Abstract
The principle of the technique presented in this chapter is illustrated in Fig. 1. As with S1 mapping or riboprobe mapping, this technique can be used to determine precisely the start site of transcription of a mRNA sequence (1–3). Since this technique is relatively easier than other techniques, it is readily used for the primary determination of the transcription start site of a target gene. A radiolabeled probe derived entirely from within the gene is hybridized to mRNA complementary to the probe and extended using reverse transcriptase (RT). The cloned probe is normally derived from a region near the 5′ end (cap site) of the gene and the extension reaction terminates at the extreme 5′ end of the mRNA. Since only a small fragment of DNA probe is required as a primer, synthetic oligonucleotides are now almost exclusively used (although it is possible to use double-stranded DNA fragments or single-stranded primers generated by restriction enzyme digestion and gel electro-phoresis) (4).
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© 1999 Humana Press Inc., Totowa, NJ
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Kitami, Y., Hiwada, K. (1999). Primer Extension Analysis to Map Transcription Start Sites of Vascular Genes. In: Baker, A.H. (eds) Vascular Disease. Methods in Molecular Medicine™, vol 30. Humana Press. https://doi.org/10.1385/1-59259-247-3:133
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DOI: https://doi.org/10.1385/1-59259-247-3:133
Publisher Name: Humana Press
Print ISBN: 978-0-89603-731-1
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