Fragmentation of oligonucleotides

Fragmentation of nucleotides can be observed also in MALDI spectra [188,189], In principle, these fragmentations could allow the sequence to be deduced. However, coupling of MALDI to a TOF spectrometer and the variable kinetics of these fragmentations result, in the absence of specific experimental conditions, in broadening of the peak of the molecular species and a loss of resolution and sensitivity. 

Only fragmentations that occur as fast as the desorption can be observed in the mass spectrum at their right m/z values in a linear TOF instrument. Dissociation that occurs later on can be observed only if the extraction from the source is delayed or if a reflection analyser is used. 

The coupling of the MALDI to a trapping analyser, either ion trap or FTICR, allows all those fragments to be detected. [190] The formation of these fragments depends on the matrix, the power of the laser beam and the sequence of the oligonucleotide submitted to the analysis. 

Negative mode FAB spectrum of an oligonucleotide with sequence UGUU. Reproduced (modified) from Grotjahn L., in Mass Spectrometry in Biomedical Research edited by Gaskell S.J., Wiley, New York, 1986, pp. 215-234, with permission. 

Electrospray ionization produces stable ions of the molecular species, except if the conditions in the source are adjusted to induce dissociation by collision in the nozzle-skimmer region. [191] 

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The availability of very high specific activity 32P- and 35S-labeled deoxynu-cleoside triphosphates has encouraged the development of more sensitive ATase assays based on end-labeled fragments of oligonucleotides containing... 

Figure 4.2 MS fragmentation of oligonucleotides. Four possible fragmentation sites of oligonucleotides and their proposed nomenclature are depicted...

Although MALDI has been highly successful in the analysis of proteins and peptides, its application to the field of nucleic acids has faced many challenges. The lack of an appropriate matrix, interference of sample impurities, and unexpected fragmentation of oligonucleotides upon bombardment with a laser beam are some of the hurdles that must be surmounted. 

Martin et al. have recently shown that sequencing information can be obtained from fragmentation of oligonucleotides occuring in the ion source using delayed-extraction MALDI. Figure 11.22 compares the delayed-extraction MALDl mass... 

All the numerous cleavage possibilities listed probably are more significant for research on nucleic acids than for in vivo processes. In the cell the decisive role is played by the nucleases that break the high molecular nucleic acids into fragments of oligonucleotide size. These enzymes are able to degrade nucleic acids swiftly and to abolish their biologic activity. 

With the use of oligonucleotide probes based on the amino acid sequences of these protease V8-obtained peptides and of cyanogen bromide fragments of the porcine H,K-ATPase P subunit, cDNA clones for the rat [12,25] and rabbit [74] H,K-ATPase P subunit were then isolated. 

Zoller, M. J., and Smith, M., Oligonucleotide-Directed Mutagenesis Using M13-Derived Vectors an Efficient and General Procedure for the Production of Point Mutations in Any Fragment of DNA. Nucleic Acids Res., 1982. 10(20) pp. 6487-6500. 

Depending on the size of the probe, either covalent (Figures 10 and 11) or electrostatic immobilization (Figure 12) may be preferred. In general, oligonucleotides and DNA fragments of approximately 20 to 70 bases are amino-modified and bound covalently to the chip surface. Complete or partially complementary DNA of up to 5000 nucleotide bases is bound to the chip by electrostatic adsorption. 

SIAB and sulfo-SIAB have been used to make a high-capacity RNA affinity column for the purification of human IRP1 and IRP2 (Allerson et al., 2003), to couple antibodies or Fab fragments to amine-modified microparticles (Harma et al., 2000), and in the attachment of oligonucleotides to surfaces for detection arrays (Adessi et al., 2000). 

PCR is a technique for in vitro amplification of DNA sequences that involves repeated cycles of denaturation, oligonucleotide annealing, and DNA polymerase extension [29], The amplified products following PCR cycles contain double-stranded DNA fragments of discrete length. These DNAs are copies of the template DNA that are bounded at the 5 -terminus by the oligonucleotide primer for the sequence extension with a heat-resistant DNA polymerase. In quantitative assays of PCR products, therefore, nonspecific products interfere with the assay. 

Hofstadler, S.A. Griffey, R.H. Pasa-Tolic, R. Smith, R.D. The Use of a Stable Internal Mass Standard for Accurate Mass Measurements of Oligonucleotide Fragment Ions Using ESI Fourier Transform Ion Cyclotron Resonance-MS With Infrared Multiphoton Dissociation. Rapid Commun. Mass Spectrom. 1998,12, 1400-1404. 

Hong SP, Kim NK, Hwang SG, Chung HJ, Kim S, et al. 2004. Detection of hepatitis B virus YMDD variants using mass spectrometric analysis of oligonucleotide fragments. J Hepatol 40 837. 

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