Abstract
In this work, a ligation-independent, fully gene-specific, nested polymerase chain reaction (PCR) method for the elucidation of 5′ cDNA sequence is described and demonstrated for the first time. Two manifestations of the method, rapid amplification of cDNA ends (RACE) by lariat-dependent nested PCR 5′ (RACE LaNe), at least as simple to perform as conventional RACE, were successfully applied to the murine housekeeping genes phosphoglycerate kinase 1 (PGK1), β-actin (β-ACT), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the alpha thalassemia mental retardation Y homolog (ATRY) gene of the marsupial, Macropus eugenii. Significantly, a new murine GAPDH 5′ exon, separated by 365 kb of intronic sequence from previously annotated GAPDH sequence, was discovered using 5′RACE LaNe.
Similar content being viewed by others
References
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.
Frohman, M. A. and Martin, G. R. (1989) Rapid amplification of cDNA ends using nested primers. Technique 1, 165–170.
Triglia, T., Peterson, M. G., and Kemp, D. J. (1988) A procedure for in vitro amplification of DNA segments that lie outside the boundaries of known sequences. Nucleic Acids Res. 16, 8186.
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.
Volloch, V., Schweitzer, B., and Rits, S. (1994) Ligation-mediated amplification of RNA from murine erythroid cells reveals a novel class of beta globin mRNA with an extended 5′-untranslated region. Nucleic Acids Res. 2, 2507–2511.
Schmidt, W. M. and Mueller, M. W. (1999) CapSelect: a highly sensitive method for 5′CAP-dependent enrichment of full-length cDNA in PCR-mediated analysis of mRNAs. Nucleic Acids Res. 27, e31.
Park, D. J. (2004) 3′RACE LaNe: a simple and rapid fully nested PCR method to determine 3′-terminal cDNA sequence. BioTechniques 36, 586–590.
Park, D. J. (2004) Methods of sequence determination. Australian Provisional Patent application number 2004900731.
Efstratiadis, A., Kafatos, F. C., Maxam, A. M., and Maniatis, T. (1976) Enzymatic in vitro synthesis of globin genes. Cell 7, 279–288.
Higuchi, R., Paddock, G. V., Wall, R., and Salser, W. (1976) A general method for cloning eukaryotic structural gene sequences. Proc. Natl. Acad. Sci. USA 73, 3146–3150.
Maniatis, T., Kee, S. G., Efstratiadis, A., and Kafatos, F. C. (1976) Amplification and characterization of a β-globin gene synthesized in vitro. Cell 8, 163–182.
Rougeon, F. and Mach, B. (1976) Stepwise biosynthesis in vitro of globin genes from globin mRNA by DNA polymerase of avian myeloblastosis virus. Proc. Natl. Acad. Sci. USA 73, 3418–3422.
Pask, A. J., Renfree, M. B., and Graves, J. A. M. (2000) The human sex-reversing ATRX gene has a homologue on the marsupial Y chromosome, ATRY: implications for the evolution of mammalian sex determination. Proc. Natl. Acad. Sci. USA 97, 13198–13202.
Siebel, A. L., Gehring, H. M., Reytomas, I. G. T., and Parry, L. J. (2003) Inhibition of oxytocin receptor and estrogen receptor-α expression, but not relaxin receptors (LGR7), in the myometrium of late pregnant relaxin gene knockout mice. Endocrinology 144, 4272–4275.
Carninci, P., Kvam, C., Kitamura, A., et al. (1996) High-efficiency full-length cDNA cloning by biotinylated CAP trapper. Genomics 37, 327–336.
Sirover, M. A. (1999) New insights into an old protein: the functional diversity of mammalian glyceraldehydes-3-phosphate dehydrogenase. BBA-Protein Struct. M 1432, 159–184.
Lei, B., Lionetti, V., Young, M. E., et al. (2004) Paradoxical downregulation of the glucose oxidation pathway despite enhanced flux in severe heart failure. J. Mol. Cell. Cardiol. 36, 567–576.
Kusner, L. L., Sarthy, V. P., and Mohr, S. (2004) Nuclear translocation of glyceraldehydes-3-phosphate dehydrogenase: a role in high glucose-induced apoptosis in retinal muller cells. Invest. Opthalmol. Vis. Sci. 45, 1553–1561.
Tsuchiya, K., Tajima, H., Yamada, M., et al. (2004) Disclosure of a pro-apoptotic glyceraldehydes-3-phosphate dehydrogenase promoter: anti-dementia drugs depress its activation in apoptosis. Life Sci. 74, 3245–3258.
Agrawal, N., Fujimoro, M., Igarashi, S., et al. (2000) The role of the interaction of Huntington with p53 and GAPDH in the pathology of Huntington’s disease. Am. J. Human Genet. 67, 226.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Park, D.J. A new 5′ terminal murine GAPDH exon identified using 5′RACE LaNe. Mol Biotechnol 29, 39–46 (2005). https://doi.org/10.1385/MB:29:1:39
Issue Date:
DOI: https://doi.org/10.1385/MB:29:1:39