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|Last Updated (September 15, 2003)|
About this Database
Malaria Full-Length cDNA project
2.Construction of Full-length cDNA Library
2-2. For Cloning of a Full-Length cDNA
In order to make a "full-length" cDNA library, we must devise some type of selection procedure for the full-length cDNA. That is, cDNAs which contain both ends of the intact mRNA should be selected. For that purpose, features which are characteristic of the 3 '-end and the 5'-end of the mRNA should be used as "tags". The full-length cDNA can then be selected through selection steps for both the 3'-end and the 5 '-end "tags".
The polyA stretch is a characteristic feature of the 3'-end of mRNA. Conventional methods use the polyA as a "sequence tag" to select the 3'-end of mRNA. In the conventional methods, the first-strand cDNA is usually synthesized using an oligo dT primer. Because the dT primer hybridizes mostly at the polyA, most of the cDNA is selectively synthesized from the 3'-end of the mRNA. Thus, the conventional methods include a selection step for the 3'-end "tag" of the mRNA. However, they include no step to select the 5'-end of mRNA. As a result, the largest part of the cDNA library consists of cDNAs which lack the 5'-end of the mRNA.
The main reason for this lies, in our view, in the fact that mRNA does not originally have a "sequence tag" at the 5'-end. The 5'-end of mRNA also has a characteristic structure, called the cap structure, but unfortunately it is not a "sequence tag". Unlike the polyA at the 3'-end, it cannot be used for the hybridization. If the 5'-end "tag" of the mRNA were also a "sequence tag", it would be easy to use it to select the 5'-end of mRNA.
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