Exploring Expressed Sequence Tags (ESTs)
Expressed Sequence Tags (ESTs) are short pieces of DNA sequences derived from cDNA sequences (complementary DNA) corresponding to transcribed regions of genes. ESTs are widely used by various industries, including pharmaceuticals, agriculture, and biotechnology. They play a significant role in understanding the function of genes by encoding proteins, gene discovery, transcriptomics, and evaluating gene expression. Understanding the fundamentals of ESTs is paramount to unlocking their potential to further advance research. In this article, we’ll be discussing ESTs, how they are identified, their significance, applications in different fields, and future potential.
The Definition and Significance of Expressed Sequence Tags (ESTs)
A sequence tag is a small sequence of nucleotides (ATCG) that identifies a specific DNA fragment. Expressed Sequence Tags (ESTs) are partial cDNA sequences that represent transcribed regions of genes. ESTs reveal information about transcribed regions of genes that may not be readily available in genomic sequences. They serve as a bridge between known genes and the unknown genomic regions. ESTs have become crucial in a wide range of applications such as gene discovery, functional genomics, and transcriptomics.
ESTs can be derived from various tissues but are commonly derived from mRNA. This is because mRNA is a single-stranded RNA molecule that has been transcribed from DNA, indicating that it is not affected by genomic DNA contamination. ESTs are incredibly cost-effective, as they provide valuable insights into gene function without the need for full genomic sequencing. Because EST sequencing relies on cDNA rather than genomic DNA, it offers some information not available from genomic sequencing, such as the presence of alternative splicing variants.
ESTs are a valuable resource for researchers in many fields. In pharmaceuticals, ESTs are used to identify novel drug targets. Additionally, they can be used for drug development, pharmacogenomics, and studying diseases by identifying the genes that cause the disease. In agriculture, ESTs can be used to study flatulent cows by identifying genes involved in methane emission. Estimating methane emissions provide critical information for environmental purposes, including greenhouse gas emissions. In biotechnology, ESTs allow for an understanding of bacteria’s metabolic function, which can influence the optimization of industrial fermentation processes.
How to Identify ESTs: Methods and Applications
ESTs are identified using several techniques, including the Sanger sequencing method, high-throughput sequencing (HTS), and computational methods. The Sanger sequencing method involves the use of fluorescently labeled nucleotides to sequence DNA fragments, which can then be used to assemble cDNA fragments. High-throughput sequencing (HTS) technologies, such as Illumina and Ion Torrent, allow the simultaneous sequencing and identification of multiple ESTs fragments.
Computational approaches employ plug-in tools that extract ESTs from raw sequence datasets, including the Velvet, Trinity, and Oases assemblers that comprehend RNA-Seq platforms. The main benefit of computational methods is their relatively low cost and fast results. However, the computational approach is prone to errors due to problems with alignment and contig assembly.
ESTs have several applications, including gene discovery, transcriptomics, and gene expression analysis. Transcriptomics utilizes global gene expression profiling, which demands extensive EST sequencing. ESTs are used to obtain gene expression profiles without quantifying noncoding RNA since they only represent portions of mRNA transcribed from genes.
ESTs vs. Genomic DNA Sequencing: A Comparative Analysis
EST sequencing is an alternative to genomic DNA sequencing. Genomic DNA sequencing involves sequencing all the DNA in an organism. EST sequencing utilizes only portions of sequences that are transcribed from the genome. The genomic DNA sequence allows for a more comprehensive analysis of the genome. However, EST sequencing provides insights into the transcriptome’s dynamic nature, including alternative splicing and gene regulatory mechanisms. It provides a better opportunity to identify gene structure and function that are not available in the genomic DNA sequence. Genomic sequencing is costly and time-consuming, while EST sequencing is cost and time-efficient.
The main use of genomic sequencing is for the identification of new genes, discovering genetic variations, and identifying the entire gene complement of a particular organism in comparison to transcript identification and comparison achieved by EST sequencing. The former works best when studying prokaryotic, single-celled eukaryotes, and highly inbred plant species but has limitations for eukaryotic organisms.
The Use of ESTs in Transcriptome Analysis
Transcriptome analysis involves the identification of all RNA sequences produced by a cell. ESTs are a versatile, cost-effective, and efficient method for identification of biochemically functional transcripts. Through successive curation, EST data can be constructed into a Transcriptome Assembly Dataset (TAD), a functional dataset that enables annotation and extraction of sequence-based queries.
ESTs assemble unique sequences found in a cDNA library, including alternative splicing variants. ESTs play an important role in identifying differentially expressed genes and alternative isoforms, therefore monitoring changes in gene expression and splicing activities due to environmental changes, tissue specificity, and disease state. In this way, ESTs provide a better understanding of gene function, protein function, and human diseases.
The Role of ESTs in Gene Discovery and Functional Genomics
ESTs play an essential role in identifying new genes and discovering their function. ESTs are used in functional genomics because they are derived from the mRNA of expressed genes, providing insight into protein function. Functional genomics identifies the function of genes and gene products in entire organisms. It involves characterizing gene expression patterns, identifying gene regulatory elements, identifying protein-protein and protein-DNA interactions, and monitoring the activity of genes and gene products.
ESTs are useful in discovering new genes because expressed genes contain valuable information on gene function. ESTs provide essential insights into the identification of novel gene sequences and comprehensive information on gene expression and protein function. EST sequencing has helped to identify novel genes in various organisms, including mammals, plants, and microbes. For instance, the discovery of the Eucalyptus gunnii gene, implicated in cold tolerance, was published following the identification of a cold-responsive EST-derived sequence tag.
EST Databases: Accessing and Utilizing the Information
The number of publicly available EST databases is continually increasing. The NCBI GenBank, the DDBJ (DNA Data Bank of Japan), and the EMBL European Bioinformatics Institute (EBI) are the three international genomics data repositories that collect and distribute the vast amounts of sequence data available. In addition, UniGene, the Composite Sequence, and TIGR’s Gene Indices are popular centralized databases that information obtained from EST sequencing projects.
EST databases provide an essential resource for functional and comparative genomic approaches among various organisms. Accessing and utilizing information from these databases generally require searching through cDNA libraries using query sequences, which can be easily retrieved and utilized for further analysis. Integrating EST sequences with genomic information can lead to a more comprehensive description of gene structure and function.
The Future of ESTs: Potential Applications and Advancements
EST sequencing has already contributed significantly to understanding gene expression, function, and evolution. It is expected that EST sequencing will continue to shape research in various industries by enabling researchers to identify novel genes, alternative splicing, and identifying tissue specificity in gene expression. With the increasing volume of genetic data continually produced, there is an urgent need to develop complete sequencing technologies.
Nanotechnology and third-generation sequencing technologies are emerging as important areas of future EST research. Nanopore sequencing is an innovative third-generation sequencing that speeds up the entire sequencing process while providing accurate and reliable results. Nanopore sequencing will play an essential role in the future of sequencing and provide further insight into gene function, expression, and protein function.
Conclusion
Understanding ESTs is essential for unlocking their potential in scientific research, including gene discovery, functional genomics, and transcriptomics. ESTs have already advanced the pharmaceutical, agriculture, and biotechnology industries in various applications, including drug development and environmental studies. With further advancements in sequencing technologies, ESTs and their application will undoubtedly play a more significant role in shaping the future of research in various industries.