NEW YORK, Oct. 8 - A novel technique for generating expressed sequence tags promises to speed up both gene discovery and DNA sequencing, according to results of a recent study.
The technique relies on open-reading frame-expressed sequence tags, or ORESTES, which differ from conventional ESTs in that they provide a high proportion of sequence data from the central protein-coding region of a gene transcript rather than from its ends.
Also, ORESTES, which is based on a low-stringency RT-PCR approach, tend not to under represent genes with low expression levels.
Writing in the Oct. 9 issue of the Proceedings of the National Academy of Sciences , a team of researchers in Brazil generated almost 700,000 ORESTES from 24 normal and malignant human tissue samples. The sequences are publicly available at GenBank, where they represent about 20 percent of all human ESTs.
Comparing their data with roughly 15,000 known full-length mRNA sequences, the scientists estimated that their sequences represent more than 80 percent of all highly and moderately expressed human genes, and around half of all rarely expressed human genes.
“ORESTES can speed up gene discovery principally as it does go a considerable way to enhancing the representation of rare transcripts in the accumulated data,” Andrew J.G. Simpson, a study author and the head of the Laboratory of Cancer Genetics at the Ludwig Institute for Cancer Research in Sao Paulo, said in an e-mail interview. “Thus, in many cases the first experimental evidence that a particular transcript exists comes from an ORESTES sequence.”
“Comparing this ORESTES sequence with the genome sequence can then pinpoint a gene for the first time,” Simpson said. “We are finding that some 10 percent of human genes are not predicted at all by current algorithms, requiring ESTs, such as ORESTES, to demonstrate their existence.”
However, the technique is limited because it requires very high-quality RNA and because of the occurrence of PCR artifacts.
Apart from finding novel genes in the human genome database, ORESTES can provide an alternative approach to full-length cDNA sequencing. Using ORESTES as a scaffold, gaps can be bridged by RT-PCR, according to the researchers.
"The transcript-finishing approach will likely provide a convenient means for delineating the boundaries of each gene and providing complete transcriptome sequences," according to an accompanying commentary.