|Home | About | Journals | Submit | Contact Us | Français|
The Nucleic Acids Research Group (NARG) believes that quality products and service are the main reasons that DNA synthesis core facilities should be able to contribute and prosper in their home institutions. Therefore, over the last three years, the NARG has focused on establishing methods for improving oligonucleotide synthesis and quality control. Avenues of communication with ABRF DNA synthesis laboratories were established via personal contact, conducting surveys, and establishment of World Wide Web and FTP sites where relevant information could be viewed.
A new focus of the NARG is the issue of quality versus economy for quantitative real-time PCR (qPCR). qPCR has become a mainstay in genomic research, and the cost, quality, and timeliness of probe delivery by the suppliers has become rate limiting on the productivity of research that could benefit from using qPCR. The NARG wanted to determine whether qPCR TaqMan®-type probes could be reasonably supplied by ABRF core facilities. In 2002 and 2003, the hypotheses tested by the NARG are (1) whether dual-labeled qPCR TaqMan probes are as easy and economical to make as regular oligonucleotides, and (2) whether crude unpurified dual-labeled probes can perform well in most qPCR experiments. After a preliminary pilot study among its own members in 2001–2002, for the 2002–2003 study the NARG designed a test probe sequence and asked member labs to synthesize and submit a 5′ FAM, 3′ BHQ1, or TAMRA labeled TaqMan-type probe, crude or purified. The NARG received 31 probes made by the constituent laboratories for analysis. About half of the probes are crude and half are purified by HPLC. A major question of interest is whether the highly purified probes will perform significantly better than the unpurified probes. Because a probe is typically only a 24mer, one made in a facility that routinely produces high-quality oligonucleotides may have little to gain by HPLC purification. In contrast, research is greatly facilitated by the availability of quality crude probes that can be delivered overnight at a fraction of the cost of HPLC-purified probes. In the 2003 NARG study, the qPCR probes will be tested for quality with capillary electrophoresis, polyacrylamide gel electrophoresis, DHPLC analysis on a WAVE instrument, and a qPCR performance test. The latter will be pipetted with the accuracy of quality liquid handling robotics and tested on the ABI 7700 qPCR instrument, using a 5-log dilution series of a synthetic template down to a target number of 200 copies. The interesting and precise findings of these analyses will be presented as a poster and further discussed at NARG’s Research Group session at the ABRF 2003 meeting. All data will be identified by the anonymous codes provided by the participants and will be accessible at the NARG website after the meeting.