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Reversed-phase HPLC separates oligonucleotides and contaminants on the basis of differences in hydrophobicity. Crude oligonucleotides contain the desired product, failure sequences and chemically modified by-products, all of which will vary in hydrophobicity. For example, any oligonucleotide strands that have not been completely deprotected will be more hydrophobic than the fully deprotected product. The HPLC chromatogram shows these impurities and gives an indication of the purity of the oligo.
Anion-exchange HPLC has the same principles as reversed-phase HPLC, but it separates the mixture depending on its ionic interactions with the column − longer, more highly charged oligonucleotides elute later than the shorter ones. The stationary phase includes anionic functional groups that interact with any positive charge on the oligonucleotide.
Capillary electrophoresis separates molecules according to their electrical charge.
Mass spectrometry has been an important analytical tool in organic chemistry for many years, but until recently its application to large biomolecules was limited. This is because the harsh ionisation methods led to decomposition of fragile biomolecules, and the required high mass ranges were inaccessible. However, new volatilisation and ionisation techniques have now established MS as an excellent tool for analysing and identifying synthetic oligonucleotides and natural DNA. It has significant advantages over techniques such as gel electrophoresis as it combines high resolution with very short analysis times. The mass spectrometry process involves ionisation of the biomolecule, detection of the resultant ions and measurement of their mass to charge ratio (m/z). The two most successful techniques are described below are electrospray ionisation and matrix-assisted laser desorption ionisation.
ESI-MS allows direct analysis of PCR products up to 100 nucleotides in length and has been used to determine the masses of modified and unmodified oligonucleotides. The technique produces a fine spray of highly charged droplets in the presence of a strong electric field. Vaporisation of these droplets results in the production of gaseous ions, which are singly or multiply charged and are accelerated by electric fields towards a mass analyser.
Sample preparation and clean-up are critical as this technique has a relatively low tolerance to salt. DNA has a high affinity for sodium and potassium ions and this can lead to complicated spectra. A major advantage of ESI over other techniques is that it can be coupled to high-performance liquid chromatography (HPLC) or capillary electrophoresis to provide in-line separation of complex mixtures.
MALDI-TOF MS is an established technique in the field of protein and peptide analysis and has proved to be the method of choice for DNA mass spectral analysis. It is a soft ionisation technique (i.e. it occurs with a minimal amount of fragmentation).
The DNA sample is prepared by embedding it within an organic matrix and allowing the mixture to co-crystallise on the sample plate. The matrix (normally 3-hydroxypicolinic acid/4-methoxy cinnamic acid) absorbs energy at the wavelength of laser irradiation. The sample plate can be loaded with hundreds of samples to be for a single run. The sample is treated with a short pulse of UV laser light that liberates and ionises the DNA sample whilst the matrix protects it from fragmentation. These ions then travel in a vacuum, along the flight tube to the detector. The time required for an ion to travel the distance from the ion source to the detector ("time of flight") is dependent on the mass of the individual ions. Small ions with greater velocity will reach the detector first.
Although protein sequencing by mass spectrometry has become routine, DNA sequencing by mass spectrometry has lagged behind, mainly because gel-based methods are well-established and reliable. However, DNA sequencing is possible by enzymatic digestion using exonuclease enzymes which generate small oligonucleotide fragments that can be separated and analysed. DNA sequencing by mass spectrometry has potentially numerous advantages over traditional methods; the mass of the fragments is unaffected by secondary structure, labelling is not required and data acquisition is fast. Single nucleotide polymorphisms (SNPs) vary by a single nucleotide and the difference in mass between A and T (9 Da) sets the lower limit for discrimination by mass spectrometry. Genotyping by mass spectrometry can be achieved by direct analysis of a PCR amplicon containing the SNP or by indirect detection via a reaction such as single base extension. Forensic applications rely heavily on the analysis of microsatellites (short, tandem-repeat polymorphisms). This is normally performed using electrophoretic separation of alleles which have different numbers of repeat sequences. The number of repeat units in microsatellites has also been rapidly and precisely determined by MALDI-TOF MS.
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