What are Expressed Sequence Tags (EST)? | Genomics

Summary

In this Spartan Tutorials video, Rubinder Sayal explains Expressed Sequence Tags (ESTs), which are small DNA fragments from cDNAs used in early gene discovery and genome mapping. He highlights their role in identifying coding genes, which make up only 2% of the human genome. The video uses Duchenne Muscular Dystrophy (DMD) as an example to illustrate the efficiency of EST sequencing over whole genome sequencing. It also discusses the process of EST sequencing, its utility in gene function prediction, and how it aids in designing probes for gene analysis across different species.

Takeaways

• 🧬 Expressed Sequence Tags (ESTs) are small sequence fragments derived from cDNAs that were crucial in early gene discovery and genome mapping.
• 🔍 ESTs are not found in the genome itself but are a technical byproduct, representing short sequences from the coding regions of the genome.
• 📊 The human genome's protein-coding genes constitute only about 2% of the total genome, making EST sequencing an efficient way to focus on these areas.
• 🕒 The Human Genome Project, which lasted from 1990 to 2001, took 11 years to complete, highlighting the need for more efficient sequencing methods like EST sequencing.
• 🧬 The Duchenne Muscular Dystrophy (DMD) gene, dystrophin, is an example used to illustrate the size and complexity of genes, with a coding portion of only 11kb out of 2500kb.
• 🧪 EST sequencing involves isolating mRNA from a tissue of interest, cloning it into vectors, and then randomly sequencing colonies to identify the most abundant mRNAs.
• 🔎 The UCSC Genome Browser is a valuable resource for browsing and analyzing genome sequences, including those used in EST sequencing.
• 🧬 Shotgun sequencing, a method of breaking DNA into many small chunks and sequencing them individually, is used in EST sequencing to piece together the whole sequence from overlaps.
• 🔗 EST sequencing can help identify new isoforms and proteins expressed in specific tissues, contributing to a better understanding of gene function.
• 📚 The dbEST database, although not directly browsable, is searchable and contains EST sequences that can be used to design probes and identify homologous genes across species.
• 🌐 EST sequencing has been instrumental in comparative genomics, helping to identify homologous genes and understand gene function across different organisms.

Q & A

What are Expressed Sequence Tags (ESTs)?

Expressed Sequence Tags (ESTs) are small sequence fragments obtained from cDNAs, which are complementary DNAs. They were instrumental in the early days of human genome sequencing and other organisms' genome mapping, aiding in gene discovery and mapping.

Why were ESTs important in the initial stages of genome sequencing?

ESTs were important because they allowed scientists to focus on the coding regions of the genome, which are more relevant for gene discovery and mapping. This was more efficient than sequencing the entire genome, especially considering that protein-coding genes make up only about 2% of the human genome.

How did EST sequencing help in mapping the Duchenne muscular dystrophy (DMD) gene?

EST sequencing helped in mapping the DMD gene by allowing scientists to sequence the coding portions of the gene more efficiently. The DMD gene is large, spanning almost 2 Mb, but the coding portion is only 11 kb. By using EST sequencing, researchers could focus on these coding regions and identify the gene's structure and function more quickly.

What is the difference between ESTs and the actual genome?

ESTs are not present in the genome; they are technical manifestations created from the coding portions of the genome. They are short fragments of sequences that are derived from the mRNA transcripts, which are then used for gene discovery and analysis.

How does the process of EST sequencing work?

The process of EST sequencing involves isolating mRNA from a tissue of interest, cloning it into suitable vectors to create a cDNA library, and then randomly sequencing colonies from this library. The most abundant mRNAs will be sequenced first, providing insights into the most active genes in that tissue.

What is the significance of the number of exons in the DMD gene?

The DMD gene has about 80 exons, which is significant because it indicates the complexity of the gene. This large number of exons means that the gene is large and complex, making it challenging to sequence and analyze using traditional methods. EST sequencing provided a way to overcome this challenge.

How does shotgun sequencing relate to EST sequencing?

Shotgun sequencing is a method where DNA is broken into many small chunks, sequenced individually, and then the sequences are assembled based on overlaps. EST sequencing also uses this approach, where random sequencing of clones is performed, and the sequences are assembled to understand the gene structure.

What is the role of the UCSC Genome Browser in genome research?

The UCSC Genome Browser is a valuable resource for genome research, providing a clean repository of genome sequences. It allows researchers to browse and analyze the sequences of various model organisms and is often relied upon for analysis in scientific papers.

How can EST sequences be used to design probes for other species?

EST sequences can be used to design probes for other species by identifying homologous genes. Researchers can use the EST sequence from one species or a protein sequence from another species to design random primers and amplify the homologous gene from another species.

What is the dbEST database, and how is it used?

The dbEST database is a collection of all public EST sequences. It is not directly browsable on NCBI's database but is searchable using command line tools and BLAST. Researchers can use dbEST to find EST sequences that can help in gene discovery and function prediction.

How have ESTs contributed to our understanding of gene function?

ESTs have contributed to our understanding of gene function by allowing researchers to compare sequences with already sequenced genomes and proteins. This comparison can reveal the domain present in the gene, providing insights into its probable function and potential homology with genes in other model organisms.