Hybrid arrest technique to test for functional roles of cloned cDNAs and to identify homologies among ion channel genes

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Abstract

DNA and RNA antisense sequences—complementary to a target mRNA—can block selectively the expression of mRNA in prokaryotic and eukaryotic cells. This chapter describes a modified method for antisense arrest of expression with oligonucleotides, applicable for messages coding for channel proteins contained in tissue-derived total RNA. This is a powerful method to test for functional roles of cloned cDNAs, which by themselves, do not direct expression of currents when injected into oocytes and also enable the identification of potential homologies between sequenced mRNAs coding for channel proteins and mRNAs than can be assayed in the oocyte. The chapter demonstrates that the expression of α-globin mRNA is arrested specifically, by its complementary DNA sequences generated from an orientation-specific construct of the α-globin clone in an expression vector. The hybrid arrest method identifies sequence similarities of channel genes and may thereby permit the isolation of homologs by screening relevant libraries with the effective oligonucleotide sequences. This method is applicable for isolating genes of unknown sequence that code for channel proteins, whose expression in mRNA-injected oocytes can be assayed.

Original languageEnglish
Pages (from-to)605-612
Number of pages8
JournalMethods in Enzymology
Volume207
Issue numberC
DOIs
StatePublished - 1 Jan 1992

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