New technique may aid Human Genome Project

Scientists at the University and Johns Hopkins University have developed a new technique that promises to significantly enhance the rate of novel gene discovery, a process that becomes increasingly difficult as the Human Genome Project moves closer to completion.

“We have identified and corrected a fundamental flaw in the current genome-wide gene studies,” noted the authors in a paper published in the April 11 issue of The Proceedings of the National Academy of Sciences. The technique they developed “provides a much higher degree of novel gene identification than the current approaches,” added lead author San Ming Wang, Assistant Professor in Medicine.

Although two-thirds, more than 90,000, of the estimated 140,000 human genes have already been identified, finding the final third could be far more challenging. These elusive, as yet undiscovered genes tend to be expressed at low levels or only in certain cell types or turned on only during specific developmental stages or growth conditions. Yet these genes may play important roles in normal processes or in the development or prevention of various diseases.

To speed the search for these unknown genes, scientists use a “subtraction” method to remove the bulk of ubiquitous, already-identified genes from a sample pool of genetic material. This method leaves behind a higher percentage of the undiscovered genes that are expressed less frequently or at low levels and is an attempt to increase the probability of discovering unknown genes.

However, after this treatment, many of the unknown genes disappear. Wang and his co-authors have discovered how and why they disappear.

Genes are located in DNA. They are expressed as messenger RNA from which protein is made. This messenger RNA is the target for gene discovery.

In order to identify genes, the messenger RNA needs to be converted back into DNA molecules called complementary DNA. The subtraction method is then applied to remove the known, highly expressed genes in order to identify the unknown genes.

Almost every messenger RNA contains a naturally long “tail,” a strand of hundreds of adenosine bases located on one end. The conventional method used in past decades converts each messenger RNA into a DNA molecule that also includes a long tail copied from the messenger RNA.

During the subtraction reaction, tangled hybrids form randomly between these long tails of unrelated DNA molecules. Because all the hybrids are removed after subtraction, many unknown genes in the sample pool become the victims; they also are removed inadvertently even before having a chance to be discovered. Genes expressed at low levels are particularly affected by this phenomenon.

The authors of the paper devised a specific method to truncate these troublesome tails, reducing them from hundreds down to tens of adenosine bases. They showed that removal of the tails resulted in a retention of 1.4 to 7.8 times more copies in four out of five colon-specific genes expressed at low levels after subtraction.

When they applied their method to fish out genes in a previously well-characterized sample of colon cells, they found that many unknown genes were still hidden within that sample, and these genes can be identified with their new method.

“This could make a real difference in the increasingly difficult process of cataloguing the human genes,” said Janet Davison Rowley, the Blum-Riese Distinguished Service Professor in Medicine, Molecular Genetics & Cell Biology and Human Genetics and director of the study. “It could also simplify the post-genome process of learning what these less common genes do and when they are expressed.”

Additional authors of the study include University researchers Scott Fears and Jian-Jun Chen and Lin Zhang of Johns Hopkins. This work was supported by the National Cancer Institute, the American Cancer Society and the G. Harold and Leila Y. Mathers Foundation.