Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification – Introduction of a polynucleotide molecule into or...
Reexamination Certificate
1999-02-25
2003-09-23
Whisenant, Ethan (Department: 1637)
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
Introduction of a polynucleotide molecule into or...
C435S006120, C435S091200, C435S091400, C435S091410, C435S091500, C435S320100, C536S024330
Reexamination Certificate
active
06623965
ABSTRACT:
BACKGROUND OF THE INVENTION
Cloning of DNA sequences encoding expressed proteins and construction of cDNA libraries from poly A+ mRNAs isolated form cells and tissues is currently performed in accordance with procedures outlined in FIG.
1
. However, the overall process is very laborious and has several technical limitations. Decreasing activity of the reverse transcriptase enzyme during first strand synthesis of the reverse complementary DNA (−) strand from the mRNA can result in yield of a product that is not full length. In addition, truncations can occur during the second round of synthesis to regenerate the corresponding “sense” coding (+) DNA sequences from the (−) DNA strand. For abundant mRNAs, a complete full length second strand is not always required as there is a greater likelihood for overlapping cDNAs that span a complete coding region. However, for smaller quantities of mRNA a full length strand may be only represented a few times.
As outlined in
FIG. 1
, the current procedure also requires homopolymeric tailing of both the cDNA sequences and the restriction digested cloning vectors, thus doubling the amount of manipulation involved. In addition, homopolymeric tailing of the vector results in loss of the original restriction site thereby limiting the ease of subsequent excision of the cloned cDNA region for the transfer to other expression or amplification vectors.
Accordingly, improvements to simplify cloning of mRNA sequences for use in the cloning of cDNAs for expression of proteins and in the construction of cDNA libraries are desired.
SUMMARY OF THE INVENTION
In the present invention a simplified method of directional cloning is provided. This method can be used, for example, in the cloning of the 5′ ends of cDNAs. The present invention differs from prior art cloning methods requiring homopolymeric tailing of both cDNA sequences and restriction enzyme digested vectors along with complete second strand synthesis before homopolymeric tailing. The method of the present invention improves the efficiency of the cloning of 5′ cDNA ends thereby increasing the likelihood of constructing full-length cDNA libraries comprised of overlapping cDNA subsequences.
The present invention uses oligonucleotides encoding restriction sites to create local double-stranded regions upon the first strand cDNA product of reverse transcriptase. The double-stranded regions are cleaved by double-strand requiring restriction endonucleases and serve to limit the regions to be replicated in a second (+) strand synthesis. Use of these oligonucleotides also increases the accuracy of replication of the entire shorter (−) cDNA strand to yield more of the 5′ (+) cDNA sequences necessary for obtaining a full representation of the entire mRNA coding sequence.
The method of the present invention also uses an oligonucleotide primer containing the same restriction site that is homopolymerically tailed to complement the homopolymerically tailed 3′ end of the (−) cDNA strand. The 5′ end of this primer hybridizes to the palindromic complement 3′ end of the restriction digested (−) cDNA strand thereby forming a more stable and replication competent gapped single-stranded circle. The resultant double-stranded product contains a unique copy of the targeting restriction site encoded by the priming oligonucleotide. Cleavage at this site yields double-stranded cDNA containing pairs that can be directly ligated into appropriate multiple cloning sites of commercial cloning and expression vectors. Since the restriction site is preserved and flanks the cDNA insert, the desired cDNA sequences can be readily excised and transferred to other vectors if necessary.
REFERENCES:
patent: 6114149 (2000-09-01), Fry et al.
Meissner, P.S., “Bacteriophase &lgr; cloning system for the construction of directional cDNA libraries”, Proc. Nat'l. Acad. SCi. USA 84; 4171-4175 1987.
Okayama, H., “High-Efficiency Cloning of Full-Length cDNA”, Mol. Cell. Biol. Feb. 1982 2;2 161-170.
Eberwine James H.
Madison Roger
Licata & Tyrrell P.C.
Trustees of the University of Pennsylvania
Tung Joyce
Whisenant Ethan
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