5′-Terminal sequences of eucaryotic mRNA can be cloned with high efficiency
Authors:
- Hartmut LandHLHartmut Land
- Manuel GrezMGManuel Grez
- Hansjörg HauserHHHansjörg Hauser
- Werner LindenmaierWLWerner Lindenmaier
- Günther SchützGSGünther Schütz
Open Access
- 1 January 1981
- journal article
- Published by Oxford University Press (OUP) in Nucleic Acids Research
- Vol. 9 (10) , 2251-2266
Abstract
A method for cloning mRNAs has been used which results in a high yield of recombinants containing complete 5′-terminal mRNA sequences. It is not dependent on self-priming to generate double-stranded DNA and therefore the SI nuclease digestion step is not required. Instead, the cDNA is dCMP-tailed at its 3′-end with terminal deoxynucleotidyl transferase (TdT). The synthesis of the second strand is primed by oligo(dG) hybridized to the 3′ - tail. Double-stranded cDNA is subsequently tailed with dCTP and annealed to dGMP-tailed vector DNA. This approach overcomes the loss of the 5′-terminal mRNA sequences and the problem of artifacts which may be introduced into cloned cDNA sequences.Keywords
This publication has 32 references indexed in Scilit:
- mRNA complexity and egg white protein mRNA content in mature and hormone-withdrawn oviductCell, 1977
- Rat Insulin Genes: Construction of Plasmids Containing the Coding SequencesScience, 1977
- Stepwise biosynthesis in vitro of globin genes from globin mRNA by DNA polymerase of avian myeloblastosis virus.Proceedings of the National Academy of Sciences, 1976
- A general method for cloning eukaryotic structural gene sequences.Proceedings of the National Academy of Sciences, 1976
- Integration of synthetic globin genes into an E. coli plasmidNucleic Acids Research, 1976
- Amplification and characterization of a β-globin gene synthesized in vitroCell, 1976
- Terminal labeling and addition of homopolymer tracts to duplex DNA fragments by terminal deoxynucleotidyl transferaseNucleic Acids Research, 1976
- Bacterial cloning of plasmids carrying copies of rabbit globin messenger RNANature, 1976
- In vitro synthesis of the E3 immunity protein directed by Col E3 plasmid deoxyribonucleic acid.Journal of Biological Chemistry, 1975
- Complementation analysis of temperature-sensitive host specificity mutations in Escherichia coliJournal of Molecular Biology, 1970