I read in an article that ESTs are partial or sub-sequences of cDNA. What are ESTs exactly? Which part of cDNA is cut in order to make ESTs? Why are they so instrumental in genomic research?
1 Answer
The question does not cite the source of the poster’s information, but it might well be the Wikipedia article on ESTs. I suspect the reason that does not answer the first question (“What are ESTs”) is the confusion in how ESTs are generated from cDNAs implicit in the second question “Which part of cDNA is cut…”. I therefore start by dealing with the source of the confusion.
Clarification 1: A Sequence need not have been Sequenced
One source of confusion lies in the word ‘sequence’. One meaning of this (noun only) is “a specific linear contiguous fragment or region of nucleic acid”. When used in this way there is no implication that even the length of this is accurately known, never mind the identity and linear arrangement of the nucleotides. The second use of the word sequence (noun) is the identity and order of nucleotides in a (linear) nucleic acid, with the corresponding verb meaning to determine this. Because today it is so cheap and quick to determine the sequence of a nucleic acid fragment, the student may not realize what a relatively slow and expensive undertaking it was in the last century. I shall use DNA fragment or region instead of the first use of ‘sequence’ in the rest of this answer.
Clarification 2: There are cDNAs and then there are cDNA clones
The second source of confusion seems to be with the cDNA clones. The question does not use the word ‘clone’, but asks “which part of cDNA is cut…”. This implies the poster is thinking in terms of individual cDNA clones in which the inserted cDNA fragment has been characterized — restriction mapped, perhaps sequenced and the protein product even identified. This is the approach adopted when the scientific problem is to identify the mRNA or gene encoding a particular protein — actin, beta-globin, etc. This is not the case with ESTs. Here the only characterization performed on a cDNA clone is a single DNA sequence determined (historically by the Sanger method) from a primer position in the cloning vector. This is because the focus is on producing a bulk library of DNA sequences.
So, what are ESTs and how are they generated?
As an alternative to the standard definition, one might say:
An EST is a single DNA sequence read derived from only part of a mRNA. Despite its uncharacterized nature, possible redundancy, limited size (perhaps 100-200 nt) and likelihood of errors it is useful in combination with many others from the same tissue as providing a reference to genes that are expressed in that tissue.
So, to reiterate, the answer to “what part of the cDNA is cut…” is “none”. One (presumably) just isolated total DNA from the recombinants in the M13 plaques and ran a single sequence determination from the primer site. And this was because one did not know which EST would be useful, but a collection or library of ESTs would likely contain some that would be useful.
The word ‘tag’ expresses the fact that they act as a handle or reference to the gene or mRNA, rather than being complete in themselves.
Why are/were ESTs important/useful?
The words the poster used originally were “Why are they so instrumental in genomic research?”, but I think that certainly exaggerates their current — if not their past — utility, so I have rephrased the question. Examples of the use of ESTs are given in the Wikipedia article already quoted, so I shall quote from Peter Goodfellow, writing in Nature in 1995.
Only 3 per cent of the human genome codes for proteins… the most important part of the genome is the protein coding genes and that sequencing these will give the largest return of biological information…
After pointing out his own scepticism towards the approach he briefly mentioned one important historical issue:
Given these disincentives, why have tens of millions of dollars been spent on ESTs? After a brief exploratory phase… a major driving force was the hope and the fear that ESTs could be used to patent any gene that had been tagged. Attempts to patent genes via ESTs raised the spectre of an international trade war and the echoes are still reverberating around courts in the United States.
No longer, I hope. Goodfellow then turned to the science:
It was hoped that large-scale EST projects would define the broad limits on the number of genes and the types of genes in the genome and provide a snapshot of their expression patterns. It was also expected that enough sequence information would be generated to be able to recognize new members of gene families of biological and commercial interest.
Coda: Yesterday and Today
Currently the method most used to investigate expression of genes as mRNA is, of course, RNAseq, which is faster, cheaper and, unlike ESTs, comprehensive.