There are huge numbers of genes in our genome yet only few of them express to synthesis mRNAs which encode different proteins. These mRNAs are collectively called as transcriptome and mRNA can be reverse transcribed into cDNA, which provides evidence for all mRNA transcripts. Hence, mRNA and cDNA are crucial for gene expression profiling and transcriptome study.
Expressed sequence tags (ESTs) are short, unverified nucleotide fragment usually of 200-800 nucleotide bases. It is randomly selected by single-pass sequencing of either the 5’- or 3’-end of cDNA derived from cDNA libraries that constructed based on mRNA of specific gene. EST data sets has been recognized as the ‘poor man’s genome’ because EST data are widely used as a substitute to the genome sequencing.
There are several steps involved in EST generation. First, mRNAs isolated from specific cell line will be reverse transcribed to double-stranded cDNAs by using reverse transcriptase enzyme. cDNAs are then ligated into plasmid vector and cloned in order to get multiple copies of the cDNA for libraries construction. After then, ESTs can be generated by random sequencing of the cDNA clones with single-pass run from both 5’- and 3’-end directions, with no full-length read. The redundancies of the ESTs set can be reduced by normalization. EST data can be retrieved from different network interface such as UniGene from NCBI, TIGR, Cancer Genome Anatomy Project, ESTree, and dbEST at NCBI.
There are 5 stages involved in EST sequence analysis as described below:
1st: EST pre-processing • Lessen overall noise in EST data and enhance efficiency of downstream analysis.
• Identify and remove vector-sequence contaminan...
... middle of paper ...
...ion of Next Generation Sequencing is definitely a better approach to make genes sequencing more efficient. EST and SAGE has similarity that they require no prior understanding of the sequences to be analyzed since they are both sequencing-based gene expression profiling approaches (Pationo et al. as cited in Yamamoto et al., 2001).
Nagaraj, S. H., Gasser, R. B., & Ranganathan, S. (2006). A hitchhiker’s guide to expressed sequence tag (EST) analysis. Briefing in Bioinformatics, 8(1), 6-21.
Patino, W. D., Mian, O. Y., & Hwang, P. M. (2002). Serial analysis of gene expression: technical consideration and applications to cardiovascular Biology. Circulation Research, 91, 565-569.
Yamamoto, M., Wakatsuki, T., Hada, A., & Ryo, A. (2001). Use of serial analysis of gene expression (SAGE) technology. Journal of Immunological Methods, 250, 45-66.
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