Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1996-11-20
2001-01-16
Schwartzman, Robert A. (Department: 1635)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S007100, C435S007500, C435S091100, C536S023100, C536S023500
Reexamination Certificate
active
06174669
ABSTRACT:
BACKGROUND OF THE INVENTION
(1) Field of the Invention
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.
(2) Related Art
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 prepared 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 working 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.
1. cDNAs covering most part of transcripts can be obtained by using a random primer. However, those cDNAs are short fragments and clones covering from the poly A site to 5′ Cap site cannot be isolated.
2. Any cDNA obtained by using oligo dT as a primer contains 3′ end. 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.
3. Efficiency of any conventional methods for isolating cDNAs in their full-lengths including those methods mentioned above is not sufficient (only 2, 000, 000 recombinant phages can be obtained from 100 &mgr;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 an 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.
{circle around (1)} Because the recognition of 5′ Cap site depends on reactions of proteins 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.
{circle around (2)} When the first strand of cDNA is synthesized by a reverse transcriptase, the synthesized strand does not extend to the 5′ Cap site.
{circle around (3)} There are also problems of the addition of primer sequences, synthesis efficiency of second strand, and cloning efficiency of double stranded cDNA after the synthesis of the first strand.
As described above, in the production of cDNA libraries in a multi-step process, there are problems such as those mentioned as {circle around (1)} to {circle around (3)} above.
Therefore, an 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.
Another object of the present invention is to provide a method for making full-length cDNA libraries utilizing the novel method of the present invention for labeling of the 5′ Cap site.
SUMMARY OF THE INVENTION
The present invention relates to a method for making full-length CDNA libraries, which is for making libraries of cDNAs having a length corresponding to full-length mRNAs and comprises the following steps of;
binding a tag molecule to a diol structure present in 5′ Cap (
7Me
G
ppp
N) sites of mRNAs,
forming RNA-DNA hybrids by reverse transcription using primers and the mRNAs connected with the tag molecule as templates, and
separating RNA-DNA hybrids carrying a DNA corresponding to full-length mRNAs from the RNA-DNA hybrids formed above by using function of the tag molecule.
REFERENCES:
patent: 5142047 (1992-08-01), Summerton et al.
patent: 9402603 (1994-03-01), None
I. Edery et al., “An Efficient Strategy to Isolate Full-Length cDNAs Based on an mRNA Cap Retention Procedure (CAPture)”, Molecular and Cellular Biology vol. 15, No. 6, Jun. 1995, p. 3363-3371.
K. Maruyama et al., “Oligo-capping: a simple method to replace the cap structure of eukaryotic mRNAs with oligoribouncleotides”, Gene, 138, 1994 p. 171-174.
S. Kato et al., “Construction of a human full-length cDNA bank”, Gene, 150, 1994, p. 243-250.
Carninci Piero
Hayashizaki Yoshihide
Schneider Claudio
Griffin & Szipl, P.C.
McGarry Sean
Schwartzman Robert A.
The Institute of Physical and Chemical Research
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