Process for selective amplification of a full-length cDNA...

Details Process for selective amplification of a full-length cDNA... Process for selective amplification of a full-length cDNA...

2001-11-21

2003-11-25

Whisenant, Ethan (Department: 1637)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Preparing compound containing saccharide radical

C435S006120, C435S091510, C435S091520

Reexamination Certificate

active

06653108

ABSTRACT:

TECHNICAL FIELD
The present invention relates to a process for the preparation of full-length complementary DNA (cDNA). More particularly, the present invention is directed to a process for selective amplification of full-length cDNA, which comprises: i) a step for preparing a hybrid composed of a messenger RNA (mRNA) strand and a cDNA strand of which three (3) or four (4) deoxycitidinemono phosphate (dCMP) are combined at 3′ end, by treating mRNA with reverse transcriptase; separately from the above step, ii) a step for adenylating single strand anchor of which biotin or phosphate group is combined at 3′ end, and phosphate group is combined at 5′ end; iii) a step for ligating said adenylated single strand anchor to 3′ end of full-length cDNA strand of said cDNA/mRNA hybrid to select full-length cDNA/mRNA hybrid; and iv) a step for amplifying only the full-length cDNA/mRNA hybrid through polymerase chain reaction (PCR) which employs a primer of which base sequence is complementary to that of said anchor.
BACKGROUND ART
Recently, new techniques for mass production of various genetic engineering products such as proteins, have been developed through identification of novel genes and determination of base sequences thereof, and then characterization of their biological properties.
In addition, some methods for the treatment of various diseases caused by inappropriate expression and/or suppression of a specific gene or by the influence of foreign substance such as carcinogen or teratogen, can be developed through the analysis of the base sequences of the gene.
To these ends, a process for the mass-production of protein having significant biological application, wherein cDNA prepared from mRNA through reverse transcription is inserted into a cloning vector to be cloned, have been developed as a basic skill in biotechnology.
Therefore, the development of more efficient and simple process for the production of full-length cDNA in large scale, have been needed for the mass-production of human cDNA library and for the study of gene expression pattern by using DNA chip.
As a prerequisite tool for the research of expression pattern of human genes and the structure of protein prepared therefrom, the method of 5′, 3′ end rapid amplification of cDNA end (RACE) is used widely, and the method for determination of complete base sequence by combining those of expressed sequence tags (ESTs) obtained by partial amplification of cDNA, has been developed.
To the present, the method for the preparation of full-length cDNA wherein the cap structure of mRNA is used as a identification marker of full-length cDNA, has been developed. The cap structure of mRNA is characteristic part of the 5′ end of eukaryotic mRNA, and contains guanidine nucleotide substituted with methyl group. In general, the cap structure of mRNA is the site recognized by initiation factor in protein synthesis process.
At present, several methods for recognizing the cap structure of the eukaryotic mRNA, for example, a method wherein a fusion protein composed of cap binding protein and solid support matrix, is bound on 5′cap site; a method wherein biotin which can recognize diol group of cap structure, is employed; a method wherein 5′cap structure of mRNA is removed by using tobacco acid pyrophosphatase and then synthetic oligonucleotide is ligated thereto, and etc., are used frequently.
However, these methods are not cost-efficient process and moreover, these methods are time-consuming process because the enzyme treatment step and purification step requires a long time and are so complicated that starting materials may be decomposed or lost during these steps.
Therefore, recently, other methods so called “CapFinder” method or “CapSelect” method are used as a typical method for identification of the cap structure of 5′ end of the eukaryotic mRNA during the reverse transcription. However, it takes a long time to add nucleotide on the cap structure of mRNA by employing reverse transcriptase in the CapFinder method, and in addition, the incomplete amplification pattern may be occurred since reverse transcriptase have to recognize template switching oligonucleotide.
The “Capselect” method overcomes partly the above problems. However, “Capselect” method also has drawbacks that it requires an additional step wherein adenine group is added through Ribo-tailing step by using terminal deoxyribonucleotidyl tranferase, and that it requires a step wherein double strand adaptor is linked again. Therefore, “Capselect” method is inappropriate to be employed as a commercial method for the production of full-length cDNA in large scale since it needs considerable time, at least more than ten hours to link double-strand anchor.
Therefore, a novel process by which full-length cDNA can be obtained through more efficient and simple procedures than before, and by which full-length cDNA can be amplified completely to produce full-length cDNA in large scale, has been anticipated in this field.
The purpose of the present invention, therefore, is to provide a novel process to produce full-length cDNA in large scale, comprising a step for obtaining full-length cDNA through only two procedures, reverse transcription of mRNA to obtain cDNA and ligation of anchor and nucleic acid; and a step for amplifying completely the full-length cDNA thus obtained.


REFERENCES:
patent: 5219989 (1993-06-01), Sonenberg et al.
patent: 5597713 (1997-01-01), Kato et al.
patent: 5659025 (1997-08-01), Engels et al.
patent: 5962272 (1999-10-01), Chenchik et al.
A. Troutt et al., “Ligation-anchored PCR: a simple amplification technique with single-sided specifity,”Proc. Natl. Acad. Sci. USA, vol. 89, pp. 9823-9825, Oct. 1992.
K. Maruyama et al., Oligo-capping: a simple method to replace the cap structure of eukaryotic mRNAs with Oligoribonucleotides,Gene, vol. 138, pp. 171-174, Elsevier Science B.V., 1994.
W. Schmidt et al., “CapSelect: A Highly sensitive method for 5′ CAP-dependent enrichment of full-length cDNA in PRC-mediated analysis of mRNAs,”Nucleic Acids Research,vol. 27, No. 21, pp. i-iv, Oxford University Press, 1999.

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