The MassEXTEND Assay

The PROBE assay has been further developed into a homogeneous assay (now termed homogeneous MassEXTEND hME) using the same purification principle, whereby all sequential enzymatic steps are performed by a simple addition of the reagents to the reaction well. No washing steps are required, and the ion- 

The issue in high-throughput processing has been addressed in various ways. 

Ivo GuL for example, introduced the concept of charge-tagging and subsequent alkylation of the phosphate backbone as a means of increasing ion-yield and avoiding analyte purification [135]. Early experiments demonstrated improvements of 

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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. 

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. 

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. 

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-... 

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. 

Numerous assays have been developed to detect DNA sequence variations such as SNPs by the MassEXTEND reaction (formerly named PROBE = PRimer OligoBase Extension) [18]. A few selected examples should be discussed here. 

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. 

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