Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
1998-11-20
2001-03-06
Horlick, Kenneth R. (Department: 1656)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S006120, C435S015000, C435S091210, C435S091200, C435S091500, C435S091510, C536S023100, C536S024100, C536S025300
Reexamination Certificate
active
06197554
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of The Invention
The present invention generally relates to the field of methods for generating full-length complementary DNA library from single cells. More particularly, the present invention relates to the field of novel methods of complete full-length cDNA library synthesis from as few as a cell.
2. Description of The Prior Art
The following references are pertinent to this invention:
1. Sambrook et.al., “
Molecular Cloning,
2nd Edition”, Cold Spring Harbor Laboratory Press, pp8.11-8.35 (1989).
2. Patanjeli et.al., “Construction of a uniform abundance (normalized) cDNA library”,
Proc. Natl. Acad. Sci. USA
88: 1943-1947 (1991).
3. O'Dell et.al., “Amplification of mRNAs from Single, Fixed, TUNEL-Positive Cells”,
BioTechniques
25: 566-570 (1998).
4. Eberwine et.al, “Analysis of gene expression in single live neurons”,
Proc. Natl. Acad. Sci. USA
89: 3010-3014 (1992).
5. Crino et.al., “Embryonic neuronal markers in tuberous sclerosis: Single-Cell Molecular Pathology”,
Proc. Natl. Acad. Sci. USA
93: 14152-14157 (1996).
6. Embleton et.al, “In-cell PCR from mRNA: amplifying and linking the rearranged immunoglobulin heavy and light chain V-genes within single cells”,
Nucleic Acid Res.
20: 3831-3837 (1992).
7. U.S. Pat. No. 5,482,854 issued to Soares et.al.
8. U.S. Pat. No. 5,637,685 issued to Soares et.al.
9. U.S. Pat. No. 5,702,898 issued to Bonaldo et.al.
The ability to generate complete complementary DNA (cDNA) library from gene transcripts of single cells has permitted the molecular investigations of intracellular gene expressions under certain special conditions, such as pathogenesis, mutation, treatment processing and developmental control. Because the formation of a full-length cDNA library requires reverse transcription of every messenger RNA (mRNA) in a cell without degradation, the content of this library preserves the whole gene expression repertoire of the cell under tested condition. When single cells from tissue are used, the full-length cDNA library will represent the tissue-specific gene expression pattern and can be used to test differentially expressed genes in vivo. Although previous methods for cDNA library synthesis (Sambrook et.al., “
Molecular Cloning,
2nd Edition”, pp8.1 1-8.35 (1989)) have succeeded in generating full-length cDNAs, the tedious procedure of reverse transcription, restriction, adaptor ligation and vector cloning usually fails to maintain the completeness of a cDNA library, resulting in loss of rare cDNAs when limited cells are used.
Prior art attempts at generation of complete cDNA library, such as U.S. Pat. No. 5,482,845 and U.S. Pat. No. 5,637,685 to Soares and U.S. Pat. No. 5,702,898 to Bonaldo, use reverse transcription and random priming polymerase chain reaction (RT-PCR) to construct normalized cDNA libraries for differential analysis (Patanjeli et.al.,
Proc. Natl. Acad. Sci. USA
88: 1943-1947 (1991)). In general, the amount of normalized cDNAs can be fully amplified by PCR to fulfill the requirement of completeness. However, the use of random-primer amplification greatly reduce the sequence integrity of the cDNAs. Because the normalized cDNA library usually lose part of sequences in the ends for cloning into a vector, this kind of low integrity may introduce significant difficulty in the sequence analysis, such as promoter detection. Moreover, the random amplification procedure increases non-specific contamination of primer dimers, resulting in false positive sequences in the cDNA library. Therefore, these disadvantages may even increase bias in experiments on minuscule samples, which require multiple amplification to bring up needed amount for analysis.
