Ammonium bicarbonate buffer system for DNA hybridization and quantification by LC-ESI-MS/MS.
Zhuo Zhen Chen, Nan Cheng, Lloyd Johnson, Jaimie Dufresne, John G Marshall
Author Information
Zhuo Zhen Chen: Research Analytical Biochemistry Laboratory, Department of Chemistry and Biology, Ryerson University, Canada.
Nan Cheng: Research Analytical Biochemistry Laboratory, Department of Chemistry and Biology, Ryerson University, Canada.
Lloyd Johnson: Research Analytical Biochemistry Laboratory, Department of Chemistry and Biology, Ryerson University, Canada.
Jaimie Dufresne: Research Analytical Biochemistry Laboratory, Department of Chemistry and Biology, Ryerson University, Canada.
John G Marshall: Research Analytical Biochemistry Laboratory, Department of Chemistry and Biology, Ryerson University, Canada. Electronic address: 4marshal@torontomu.ca.
High salt buffer may be used for the UV/VIS or radiometric based detection of trihybrid DNA but would contaminate the electrospray mass spectrometer. Colorimetric DNA hybridization assays with the substrate BCIP/NBT reacted with the alkaline phosphatase-streptavidin (APSA) enzyme conjugate that showed a linear range for HIV DNA from 1 pM to 100 pM DNA in presence of high salt concentrations. Ammonia bicarbonate (AMBIC) or ethanolamine resulted in strong DNA hybridization similar to NaCl but was compatible with specific and sensitive mass spectrometry. Linear and Gaussian analysis of HIV DNA with 10 % error was achieved across the pico Molar range from APSA amplification that converted the substrate AMP to adenosine for detection by monitoring the precursor ion at m/z 268 and plotting the fragment intensity at m/z 136 (m/z 268��� m/z 136) that was linear to 100 fM after log transformation. The novel observation that specific DNA hybridization in NaCl may be substituted with AMBIC permitted the direct analysis of a target DNA in femto molar to pico molar range by enzyme linked mass spectrometric assay (ELiMSA) using as little as 0.1 ��L (100 nL) of sample reaction injected on column.
