Determination of the capped site sequence of mRNA based on the detection of cap-dependent nucleotide addition using an anchor ligation method.

The sequence analysis of the 5' ends of cDNAs prepared using the anchor ligation method has revealed that most of the full-length cDNAs have an additional dGMP at their 5' end that is absent in the corresponding genome sequence. Using model RNA transcripts with cap analogues possessing 7-methylguanosine and adenosine, the base of the added nucleotide has been shown to be complementary to the base of the cap analogue, suggesting that the cDNAs possessing an additional dGMP are derived from intact mRNAs with the cap structure. On the other hand, cap-free RNA did not produce cDNA with an extra dGMP. These findings suggest that we can determine whether or not the cDNA starts from the capped site sequence of mRNA based on the presence or absence of an additional dGMP at the 5' end of the cDNA synthesized using the anchor ligation method. This approach will be useful to determine the capped site sequence of mRNA, thus, to identify transcription start sites.