MassEXTEND

Figure 3 The MassEXTEND reaction. Following PCR amplification of the locus of interest, a primer-extension reaction is performed using an extend primer (ME) that is designed to anneal next to the SNP. The key feature of this scheme is the use of a terminator mixture that yields extension products that differ in length in an allele-specific manner, thus creating mass separations between alleles equal to the mass of a nucleotide. In this example, a normal dG base is used along with ddA, ddC and ddT. For the A allele (A), a ddA is incorporated, extending the primer to 20 mer. For the G allele (G), the SNP calls for incorporation of the normal dG residue prior to incorporation of a ddT, extending the 19-mer primer to 21-mer.

Figure 4 Example of a spectrum showing MassEXTEND genotyping products at a 15-plex multiplexing level.

Table 1 Comparison of PCR Conditions Changes for High-Level Multiplexing MassEXTEND...

Figure 6 Performance of 12-plexed MassEXTEND reactions under different conditions.

Figure 7 This diagram is a simplified representation of a MALDI-TOF MS spectrum. Relative concentrations of MassEXTEND primers in assay cocktails can be adjusted to even out primer peak heights. The fractions to add are indicated.

A major factor which needs to be taken into account is the cost of a whole genotyping project - not just the cost of individual SNP typing reactions. A major factor in these costs of course is data analysis by highly trained specialists. Furthermore, every new SNP assay needs a considerable time for implementation using standard curves and statistics. These are all certain drawbacks in a high throughput environment. Assay design for MassEXTEND reactions can be done automatically and implementation steps as necessary for fluorescence-based assays are obsolete. 

When compared to the analysis of hybridization events by detecting labels -even on arrays, the DNA MassARRAY approach differs significantly. The MassEXTEND assay is designed to give only the relevant information. The mass spec-trometric approach enables a direct analyte detection with 100% specificity and needs no redundancy. This accuracy and efficacy is combined with sample miniaturization, bioinformatics and chip-based technologies for parallel processing of numerous samples. 

Using a proprietary algorithm, masses as well as signal-to-noise ratios are automatically analyzed and interpreted. After completion of analysis, the results are transferred to a data base and stored as accessible genetic information (see Eig. 6). The database also provides a tool for visual control and comparison of spectra with theoretically expected results. MassEXTEND assays can easily be designed in a high throughput mode with a computer aided assay development module. 

A very promising tool in combination with Mass ARRAY is the analysis of allelic frequencies by means of pooled DNA samples. In this approach a significant number of different genomic DNA samples (e.g. 100 individual DNAs) are combined in equimolar amounts to generate a DNA pool. This pooled population is subjected to PCR amplification and analyzed in a single MassEXTEND reaction. If carefully analyzed, the outcome of such an experiment is information about the... 

Sequenom Information on MassEXTEND MALDI-TOF-based SNP genotyping approaches, fiberoptic bead arrays—http //www.sequenom.com/... 

SNP genotyping by MALDI-TOF MS takes advantage of mass differences between allele-specific primer extension products. At present, three related assays are used, the PROBE— primer oligo base extension assay, which was further developed to the MassExtend assay by SEQUENOM— the PinPoint, and the GOOD assay (8). A representative scheme is depicted in Fig. 1. 

The original PROBE assay has been replaced now by a bead-free, homogeneous assay called the MassExtend (hME) assay. Before the primer extension reaction, shrimp alkaline phosphatase (SAP) is added to the PCR product. This dephosphorylates any residual deoxynucleotides that otherwise would inter-... 

MassEXTEND primers (obtain from oligonucleotide supplier). 

Desalting the MassEXTEND products with CLEAN resin is a crucial step with strong impact on the data quality. It is important that the resin particles stay in suspension during the 5-min incubation step and do not settle. A rotation where plates are turned upside down usually provides best performance. Increased incubation temperature is not recommended. 

Fig. 2. 12-plexed MassEXTEND reaction results. To exemplify the concept, the assay definitions for one of the 12 assays (primer, potential pausing signals, and predicted alleles) are indicated with dotted lines. In the depicted case, the individual is homozygous A. The MassEXTEND primer is fully extended. Each mass signal provides a unique identifier for the presence of an allele in a specific assay. 

Figure 1. Schematic representation of the MassEXTEND reaction for SNP analysis.

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