ESTs are short fragments of DNA that represent genes expressed in certain cells or tissues. They are generated by sequencing portions of cDNA, which is synthesized from mRNA to create a more stable representation of expressed genes. This process produces either 5' ESTs from the beginning of the cDNA or 3' ESTs from the end. EST libraries provide an extensive survey of the transcribed portions of genomes and are useful for gene discovery, structure prediction, and mapping, though individual ESTs only represent partial gene sequences and have some limitations in quality and redundancy.
ESTs are short fragments of DNA that represent genes expressed in certain cells or tissues. They are generated by sequencing portions of cDNA, which is synthesized from mRNA to create a more stable representation of expressed genes. This process produces either 5' ESTs from the beginning of the cDNA or 3' ESTs from the end. EST libraries provide an extensive survey of the transcribed portions of genomes and are useful for gene discovery, structure prediction, and mapping, though individual ESTs only represent partial gene sequences and have some limitations in quality and redundancy.
ESTs are short fragments of DNA that represent genes expressed in certain cells or tissues. They are generated by sequencing portions of cDNA, which is synthesized from mRNA to create a more stable representation of expressed genes. This process produces either 5' ESTs from the beginning of the cDNA or 3' ESTs from the end. EST libraries provide an extensive survey of the transcribed portions of genomes and are useful for gene discovery, structure prediction, and mapping, though individual ESTs only represent partial gene sequences and have some limitations in quality and redundancy.
ESTs are short fragments of DNA that represent genes expressed in certain cells or tissues. They are generated by sequencing portions of cDNA, which is synthesized from mRNA to create a more stable representation of expressed genes. This process produces either 5' ESTs from the beginning of the cDNA or 3' ESTs from the end. EST libraries provide an extensive survey of the transcribed portions of genomes and are useful for gene discovery, structure prediction, and mapping, though individual ESTs only represent partial gene sequences and have some limitations in quality and redundancy.
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expressed sequence tags
What are ESTs & How are they made?
ESTs are small pieces of DNA sequence (usually 200 to 500 nucleotides long)
Generated by sequencing either one or both ends of an expressed gene.
expressed sequence tags
The idea is to sequence bits of DNA that represent genes expressed in certain cells, tissues, or organs from different organisms.
And use these "tags" to fish a gene out of a portion of chromosomal DNA by matching base pairs.
expressed sequence tags
Why use EST?
Gene identification is very difficult in organisms.
Because most of our genome is composed of introns interspersed with a relative few DNA coding sequences, or genes.
These genes are expressed as proteins.
Each gene (DNA) must be converted, or transcribed, into messenger RNA.
The resulting mRNA guides the synthesis of a protein.
Interestingly, mRNAs in a cell do not contain sequences from the regions between genes, nor from the non-coding introns that are present within many genes.
Therefore, isolating mRNA is key to finding expressed genes in the vast expanse of the human genome. Next problem:
mRNA is very unstable outside of a cell
Convert it to complementary DNA (cDNA).
cDNA is a much more stable compound and, importantly, because it was generated from a mRNA in which the introns have been removed, cDNA represents only expressed DNA sequence. From cDNAs to ESTs
Once cDNA is made, we can then sequence a few hundred nucleotides from either end of the molecule to create two different kinds of ESTs.
Sequencing only the beginning portion of the cDNA produces what is called a 5' EST.
Sequencing the ending portion of the cDNA molecule produces what is called a 3' EST. An overview of how ESTs are generated A cDNA library is constructed from a tissue or cell line of interest.
The libraries are constructed by isolating mRNA from the tissue or cell line of interest.
The mRNA is then reverse-transcribed into cDNA.
The resulting cDNA is cloned into a vector. Individual clones are picked from the library, and one sequence is generated from each end of the cDNA insert.
Thus, each clone normally has a 5' and 3' EST associated with it.
The sequences average ~ 400 bases in length.
Because the ESTs are short, they generally represent only fragments of genes, not complete coding sequences. How to Access ESTs ? Submitted to all three international sequence databases (GenBank, EMBL, and DDBJ), under the data-sharing agreement .
All ESTs can be accessed through all of these databases, regardless of where the sequence was originally submitted.
The same ESTs are also available from the NCBIs dbEST, the database of Expressed Sequence Tags. Like other sequences in GenBank, ESTs can be accessed through Entrez.
Single ESTs are retrieved by accession or gi number.
Advanced searches with multiple search terms can be limited to ESTs by selecting the Properties limit and entering EST. How to Access ESTs ? Interest for ESTs
ESTs represent the most extensive available survey of the transcribed portion of genomes.
ESTs are indispensable for gene structure prediction, gene discovery and genomic mapping.
Characterization of splice variants and alternative polyadenylation.
High-volume and high-throughput data production at low cost.
There are 69,713,950 of EST entries in GenBank (dbEST) (June 1, 2011): 8,315,231 entries of human ESTs; dbEST release 060111
Limitations of EST Data Data are not of as high a quality as sequences determined by conventional means.
High error rates (~ 1/100) because of the sequence reading single-pass.
ESTs may contain substitutions, deletions, or insertions compared with the parent mRNA sequence.
ESTs may contain bacterial, mitochondrial, or vector sequence contamination.
A single EST represents only a partial gene sequence.
Not a dened gene/protein product.
Not curated in a highly annotated form.
High redundancy in the data)huge number of sequences to analyze.