Biomedical subjects

C M Bentzley

Publications and source records attributed to C M Bentzley.

4 recordsLinked to original sources

PubMed2000

Analysis of the degradation of oligonucleotide strands during the freezing/thawing processes using MALDI-MS.

Synthetic oligonucleotide strands ranging from 5 to 25 units in length are commonly used as standards, probes, and templates in various bioanalytical applications. Until recently, their preparation, storage, and handling were regarded as unimportant, but this work provides valuable information to the contrary. The systematic degradation of oligonucleotide strands during sample preparation is investigated by repeatedly freezing/thawing short strands followed by matrix-assisted laser desorption ionization mass spectrometric (MALDI-MS) analysis. It is shown here that the longevity of an oligonucleotide strand is dependent on several factors including base composition, solution concentrations, and strand length as well as thawing conditions. Several trends in strand robustness were established. Our studies reveal that the robustness of strands is base-dependent: T-mer > A-mer > C-mer > G-mer. Likewise, an increase in the length of the strands increases the tendency of a sample to degrade. Another observation included that samples of mixed bases degrade according to structural conformations. All of these observations are attributed to the fact that the samples undergo degradation during sample/solvent isolation during freezing.

Base Sequence↗

PubMed1999

Identification of single stranded regions of DNA by enzymatic digestion with matrix-assisted laser desorption/ionization analysis.

Elucidating structure function relationships of DNA in cellular processes requires fast, reliable methods that can be applied to picomole amounts of sample. Higher order structure can be inferred by distinguishing paired and unpaired regions. It is shown here that enzymatic digestion coupled with product analysis by matrix-assisted laser desorption ionization (MALDI) is able to identify unpaired bases within structured DNA regions. The method is demonstrated with DNA duplexes having a five nucleotide mismatch as a 5' overhang, a 3' overhang, and an internal loop. Exo- and endonuclease digestions are performed under solution conditions (temperature, annealing, and enzyme buffers) which promote base pairing and specific enzyme activity. For each type of mismatch, the length and sequence of the single stranded region can be inferred from MALDI spectra taken as a function of digestion time.

Animals↗

PubMed1998

Base specificity of oligonucleotide digestion by calf spleen phosphodiesterase with matrix-assisted laser desorption ionization analysis.

Calf spleen phosphodiesterase cleaves oligonucleotide strands in a stepwise manner from the 5' end and can be used in combination with matrix-assisted laser desorption ionization (MALDI) mass spectrometry to perform ladder sequencing. The relative intensities of ladder peaks in the mass spectra of a series of 5-mers and 7-mers show that the rate of digestion is influenced by strand sequence. Sequences terminating in A or G at the 5' end are found to react two to three times faster than sequences terminating in C or T. The reactivity of the terminal base is also influenced by the sequence beyond the 5' end. When the third base from the 5' end is A or G, removal of the first and second bases is faster than when the third base is C or T. A method is described which permits reaction rates to be quantitatively determined from the time dependences of ladder peaks in the MALDI spectra. A similar approach could be used for mechanistic studies.

Animals↗

PubMed1996

Oligonucleotide sequence and composition determined by matrix-assisted laser desorption/ionization.

Molecular weight measurements of several oligonucleotides ranging in size from 12 to 60 bases were performed by matrix-assisted laser desorption/ionization with a time-of-flight mass spectrometer (MALDI-TOF). In each case, the mass accuracy was better than 0.1%. Sequences for two 12-base oligonucleotides and a 24-base oligonucleotide were determined using calf spleen phosphodiesterase to sequentially cleave from the 5' end. A MALDI-TOF spectrum of the digest mixture shortly after the addition of the enzyme produced a characteristic oligonucleotide ladder. Molecular ions in the mass spectrum corresponded to the products of enzymatic cleavage, and the mass differences between these peaks identified the individual nucleotides. The resolution and mass accuracy of MALDI-TOF were sufficient to unambiguously identify the individual nucleotides in the 12- and 24-base strands.

Base Sequence↗