Nucleic acid fragments sample preparation

The routine analysis of picomole quantities of nucleic acid fragments requires an increased compatibility between prechromatographic sample preparation procedures and sensitive liquid chromatographic detection... 

Further performance improvements in analysing nucleic acids could be achieved by the introduction of 3-hydroxypicolinic add as matrix [8] and the introduction of delayed extraction in a linear time-of-flight mass spectrometer [9]. If, for MALDI Fourier transform mass spectrometry, the molecular weight range in analysing nucleic add fragments could be extended further this type of MALDI MS would become of significant value due to the extraordinary resolution possible [10, 11]. In order to reach the sensitivity level necessary for MALDI-TOF MS analysis an amplification step has to be incorporated into the sample preparation process for... 

A wide range of nucleic acids including RNAs, DNA fragments, plasmids, and oligonucleotides can be separated effectively by SEC on the basis of molecular size. Accordingly, it is possible to adopt SEC as an alternative to gel electrophoresis for analytical purposes. Furthermore, because the separated components in samples can be recovered easily and yet almost quantitatively by collection of column effluent, SEC should be superior to gel electrophoresis for preparative purposes. Consequently, SEC seems to be a useful technique for the separation and purification of nucleic acids. 

Fig. 1 Nucleic acid samples available for electrochemical experiments, (a, b) naturally occurring DNAs (bl) dsDNA fragments of defined lengths and nucleotide sequences can be conveniently prepared by cleavage with restriction endonucleases (c) NAs (both DNA and RNA) synthesized by enzymes (d) fully synthetic DNAs and RNAs of limited lengths (e) PCR, can amplify the desired DNA segment from template DNA. ds, double-stranded ss, single-stranded, kbp, kilobase pairs. Covalently closed circles of sc, supercoiled and rel, relaxed DNA. oc, open circular DNA (containing at least one interruption of the sugar-phosphate backbone) lin, linear DNA. See text for more details.

Finally, the transcripts were completely digested by RNase Ti at 37°C for 10 min with MALDI matrix (3-HPA) added as a denaturant. Briefly, 5 pL of RNA transcript (up to 20 pg) is added to 4 pL 3-HPA in 50% ACN/water and 1 pL of RNase Tj (1000 units) and reacted for 10 min and then placed on ice for MALDI preparation. For highest quality spectra, samples are desalted by reverse-phase purification in ZipTips according to manufacturer s directions for nucleic acid purification [96]. The final step in this process is elution of purified RNA oligonucleotide fragments onto the MALDI target using 2 pL of the MALDI matrix itself. Samples (MALDI spots ) are allowed to air dry or may be rapidly dried under vacuum. 

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