| ABSTRACT:
Berkeley Lab Scientist Roger Hoskins and colleagues have developed a technique that enables the recovery of plasmid cDNA clones for previously uncharacterized genes. The technique is simpler, faster and more efficient than currently available library screening methods.
The currently used EST sequencing approach identifies cDNAs for relatively abundantly expressed genes. Large EST projects in humans and several model organisms have recovered cDNAs for about 70% of the total number of genes identified in genome sequences. Many genes involved in regulatory and signaling pathways are expressed at relatively low levels, and cDNAs have not been identified by the EST approach. The Berkeley Lab technique can be used to isolate cDNAs for many such genes.
The widely used RT-PCR technique requires knowledge of the sequences at both the start (5’) and end (3’) of the gene of interest. If these sequences are based on gene prediction rather than experimental evidence, as is often the case, and the predictions are incorrect, then the sequence information obtained with RT-PCR will represent an incomplete gene. In contrast, the Berkeley Lab technique can be successfully applied with limited knowledge of the gene sequence. Primers can be designed to anneal to any sequence within the gene. Furthermore, the Berkeley Lab technique can provide more complete information about the gene transcript structure, including the 5’- and 3’-UTRs that are often involved in the regulation of gene and protein expression.
The Berkeley Lab technique can be performed in multi-well format and does not require gel purification steps. Hence, it can be easily adapted to a high-throughput setting. |
