Determination of 5' ends of specific mRNAs by DNA ligase-dependent amplification. | Zendy

Wolf Bertling | Zendy; Frank Beier | Zendy; Ernst Reichenberger | Zendy

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Determination of 5' ends of specific mRNAs by DNA ligase-dependent amplification.

Author(s) -

Wolf Bertling,

Frank Beier,

Ernst Reichenberger

Publication year - 1993

Publication title -

genome research

Language(s) - English

Resource type - Journals

SCImago Journal Rank - 9.556

H-Index - 297

eISSN - 1549-5469

pISSN - 1088-9051

DOI - 10.1101/gr.3.2.95

Subject(s) - biology , primer (cosmetics) , microbiology and biotechnology , dna ligase , complementary dna , ligase chain reaction , gene , genetics , dna , in silico pcr , rapid amplification of cdna ends , polymerase chain reaction , multiplex polymerase chain reaction , molecular cloning , chemistry , organic chemistry

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.

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