Research at Fox Chase »Research Facilities »Biotechnology

• DNA Synthesis
• Real time PCR
• Mass Spectrometry
• Proteomic services
• HPLC service

Function

Fox Chase has obtained a license from Biosearch Technologies Inc. to synthesize and use FRET (Fluorescence Resonance Energy Transfer) probes that use the BHQ (Black Hole Quencher™). These BHQ probes we made can be used in Real Time PCR Taqman® reactions with minimal probe purification. The cost of these probes to Fox Chase researchers is a fraction of the cost of commercial probes, and much more reliable because of our quality control process.

We provide Fox Chase researchers with quality oligonucleotides in a timely manner. Volume in the facility is 500-700 oligonucleotides per month. DNA is synthesized at scales of 0.1 to 6 micromoles. The completion of synthesis coupling is monitored. Particular attention is paid to the proper functioning of the synthesizers and the specialized chemicals used. The oligonucleotides are deblocked and analyzed by FPLC Mono Q anion-exchange at pH 12.5 to ascertain the completion of the deblocking reactions, the absence of abnormal synthesis, and the absence of excessive alkali damage to the DNA. Quantitation, FPLC analysis, and interpretation of the final products are provided to the user. DNA/RNA oligonucleotides modified with a variety of probes and chemical adducts are synthesized at the highest quality and economy possible on demand. The facility evaluates different DNA synthesis chemistries and develops new protocols for quality analysis. MALDI-TOF Mass Spectrometry is used for quality control as needed. The current synthesis turnaround time is 24 hours during normal operation, even for long oligonucleotides, small or large orders, without compromising quality. This fast service was made possible by the addition of an evening shift in the facility. HPLC purification of oligonucleotides to crystallographic experiment quality is provided as needed. Designer gene assembly is performed with recursive PCR from sets of 180 mers.

Description

This facility has a simultaneous capability of 16 columns on four Applied Biosystems DNA synthesizers (Model 394). These synthesizers are able to make oligonucleotides of RNA and DNA, which are unmodified or modified with a large variety of chemical groups. Oligonucleotide sequencing and quality control protocols can be found in Yeung, et al., Nucleic Acids Res. 16:4539, 1988; Yeung and Miller, Anal. Biochem. 187:66, 1990. Mass spectrometry can also be used to determine the sequence of oligonucleotides, even for modified oligonucleotides. Oligonucleotide purification is done by the HPLC facility. The staff interacts with users on oligonucleotide technologies and will provide users with assistance whenever possible. Routine synthesis requests can be dropped off at the facility in Room R150, faxed, or e-mailed to oligo@fccc.edu. Please contact Glenn Miller or Philip Do for questions on synthesis orders.