Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-11-28
2004-08-17
Siew, Jeffrey (Department: 1637)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200, C435S091510, C536S023100, C536S026740, C536S024330
Reexamination Certificate
active
06777180
ABSTRACT:
Throughout this application, various publications are referenced by author and date within the text. Full citations for these publications may be found listed alphabetically at the end of the specification immediately preceding the claims. All patents, patent applications and publications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art as known to those skilled therein as of the date of the invention described and claimed herein.
BACKGROUND OF THE INVENTION
The human genome is estimated to contain 100,000 genes, the expressions of which define the functionality of a cell (1). Current technological advances, including large-scale DNA sequencing, efficient library construction and manipulation and PCR-based gene expression monitoring, have resulted in the identification of more than 87,000 unique expression sequence tags (ESTs) in diverse cell types and under various physiological conditions (1). Approximately 12% of the ESTs have significant homology with previously identified genes and the remainder require further investigation to define their identity and biological relevance (1). However, ESTs, short stretches of expressed genes, can only provide limited information as to the identity and biological role of specific genes. A more thorough analysis of the ESTs requires a determination of the full protein coding sequences for these expressed genes.
Several approaches are routinely used to obtain cDNAs containing protein-encoding sequences from ESTs. These include, library screening (2) and the PCR-based rapid. amplification of cDNA ends (RACE) strategy (3). A less frequently employed scheme, exon trapping is also amendable to cDNA cloning from genomic fragments (4,5).
A number of cDNA libraries from diverse sources are commercially available. This can in specific instances reduce the burden of producing cDNA libraries that are required for screening for cDNAs. However, even with well-constructed cDNA libraries, several rounds of screening and verification are often required to obtain even a single complete cDNA (2). This process is laborious and can require months of intensive effort. What exacerbates the situation is that cDNA library screening occasionally results in incomplete cDNAs lacking full protein coding information. This occurs primarily because of premature termination of reverse transcription and the self-priming procedure during second strand cDNA synthesis (2, 6, 7). Additionally, obtaining cDNA of low abundance mRNA is rarely achievable unless the cDNA library is high titer and minimally amplified (2). In these contexts, the current approach of cDNA library screening to obtain full protein coding sequence is often costly, laborious and inefficient.
SUMMARY OF THE INVENTION
The present invention provides a method for isolating a double-stranded cDNA having a nucleotide sequence of a complete open reading frame which comprises: (A) admixing (i) an isolated single-stranded cDNA, (ii) a first primer capable of forming a stem-loop structure, comprising (a) at the 3′ end of the primer, a first random sequence, linked to (b) a second sequence, linked to (c) a third sequence which forms a loop structure, linked to (d) a fourth sequence, at the 5′ end of the first primer, which is complementary to the second sequence, under hybridization conditions sufficient for annealing the first sequence of the first primer to the sequence at the 3′ end of the single-stranded cDNA, and (iii) a polymerase; (B) incubating the mixture from step (A) under suitable conditions for DNA synthesis; and (C) performing a polymerase chain reaction by admixing (i) an aliquot of the mixture from (B), (ii) a second primer which specifically binds to the single-stranded cDNA, (iii) a third primer which comprises (a) a fifth sequence identical to the third sequence of the first primer, linked to (b) a sixth sequence identical to a portion of the second sequence of the first primer, and (iv) a polymerase, under conditions suitable for a polymerase chain reaction so as to produce a double-stranded cDNA reaction product, thereby isolating the cDNA having the sequence of the complete open reading frame.
REFERENCES:
patent: 95/33853 (1995-12-01), None
Kang DC, LaFrance R, Su ZZ, Fisher PB (1998). Reciprocal subtraction differential RNA display: an efficient and rapid procedure for isolating differentially expressed gene sequences. Proc Natl Acad Sci USA 10(23):13788-13793.
Hooft van Huijsduijnen R (1998). PCR-assisted cDNA cloning: a guided tour of the minefield. Biotechniques 24(3):390-392.
Lin JJ, Jiang H, Fisher PB (1998). Melanoma differentiation associated gene-9, mda-9, is a human gamma interferon responsive gene. Gene 207(2):105-110.
Carninci P, Nishiyama Y, Westover A, Itoh M, Nagaoka S, Sasaki N, Okazaki Y, Muramatsu M, Hayashizaki Y (1998). Thermostabilization and thermoactivation of the thermolabile enzymes by trehalose and its application for the synthesis of full length cDNA. Proc Natl Acad Sci USA 95(2):520-524.
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Fromont-Racine M, Bertrand E, Pictet R, Grange T (1993). A highly sensitive method for mapping the 5′ termini of mRNAs. Nucleic Acids Res 21(7):1683-1684.
Hirzmann J, Luo D, Hahnen J, Hobom G (1993). Determination of messenger RNA 5′-ends by reverse transcription of the cap structure. Nucleic Acids Res 21(15):3597-3598.
Jiang H, Fisher PB (1993). Use of a sensitive and efficient subtraction hybridization protocol for the identification of genes differentially regulated during the i
Fisher Paul B.
Kang Dong-Chul
Baker & Botts L.L.P.
Siew Jeffrey
Trustees of Columbia University in the City of New York
Tung Joyce
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