Backbone research
Normalized emission spectra af5-pM topotecan in plasma (820 nm) )nd phosphate-buffered sallne (410 nm). (Adapted with permission ffom Academic Press.)
The signal intensity was linearly dependent on the topotecan concentration with detection limits of ~ 0.05 uM and 1 pM in plasma and whole blood, respectively. Fluorescence lifetime measurements indicated that the topotecan was responsible for the observed emission. The low levels of quantitation suggest the possible use of two-photon excitation for the noninvasive detection of camptothecin drugs such as topotecan or other analytes. (Anal. Biochem. 1196,242,266-70)
Quantifying DNA by amplifying repetitive sequences When PCR is used to quantitatively measure DNA, the standards must provide binding sites for the primer that are similar or identical to those of the nucleic acid of interest. This prerequisite cannot always be fulfilled in cases involving the detection of heterogeneous nucleic acids. Thomas Hammerle and co-workers at Immuno AG (Austria) have developed an assay for the quantitation of chicken genomic DNA with chicken repeat 1 (CR1), a family of sequences that belongs to the non-long-terminal repeat class of retrotransposons present in about 10 000 to 30 000 copies per haploid genome. After the CR1 sequences were amplified, they were separated by polyacrylamide gel electrophoresis and detected by fluorescence. Quantitation was based on
replaced by an uncharged methylphosphonate (MP) linkage. Experiments were carried out using a triplequadrupole MS. In all the model compounds, charging of more than 90% of the phosphate groups was observed, compared with about 60% in typical all-phosphodiester oligonucleotides. This unexpected result was attributed to charge stabilization through the interactions of charged sites with uncharged residues. Two generic pathways were proposed for the backbone cleavage, leading the authors to suggest that backbone cleavage reactions in conventional phosphodiester oligonucleotides occur at nonionizable sites (I. Mass Sbectrom 1996 31 1277-83)
Studies of nucleic acids, the most polar of the major classes of biopolymers, have benefitted significantly from MS methods. One arena of MS work has been the collision-induced dissociation of multiply charged oligonucleotides produced by electrospray, which has as its principal dissociation pathway the chain cleavage of the polynucleotide backbone. Two sets of ions are producedfromthis cleavage: the a-base series and the w series, representing sequence in the 5'3' and 3'5' directions, respectively. Common to all the proposed mechanisms for the decomposition is the initial involvement of the negatively charged phosphate at the cleavage site James A McCloskey and colleagues from the University of Utah and ISIS Pharmaceuticals investigated the role of backbone charge on the dissociation reaction by examining three different model compounds in Proposed mechanism for charge transfer from a remote which the normal phosphate to methylphosphonate wf; the second reaction phosphodiester linkproduct is an intermediate that may further dissociate. age was partially (Adapted with permission from John Wiley & Sons.)
the intensity of the reaction products and the known quantity of internal standard molecules that were coextracted, coampli-
i ne number of copies detected in the chicken genomic assay was linear from 0 to 25 pg/mL DNA added, after which the assay started to reach saturation. (Adapted with permission from Academic Press.)
fied, and coanalyzed. A callbration curve of numerous copies detected versus pg/mL of genomic chicken DNA added was linear between 0 and 25 pg/mL The researchers were able to detect 50 pg/mL of added DNA and after making several assumptions, they estimated that the lowest detectable amount of DNA is 28 fg/mL An analysis of chicken, mouse, hamster, African green monkey, and human genomic DNA indicated that only chicken DNA was detected with CR1 primers, even though the other species' DNA were present at 20-fold higher concentrations. The authors predicted that assays for repetitive sequences will provide sensitive and precise quantitation of genomic DNA, which is necessary in the quality control of biological products, where 100 pg of residual DNA per dose is considered an acceptable level of risk. (Anal. Biochem. 1996,242, 240-47)
Analytical Chemistry News & Features, February 1, 1997 77 A
