Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Patent
1997-10-31
2000-02-08
Horlick, Kenneth R.
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
536231, 536 254, 530350, 530413, 435 6, C12Q 168, C12P 1934, C07H 2102, C07H 2100
Patent
active
06022715&
DESCRIPTION:
BRIEF SUMMARY
The present invention relates to a method for the specific coupling of the cap of the 5' end of eukaryotic messenger RNAs (mRNAs). The present invention also relates to a method for isolating mRNA from eukaryotes comprising the cap at the 5' end. In addition, the present invention relates to a method for the preparation of single-stranded (ss) cDNA complementary to the 5' end of mRNA or of double-stranded (ds) cDNA corresponding to the 5' end of RNA as well as of complete cDNA. The present invention comprises, in addition, reagent kits for carrying out the said methods. The present invention also relates to a method for capturing protein recognizing messenger RNAs. Finally, the present invention also relates to mRNA fragments coupled specifically by the 5' cap to a molecule.
The activity of genes is governed by nucleic domaines situated upstream of the site of initiation of transcription. It is generally accepted that the region of genomic DNA situated within the first 500 nucleotides upstream of the site of initiation of transcription constitute the proximal promoter. The latter contains a large part of the information necessary for regulating transcription. Some sequences facilitating or inhibiting the transcription of genes are indeed situated upstream (5' side) or downstream (3' side) of the proximal promoter. Knowledge of the sequences regulating the activity of genes is crucial for understanding and, in the long term, modifying the mechanisms of expression of genes. The determination of these regulatory domaines is greatly facilitated by the isolation and the characterization of the 5' ends of the messengers. The said ends may indeed be used as point of anchorage in the human genome for the subsequent isolation of the promoters with the aid of conventional molecular biology techniques or alternatively for allowing identification of genes and the location of promoters by computer analysis (homologous sequences) of genomic DNA sequences.
Although the sequence of the 5' ends of mRNAs is of great importance, the conventional techniques used to characterize and isolate specific mRNAs involve the isolation of the corresponding cDNA clones. Now, upon construction, these clones are truncated to a greater or lesser extent in the parts corresponding to the 5' ends of the mRNAs. Definite improvements have recently been made in the isolation and characterization of the 5' ends. Frohman et al. (1) and Delort et al. (2) have described the isolation of the 3' ends of cDNAs corresponding to the 5' ends of mRNAs by means of the addition of a homopolymer to their ends in order to facilitate the cloning of the complete cDNA after amplification by the polymerase chain reaction (PCR). However, the examples of application provided with these methods applied only to relatively abundant messenger RNAs. Delort et al. (2) have shown that the method was difficult to apply to weakly expressed molecular species. The ineffectiveness of the method is due to the addition of homopolymeric sequences to the 3' end of cDNAs through the activity of terminal transferase. Indeed, in this case, the primer used to initiate the synthesis of the second strand (called return primer) and then optionally for the subsequent PCRs necessarily contains a homopolymeric tail at its 3' end. This homopolymeric tail, of sequence anticomplementary to that added to the 3' end of cDNAs can hybridize with internal homopolymeric sequences, thus contributing to the genesis of DNA fragment shorter than the expected fragment. A second factor limiting the sensitivity of the method is linked to the amplification of cDNA fragments synthesized by nonspecific priming. All these cDNA fragments are possible targets for the return primer provided that they have a homopolymeric sequence favourable to the hybridization.
To overcome the disadvantages linked to the addition of a homopolymeric tail, Dumas Milne Edwards et al. (3) ligated to the 3' end of cDNAs a functionalized oligonucleotide (tag) through the activity of T.sub.4 phage RNA ligase. The functionalization of th
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Dumas Milne Edwards Jean-Baptiste
Merenkova Irena Nicolaevna
Genset S.A.
Horlick Kenneth R.
Siew Jeffrey
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