On the other hand, the generation of amplified antisense RNA (aRNA) has been developed to increase transcriptional copy of specific mRNAs from limited amount of cDNA library. The aRNA can be used for characterization of the expression pattern of certain gene transcripts in cells (O'Dell et.al.,
BioTechniques
25: 566-570 (1998)). By incorporating an oligo(dT)n primer coupled to a T7 RNA polymerase promoter sequence (oligo(dT)n-promoter) during reverse transcription (RT), the single copy mRNA can be amplified up to two thousand folds by aRNA amplification (Eberwine et.al ,
Proc. Natl. Acad. Sci. USA
89:3010-3014 (1992)). The aRNAs prepared from single live neuron has been reported to cover 50-75% of total intracellular mRNA population (Eberwine et.al, (1992); Crino et.al.,
Proc. Natl. Acad. Sci. USA
93: 14152-14157 (1996)), indicating that the prevention of mRNA degradation in cells is required to achieve 100% coverage of mRNA amplification. Although these aRNA synthesis methods lead to the identification of some abundant mRNA markers from single cells, the rare mRNAs may not be assessable by the current aRNA methods (O'Dell et.al. (1998)), resulting in low completeness of cDNA library.
In summary, it is desirable to have a fast, simple and specific method for generating complete full-length cDNA libraries from single cells, of which the results may be applied to screen differentially expressed genes, to test functional domain for gene regulation, and to design a therapy for diseases.
SUMMARY OF THE INVENTION
The present invention is a novel cDNA library synthesis method which generates a complete full-length cDNA library from as few as a cell.
Described in detail, a preferred embodiment of the present invention method includes the following steps:
a. providing a plurality of fixed cells, wherein said fixed cells inhibit intracellular mRNA degradation and also increase the permeabilisation of said cells for enzyme penetration;
b. incubating said fixed cells in a reverse transcription reaction with a plurality of oligo(dT)n-promoter sequences, wherein said reverse transcription reaction is reverse transcription of a plurality of mRNAs by using said oligo(dT)n-promoter as primer, to form a plurality of complementary DNAs from said mRNAs;
c. permitting said complementary DNAs in a cDNA tailing and double stranding reaction to form a plurality of poly(N)-tailed cDNAs, wherein said cDNA tailing and double-stranding reaction is a DNA polymerase and terminal transferase reaction capable of adding multiple copies of the same nucleotide to the tails of said complementary DNAs and then double-stranding said complementary DNAs from the tails;
d. incubating said poly(N)-tailed cDNAs in an in-vitro transcription reaction to generate a plurality of full-length aRNAs, wherein said in-vitro transcription reaction is an RNA polymerase reaction capable of synthesizing said full-length aRNAs from said poly(N)-tailed cDNAs;
e. incubating said full-length aRNAs in said reverse transcription reaction with a plurality of oligo(anti-poly(N))-promoter sequences to form a plurality of full-length cDNAs; wherein said oligo (anti-poly(N))-promoter sequences are complementary to the poly (N) tails of said poly(N)-tailed cDNAs; and
f. amplifying said full-length cDNAs with a template-dependent extension of specific primers attached to the poly(dA)-tail and complementary promoter regions of said full-length cDNAs, and thereby providing a complete library enriched in full-length cDNAs from said fixed cells.
In one aspect of this embodiment, the further cycling steps of (d), (b), (d) and then (e) can be repeated at least one time on said full-length aRNA. According to another aspect of this preferred embodiment, the final nucleotide sequences are amplified, preferably, by PCR in the step (f).
The fixed cells can be prepared from cultured cells, frozen fresh tissues, fixed tissues or tissues in slides. To increase the production of said full-length aRNAs from said poly(N)-tailed cDNAs, the oligo(dT)n-promoter sequence is preferably added to the 5′-heads of said complementary DNA sequences in the step (b) for the in-cell transcription in the step (d). The promoter region of said oligo(dT)n-promoter can be recognized by an specific RNA polymerase and further transcribed into full-length aRNAs, such promoters including T3, T7, SP6, M13 and so on.
Chuong Cheng-Ming
Lin Shi-Lung
Ying Shao-Yao
Chan Raymond Y.
David and Raymond
Horlick Kenneth R.
Taylor Janell
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