Abstract
Several approaches, generally referred to as rapid amplification of cDNA ends, are currently used as a means of obtaining full-length cDNA clones by PCR. However, these protocols are not infallible and in specific instances they have proven unsuccessful, emphasizing a need for further refinement. A novel method, the complete open reading frame (C-ORF) technique, is presently described, which has proven successful in cases, where standard rapid amplification of cDNA ends (RACE) has not worked. In C-ORF, the 5′ PCR primer site is provided by a degenerative stem-loop annealing primer, which consists of a stem-loop structure and a 3′ random 12-mer. degenerative stem-loop annealing primer is designed to anneal at random sites of the first strand cDNA, while promoting second strand synthesis from the end of given cDNA. Although this technique manifests weak sequence preference for GC-rich regions, in practice it has been successfully applied to clone both known and unknown genes with varying regions of GC-rich content. C-ORF does not use additional enzymes other than reverse transcriptase and Taq polymerase making it a cost-effective and relatively simple method that should be of general utility for gene cloning in multiple laboratories.
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References
Ota, T., Suzuki, Y., Nishikawa, T., et al. (2004) Complete sequencing and characterization of 21,243 full-length human cDNAs. Nat. Genet. 36, 40–45.
Zhu, Y. Y., Machleder, E. M., Chenchik, A., Li, R., and Siebert, P. D. (2001) Reverse transcriptase template switching: a SMART approach for full-length cDNA library construction. Biotechniques 30, 892–897.
Maruyama, K. and Sugano, S. (1994) Oligo-capping: a simple method to replace the cap structure of eukaryotic mRNAs with oligoribonucleotides. Gene 138, 171–174.
Carninci, P., Kvam, C., Kitamura, A., et al. (1996) High-efficiency full-length cDNA cloning by biotinylated CAP trapper. Genomics 37, 327–336.
Matz, M., Shagin, D., Bogdanova, E., et al. (1999) Amplification of cDNA ends based on template-switching effect and step-out PCR. Nucleic Acids Res. 27, 1558–1560.
Frohman, M. A., Dush, M. K., and Martin, G. R. (1988) Rapid production of full-length cDNAs from rare transcripts: amplification using a single gene-specific oligonucleotide primer. Proc. Natl. Acad. Sci. USA 85, 8998–9002.
Liu, X. and Gorovsky, M. A. (1993) Mapping the 5′ and 3′ ends of Tetrahymena thermophila mRNAs using RNA ligase mediated amplification of cDNA ends (RLM-RACE). Nucleic Acids Res. 21, 4954–4960.
Hooft van Huijsduijnen, R. (1998) PCR-assisted cDNA cloning: a guided tour of the minefield. Biotechniques 24, 390–392.
Wathelet, M., Moutschen, S., Defilippi, P., et al. (1986) Molecular cloning, full-length sequence and preliminary characterization of a 56-kDa protein induced by human interferons. Eur. J. Biochem. 155, 11–17.
Lin, J. J., Jiang, H., and Fisher, P. B. (1996). Characterization of a novel melanoma differentiation associated gene, mda-9, that is down-regulated during terminal cell differentiation. Mol. Cell. Different. 4, 317–333.
Lin, J. J., Jiang, H., and Fisher, P. B. (1998). Melanoma differentiation associated gene-9, mda-9, is a human gamma interferon responsive gene. Gene 207, 105–110.
Sarkar, D., Boukerche, H., Su, Z. Z., and Fisher, P. B. (2004) mda-9/Syntenin: Recent insights into a novel cell signaling and metastasis associated gene. Pharm. Ther. 104, 101–115.
Kang, D.-C., Gopalkrishnan, R. V., Wu, Q., Jankowsky, E., Pyle, A. M., and Fisher, P. B. (2002) mda-5: an interferon-inducible putative RNA helicase with double-stranded RNA-dependent ATPase activity and melanoma growth-suppressive properties. Proc. Natl. Acad. Sci. USA 99, 637–642.
