The GOOD Assay

A streamlined version of the above-described concept has been implemented as the GOOD assay for SNP analysis [162]. The assay was adapted to cope with the pecuUarities of ohgonucleotide synthesis, enzymatic reactions and alkylation efficiencies. The positive-ion MALDI-MS versions of the GOOD assays will be 

One limitation of the original GOOD assay was the limited availability of modified nucleotides for subsequent coupling of positive charge-tags. However, this issue has been addressed with the introduction of new P-cyanoethyl phospho-ramidites, which allows for easier charge-tagging [163]. 

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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. 

As the third step of the GOOD assay is a primer extension with a specifically tailored set of a-S-dNTPs and a-S-ddNTPs, dNTPs of the PCR have to be removed. This is done enzymatically by degradation of the dNTPs in the second step by shrimp alkaline phosphatase (SAP). 

Figure 2. Flowchart of the GOOD assay. SAP stands for shrimp alkaline phosphatase digestion, PE for primer extension, PDEfor phosphodiesterase. Alkyl for alkylation and MS...

Figure 4. Principle of the GOOD assay. CT synibolises the charge-tag, N" a DNA base and N a quartemary ammonium.

The GOOD assay is a single tube procedure. The five reaction steps are done without transferring the samples to new reaction vials. Reagents are simply added to the reaction and the samples either placed in an incubator or thermocycler. This makes it amenable for automation. 

The main benefit of the GOOD assay is that the introduction of sensitivity enhancing chemistry circumvents the need to purify and concentrate the products prior to MALDI analysis, making it very economical for SNP genotyping. 

The GOOD assay with negative ion-mode detection is easily accessible. The required primers can be obtained from most manufacturers of ohgonucleotides. The (jOOD assay with positive ion-mode detection, on the other hand, gives options for further derivatisations of the product oligomer. 

For the GOOD assay in the positive ion mode version, amino-modified phosphoramidites A , and are integrated into the extension... 

Due to the facile processing of the GOOD assay, it is well suited for automation (Figure 8). All of the reaction steps in the preparation can be done by standard liquid handling robots. 

Figure 8. Reaction sequence scheme of the GOOD assay. The method consists of pipetting, incubation, and thermocycling steps and is therefore easy to automate.

A major drawback in the GOOD assay is the alkylating agent that is used for the chemical backbone neutralisation. Even though methyliodide is used in very low quantities, it is a hazardous reagent. We are currently investigating methods to avoid this toxic agent... 

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