Expressed Sequence Tags are generated from cDNA cloned from mRNA of any particular species (refer to the Figure 1). As the cDNA used is complementary to mRNA, the ESTs represent portions of expressed genes.

The ESTs can be generated by following steps:

Transcription of Genomic DNA: Genomic DNA is first transcribed to generate Nascent mRNA followed by splicing of synthesize perfect mRNA.

Reverse transcription of mRNA: mRNA can also be directly isolated from the species by using different kits (e.g. RNAgent Promega). mRNA synthesized undergoes reverse transcription to form cDNA library.

Generation of ESTs: From the cDNA library 5' or 3'-ESTs are generated by cDNA end sequencing. 5' EST is formed from a region of transcript which forms protein whereas the ending portion of cDNA forms 3'EST.

Assembly and organization of ESTs: The constructed ESTs can then be assembled separately in multimember sequence assembly, Bridged sequence assembly and small clusters on the basis of size of ESTs.

Figure 1 : Method of construction of ESTs from nascent DNA. The process involves transcription of nascent DNA, reverse transcription of mRNA and finally EST synthesis and clustering.