Complementary DNA Sequencing:

Expressed Sequence Tags and Human Genome Project

Adams MD, Kelly JM, Gocayne JD, Dubnick M, Polymeropoulos MH, Xiao H, Merril CR, Wu A, Olde B,

Moreno RF, Kerlavage AR, McCombie WR, and Venter JC*

Presented by Malva “Lisa” Severios

Background

• Partial DNA sequencing done on 600 human cDNA clones to generate expressed sequence tags (ESTs).

• With up to 100,000 genes present in the human genome, 30,000 are expected to be expressed in the brain.

Expressed Sequence Tags

• Markers for Genome mapping.• Obtained by randomly acquiring cDNA

sequences.• Quick method that’s able to give information

about the diversity of genes expressed.• Can be mapped to chromosomes by FISH,

RH panels, BAC contigs, and Polymorphic STS.• Uses include: determining relationships between

genes and forensic analysis.

Purpose

• Promote a faster approach to cDNA characterization.

• Tag most of the human neurological genes expressed in the brain.

• Up to 1/4th of all genetic diseases affect neurological functions, indicating the importance of the genes expressed in the brain.

• Provide a less expensive method to find human genes.

Purpose…cont.

• Examine diversity of representative cDNA libraries.

• Identify advantages / disadvantages of cDNA libraries.

• Determine info content and accuracy of single-run sequence reactions.

Experimental Goal

Of cDNA libraries, random-primed and partial cDNA clones are more informative in identifying

genes and constructing a more useful EST database than sequencing from the ends of

full-length cDNAs. Therefore, obtain coding sequences in order to

take advantage of more sensitive peptide sequences and for nucleotide sequence

comparisons.

Experiment

• 3 commercial human brain cDNA libraries~ made from isolated mRNA in the hippocampus and temoral cortex of a

2 year old and fetus~• Single-run DNA sequence data obtained

from randomly chosen cDNA clones.• Tested subtractive hybridization to

increase the # of brain-specific clones.

EST Matches to Human Genes

EST Matches to Human Genes…cont.

Map ESTs to Human Chromosomes

• PCR used to screen somatic cell hybrid cell lines with defined sets of human chromosomes → hybrids with human gene corresponding to a specific EST yields an amplified fragment.

• The chromosome present in all hybrids that yielded an amplified fragment is the location of the EST.

Chromosome Segregation of ESTs

GenBank

• Able to analyze accuracy of ds automated DNA sequencing by matching ESTs with human sequences in GenBank.

• NIH genetic sequence database, providing a collection of DNA sequences to the public.

• Avg accuracy rate: 97.7% for 150 - 400 bases, having < 3% ambiguous base calls.

• 348 ESTs met this criteria and were submitted to GenBank.

EST Similarities in GenBank and PIR Databases

Conclusions

• Single-run DNA sequencing is efficient in obtaining preliminary data on cDNA clones.

• Found 230 ESTs, representing new genes• Random selection approach yields a high

amount of highly represented clones in the cDNA libraries used ~ NOT GOOD!!

• EST and physical mapping → high resolution map of the location of genes on chromosomes ~ more efficient and cheaper than genomic sequencing.

Conclusions…cont.

• Using ESTs will provide a better way of anayzing chromosomes and discovering more human genes.

• EST method will result in partial sequencing of most human brain cDNAs in a couple years → further identification of genes involved in neurological diseases.

What’s next?

• Characterize the new 230 genes found by:~ complete sequencing and

expression~ chromosome mapping~ tissue distribution~ immunology

Debate against cDNA for the Human Genome Project

• With cDNAs, can only find sequence of protein encoding information (ie. Know sequence of exons, not introns ~ which is important for regulation and control).

• Difficulty finding every single mRNA expressed in all tissues, cell types, and developmental stages.

• Gene coding regions, and therefore mRNA sequences, are NOT predictable from genomic sequences → don’t need large-scale cDNA sequencing.