Poly A polymerase method

Dissolve 100 pCi lyophilised [a-32P] cordycepin-5 -triphosphate with the denatured RNA solution. 

Site-specific modification of large RNAs ( 20 nucleotides) has until recently been a difficult task which involved an RNA ligase-mediated ligation of two RNAs. An alternative method has been developed by Moore and Sharp19 which has facilitated this reaction. 

The basic principle is outlined in Fig. 2.3A Two RNAs, a 5 -RNA and a 3 -RNA, containing the sequences 5 and 3 of the site to be modified, respectively, are synthesised so that the 3 -RNA contains the 5 -phosphorylated modified nucleotide at the 5 -end. A complementary DNA oligonucleotide is annealed to the two RNA halves, and the ligation step is catalysed by T4 DNA ligase, which will join the 3 -OH of the 5 -RNA to the 5 -terminal monophosphate of the 3 -RNA. The 5 -RNA may be synthesised by 

RNA will therefore lower the yield of ligated product but it will not influence the homogeneity of the product. 

In the Moore and Sharp19 procedure outlined below a radioactive phosphate is inserted at the junction by labelling the 3 -RNA using T4 polynucleotide kinase and [y-32P]ATP, but the ligation approach is useful for many other types of modifications. Other applications include  

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