Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
2001-04-11
2003-01-21
Whigenant, Ethan C. (Department: 1634)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S006120, C435S091100, C435S091200, C436S094000, C536S023100, C536S024300, C536S024330
Reexamination Certificate
active
06509175
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to the construction of cDNA libraries, and in particular to methods of constructing full-length cDNA libraries.
BACKGROUND OF THE INVENTION
Methods for constructing complementary DNA (cDNA) libraries from mRNA are well known in the art. In a typical procedure, poly(A)+ mRNAs are isolated from cells, preferably a cell type in which the mRNA encoding the desired polypeptide is produced in large quantities. The mRNAs are then converted into cDNA in vitro using the enzyme reverse transcriptase to synthesize complementary cDNA strands from the mRNA template. General protocols are, for example, described in Chapter 5 of Ausubel et al.,
Current Protocols in Molecular Biology,
Volume 1 (1991). Two commonly used methods of producing cDNA from mRNA are described in Okayama and Berg,
Mol. Cell Biol.
2:161-170 (1982) and Gubler and Hoffman,
Gene
25:263-269 (1983).
In the conventional process of converting mRNA into double stranded cDNA in vitro, a first cDNA strand is synthesized by the reverse transcriptase and separated from the mRNA by treatment with alkali or using a nuclease such as the enzyme RNase H.
E. coli
DNA polymerase then uses the first cDNA strand as a template for the synthesis of the second cDNA strand, thereby producing a population of double stranded cDNA molecules from the original poly(A)+ mRNA. After converting the 5′ and 3′ ends into blunt ends, the cDNA can be ligated to linkers/adaptors and subsequently ligated into suitable vectors and transformed or packaged into a cell line to form the library. The library can then be screened for cells transformed with nucleic acid encoding the desired polypeptide.
While the conventional methods have been used to successfully create cDNA libraries, and to identify a large number of polypeptides, they do have certain disadvantages. For example, an intrinsic problem in the construction of high quality full-length cDNA libraries is that, under in vitro conditions, the reverse transcriptase very often does not extend the first strand cDNA up to the 5′ end of the mRNA, with the result that some mRNA sequences (often longer sequences) are not represented in the library. This is thought to occur in part due to misincorporation of an incorrect base by the reverse transcriptase, which destabilizes the cDNA/mRNA duplex. Enzymes or proteins present in the cell that normally repair nicks or correct mistakes during DNA synthesis are not present when the cDNA is synthesized in vitro.
In addition, hairpin formation in the mRNA can lead to early termination in the conversion to cDNA. This is especially a problem in the cloning of polypeptides having a signal sequence located at the 5′ end of the gene, as these libraries are often screened by detecting polypeptide exported from the transformed cells. Thus, these methods require full-length cDNA, including the signal sequence.
A number of methods have been developed to attempt to address these problems. For example, a different method of synthesizing cDNA in vitro selects full length poly(A)+ mRNA by treatment with bacterial alkaline phosphatase and tobacco acid pyrophosphatase, and subsequently ligating the 5′ end of the mRNA to a chimeric DNA-RNA linker containing a restriction site. See, Kato et al.,
Gene
25:243-250 (1994). The poly(A) 3′ end of the mRNA is then hybridized to an oligo d(T) sequence of and the oligo d(T) used to prime cDNA synthesis. This procedure is also limited, however, by the efficiency of the phosphatases and the ligation procedure. Moreover, the ligation procedure can work with mRNA in which the 5′ end has degraded, since the method does not distinguish between full-length and partial mRNA.
In standard methods currently used for the preparation of cDNA libraries, the mRNA in the cell is isolated by virtue of the presence of a polyadenylated tail present at its 3′ end, which binds to a resin specific for this structure (oligo dT-chromatography). The purified mRNA is then copied into cDNA using a reverse transcriptase, which starts at the 3′ end of the mRNA and proceeds towards the 5′ end. Second strand synthesis is then performed. Linkers are added to the ends of the double stranded cDNA to allow for its packaging into virus or cloning into plasmids. At this stage, the cDNA is in a form that can be propagated.
One disadvantage observed with current cDNA library synthesis protocols is that current methods tend to produce libraries having a significant proportion of incomplete cDNAs, which results from inefficiencies in the reverse transcriptase employed to generate the library. To compensate for the incomplete cDNA constituents of the library, investigators must perform many rounds of isolation (screenings) and construct a “full-length” cDNA from the accumulated pieces. Such processes are resource intensive and do not ensure that each initial mRNA is represented in the cDNA library.
In addition, there is significant under-representation of sequences close to the 5′ end of mRNAs in cDNA libraries produced by conventional methods. This under-representation results from the fact that the reverse transcriptase will usually “fall off” before reaching these sequences. In many instances, the information located at the 5′ end is of great interest.
Thus, there remains a need in the art for improved cDNA libraries, and in particular for cDNA libraries that are enriched for full-length cDNAs.
SUMMARY OF THE INVENTION
The present invention provides methods for identifying cDNAs comprising sequences corresponding to the 5′ end of a transcript, methods of producing libraries comprising cDNAs, methods of verifying the presence of a 5′-end in a cDNA, and producing libraries particularly rich in 5′ ends and full length cDNAs (40-70%) relative to libraries produced from conventional technologies (10-30%).
In one embodiment, the present invention provides an improved method for producing full-length cDNAs comprising 1) relaxing the mRNA secondary structure, e.g., by adding an agent such as dimethyl sulfoxide (DMSO) to the first-strand synthesis reaction mixture and 2) utilizing a thermostable enzyme that exhibits 3′ to 5′ exonuclease activity for template driven enzymatic deoxynucleotide synthesis during first strand synthesis.
In one embodiment, the invention provides a method of isolating a full-length cDNA by: 1) contacting a ribonucleic acid molecule with a primer under conditions sufficient for template driven enzymatic deoxyribonucleic acid synthesis to occur to form a hybrid RNA:DNA molecule; 2) contacting the hybrid molecule with a detectably labeled oligo-dV primer under conditions sufficient for template driven enzymatic deoxyribonucleic acid synthesis to occur to produce hybrid primer:RNA sequences, provided the deoxyribonucleic acid of the first hybrid molecule does not extend to the 5′ end of the ribonucleic acid; 3) isolating a hybrid molecule that does not contain label; and 4) converting the unlabelled hybrid molecule to a first double-stranded deoxyribonucleic acid molecule. In a particular embodiment, the single-stranded ribonucleic acid molecule is an mRNA. Following cDNA production, the double-stranded cDNA molecule can be introduced into a vector.
The primer used for first strand cDNA synthesis may be any primer that allows for directed synthesis of a deoxyribonucleic acid from a ribonucleic acid (including a gene specific primer and/or a random primer), but is preferably an oligo-dT primer. The detectably labeled oligo-dV primer can be labeled with anything known in the art, including but not limited to, biotin, digoxygenin, radioactivity, and the like.
The present invention also features a method of identifying a full-length first strand cDNA including the steps of contacting an RNA-cDNA hybrid with a detectably labeled primer under conditions sufficient for template driven enzymatic deoxyribonucleic acid synthesis to occur, where the primer is composed of a plurality of deoxyadenosin
Fu Glenn K.
Lee Walter H.
Ni Irene
Stuve Laura L.
Bozicevic Field & Francis LLP
Francis Carol L.
Incyte Genomics Inc.
Lu Frank
Whigenant Ethan C.
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