Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Patent
1999-07-16
2000-11-07
Riley, Jezia
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
435 5, 435 6, 435 912, 536 221, 536 231, C12P 1934
Patent
active
061435287
DESCRIPTION:
BRIEF SUMMARY
TECHNICAL FIELD
The present invention relates to a method for making full-length cDNA libraries. More in detail, it relates to a method for making full-length cDNA libraries by a method for purification of full-length cDNAs utilizing chemical modification of mRNAs.
TECHNICAL BACKGROUND
Methods for synthesizing cDNAs are essential techniques for researches in the fields of medical science and biology as an indispensable method for analyzing gene transcripts. Any DNA genetic information manifests physiological activity through transcripts and a potential means for analyzing such transcripts is cDNA cloning. In cDNA syntheses according to conventional methods, clones are ultimately isolated from a cDNA library synthesized from poly A sites by using oligo dT as a primer. However, in most cases using such a method, whole structures of transcription units cannot be analyzed since the transcription units are not synthesized in their full-lengths. Therefore, when using a conventional cDNA library, it is essential for analysis of gene structures in their full-lengths to synthesize 5' upstream regions by the primer elongation method, or perform gene-walking of the 5' upstream regions by cDNA synthesis using a random primer.
However, such conventional methods for synthesizing cDNAs as described above have, for example, the following problems. random primer. However, those cDNAs are short fragments and clones covering from the poly A site to 5' Cap site cannot be isolated. However, because the reverse transcriptase cannot reach the 5' Cap site, the 5' upstream should be further isolated and analyzed by the primer elongation method and 5' RACE or the like. full-lengths including those methods mentioned above is not sufficient (only 2,000,000 recombinant phages can be obtained from 100 .mu.g of mRNA). Therefore, more efficient techniques are desired for practical purposes.
As conventional methods for synthesizing full-length cDNAs, the following methods can be mentioned;
the method utilizing a Cap binding protein of yeast or Hela cells for labeling the 5' Cap site (I. Edery et al., "An Efficient Strategy To Isolate Full-length cDNAs Based on a mRNA Cap Retention Procedure (CAPture)", MCB, 15, 3363-3371, 1995); the method where phosphates of incomplete cDNAs without 5' Cap are removed by using alkaline phosphatase and then the whole cDNAs are treated with de-capping enzyme of tobacco mosaic virus so that only the full-length cDNAs have phosphates (K. Maruyama et al., "Oligo-capping: a simple method to replace the cap structure of eukaryotic mRNAs with oligoribonucleotides", Gene, 138, 171-174, 1995., S. Kato et al., "Construction of a human full-length cDNA bank", Gene, 150, 243-250, 1995) and the like.
The reasons why efficiency of these conventional methods for synthesizing full-length cDNAs is not sufficient include, for example, the followings. like adenovirus Cap binding protein and the de-capping enzyme of tobacco mosaic virus, high efficiency of the selection of full-length cDNAs (RNAs) cannot be expected. the synthesized strand does not extend to the 5' Cap site. efficiency of second strand, cloning efficiency of double stranded cDNA after the synthesis of the first strand, and of a host vector system for cloning.
As described above, in the production of cDNA libraries in a multi-step process, there are problems such as those mentioned as 1 to 3 above.
Therefore, the first object of the present invention is to provide a novel method in which 5' Cap site can be more efficiently labeled compared with the labeling by the proteins reactions such as those by the conventional adenovirus Cap binding protein and the de-capping enzyme of tobacco mosaic virus which is directed to isolation of full-length cDNAs.
The second object of the present invention is to provide a method for making full-length cDNA libraries utilizing the novel method for labeling of the 5' Cap site. The inventors of the present invention have found a novel method for preparing a full-length cDNA libraries, and have applied for patent ahead (Japan
REFERENCES:
patent: 5891637 (1999-04-01), Ruppert
Carninci et al. "High-efficiency full-length cDNA cloning by biotinylated CAP trapper" Genomics, vol. 37, pp. 327-336, 1996.
Edery et al., Mol. Cell. Biol. (1995), vol. 15, No. 6, p. 3363-3371 "An Efficient Strategy to Isolate Full-Length cDNAs Based on an mRNA Cap Retention Procedure (capture)".
Maruyama et al., Gene (1995), vol. 138, p. 171-174, "Oligo-capping: a simple method to replace the cap structure of eukaryotic mRNA with oligoribonucleotides".
Kato et a., Gene (1995), vol. 150, p. 243-250, "Construction of a human full-length cDNA bank".
Riley Jezia
The Institute of Physical and Chemical Research
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