Mass Spectrometry

SNP genotyping by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) takes advantage of mass differences between allele-specific primer extension products (Fig. 3). At present, three related assays are used, the PROBE

variant c t

dNTP complementary to wild type ptus mix of all other ddNTPs plus polymerase d=>

Laser excitation of extension products time of flight recording

Figure 3 Primer extension and MALDI-TOF mass spectrometry. Abbreviation: MALDI-TOF, matrix-assisted laser desorption/ionization time-of-flight.

Laser excitation of extension products time of flight recording

Spotted on matrix covered chips

Figure 3 Primer extension and MALDI-TOF mass spectrometry. Abbreviation: MALDI-TOF, matrix-assisted laser desorption/ionization time-of-flight.

(primer oligobase extension assay further developed to the MassEXTEND® assay by Sequenom), the PinPoint, and the GOOD assay.

The PROBE assay involves annealing of a primer to previously generated PCR amplicon immediately up- or downstream of a SNP position. Prior to the primer extension reaction shrimp alkaline phosphatase (SAP) is added to the samples, which dephosphory-lates any residual nucleotides, because the extension may be hampered by PCR components. The heat-labile SAP is then easily inactivated. A reaction cocktail containing the extension primer, a mix of dNTPs and ddNTPS, along with a thermostable DNA poly-merase is added to the template. DNA polymerase adds the available nucleotides, which results in linear amplification of the extension primer. The reaction terminates if a single dideoxynucleotide is incorporated. The size of allele-specific extension products generated can then be used to identify the possible variants. After purification of the primer extension product, only a few nanoliters of products are transferred onto a matrix-loaded chip with a pintool spotting device. Chips can carry either 96 or 384 samples that are analyzed by high-resolution TOF MS. As the preparations are very small, there is little to no sample preparation heterogeneity.

The major strengths of mass spectrometric analysis are the accuracy of detection, automatic data accumulation, and HT capacity.

Recent commercially available fully automated systems allow up to 20,000 analytical reactions during 12 hours. Despite high initial set-up costs for instrumentation, the effort required for the assay development is low. Therefore, MS appears to be particularly suitable for analysis of a large number of markers. Apart from the genotyping capacity, MALDI-TOF MS provides the possibilities of multiplexing and quantification of allele frequencies in DNA.

A disadvantage of MALDI-TOF is the need for rigorous purification of extension products due to the higher sensitivity of the analysis to impurities than for other genotyping techniques. To circumvent this problem several strategies were established. The first available protocol (PROBE assay) included magnetic bead purification with a biotin-/ streptavidin-binding system. The PinPoint assay uses reversed-phase tip purification, whereas the GOOD assay does not require purification due to the introduction of thiol groups into the 3'-region of the primer. For the MassExtend assay, a bead-free, homogeneous version of the MassExtend assay has now been developed, which allows a singletube procedure with resin purification that can be used in automated sample preparation.

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