence of individual cDNAs. T4 DNA ligase is employed to ligate an annealed duplex of complementary primers, one of them with a 4-nucleotide-long randomized overlap, to first-strand cDNA, generating a new 5' end. Subsequent PCR with a down-stream primer and a primer with specificity for this new 5' end leads to products that can easily be cloned and sequenced. Considerations for the choice of primers for ligation and amplification are given. We have used this method to determine the 5' sequences of three independent mRNAs: the human collagen type-X gene, the chicken anchorin CII gene, and the human cytidine deaminase gene. We will discuss this method in comparison with other methods published for the amplification of unknown 5' ends of mRNA species.
Bertling
W M
WM
Paul-Ehrlich-Institute, Unit of Molecular Pathology, Langen, Germany.
Beier
F
F
Reichenberger
E
E
D000818:Animals; D017304:Annexin A5 / Q000096:biosynthesis / Q000235:genetics*; D001483:Base Sequence; D002356:Cartilage / Q000378:metabolism; D002645:Chickens; D003094:Collagen / Q000096:biosynthesis* / Q000235:genetics; D003564:Cytidine Deaminase / Q000096:biosynthesis / Q000235:genetics*; D011088:DNA Ligases / Q000378:metabolism*; D017931:DNA Primers; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000737:chemistry* / Q000378:metabolism
D017304:Annexin A5; D017931:DNA Primers; D012333:RNA, Messenger; D003094:Collagen; D003564:Cytidine Deaminase; D011088:DNA Ligases