Kang, D.-C., Gopalkrishnan, R. V., Lin, L., et al. (2004) Expression analysis and genomic characterization of human melanoma differentiation associated gene-5, mda-5: a novel type I interferon apoptosis-inducing gene. Oncogene 23, 1789–1800.
Leszczyniecka, M., Kang, D.-C., Sarkar, D., et al. (2002) Identification and cloning of human polynucleotide phosphorylase, hPNPase old-35, in the context of terminal differentiation and cellular senescence. Proc. Natl. Acad. Sci. USA 99, 16,636–16,641.
Leszczyniecka, M., Kang, D.-C., Su, Z.-Z., Sarkar D., and Fisher, P. B. (2003) Expression regulation and genomic organization of human polynucleotide phosphorylase, hPNPase old-35, a type I interferon inducible early response gene. Gene 316, 143–156.
Sarkar, D., Leszczyniecka, M., Kang, D.-C., et al. (2003) Downregulation of Myc as a potential target for growth arrest induced by human polynucleotide phosphorylase (hPNPase old-35) in human melanoma cells. J. Biol. Chem. 278, 24,542–24,551.
Leszczyniecka, M., DeSalle, R., Kang, D.-C., and Fisher, P. B. (2004) The origin of polynucleotide phosphorylase domains. Mol. Phylogenet. Evol. 31, 123–130.
Sarkar, D., Lebedeva, I. V., Emdad, L., Kang, D.-C., Baldwin, A. S. Jr., and Fisher, P. B. (2004) Human polynucleotide phosphorylase (hPNPase old-35): a potential link between aging and inflammation. Cancer Res. 64, 7473–7478.
Kang, D.-C., LaFrance, R., Su, Z.-Z., and Fisher, P. B. (1998) Reciprocal subtraction differential RNA display: an efficient and rapid procedure for isolating differentially expressed gene sequences. Proc. Natl. Acad. Sci. USA 95, 13,788–13,793.
Su, Z. Z., Lin, J., Shen, R., Fisher, P. E., Goldstein, N. I., and Fisher, P. B. (1996) Surface-epitope masking and expression cloning identifies the human prostate carcinoma tumor antigen gene PCTA-1 a member of the galectin gene family. Proc. Natl. Acad. Sci. USA 93, 7252–7257.
Gopalkrishnan, R. V., Roberts, T., Tuli, S., Kang, D.-C., Christiansen, K. A., and Fisher, P. B. (2000) Molecular characterization of prostate carcinoma tumor antigen-1, PCTA-1, a human galectin-8 related gene. Oncogene 19, 4405–4416.
Su, Z.-Z., Kang, D.-C., Chen, Y., et al. (2002) Identification and cloning of human astrocyte genes displaying elevated expression after infection with HIV-1 or exposure to HIV-1 envelope glycoprotein by rapid subtraction hybridization, RaSH. Oncogene 21, 3592–3602.
Su, Z.-Z., Chen, Y., Kang, D.-C., et al. (2003) Customized rapid subtraction hybridization (RaSH) gene microarrays identify overlapping expression changes in human fetal astrocytes resulting from human immunodeficiency virus-1 infection or tumor necrosis factor-alpha treatment. Gene 306, 67–78.
Kang, D.-C. and Fisher, P. B. (2005) Complete open reading frame (C-ORF) technology: simple and efficient technique for cloning full-length protein-coding sequences. Gene 353, 1–7.
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© 2007 Humana Press Inc., Totowa, NJ
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Kang, Dc., Fisher, P.B. (2007). Complete Open Reading Frame (C-ORF) Technique. In: Fisher, P.B. (eds) Cancer Genomics and Proteomics. Methods in Molecularbiology™, vol 383. Humana Press. https://doi.org/10.1007/978-1-59745-335-6_8
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DOI: https://doi.org/10.1007/978-1-59745-335-6_8
Publisher Name: Humana Press
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Online ISBN: 978-1-59745-335-6
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