chapter  12
8 Pages

The GOOD Assay: A Purification-Free Assay for Genotyping by MALDI Mass Spectrometry

Single nucleotide polymorphisms (SNPs) have gained great interest in the last few years. There are now more than 2 million SNPs available in public databases.1,2 Some of the interest in SNPs is founded on the hope that they may help unravel complex genetic traits such as multifactorial disorders through association studies and linkage disequilibrium mapping.3 However, their power to detect responsible genetic variations is still controversial.4 SNPs have several advantages compared to other markers like short tandem repeats (STRs). They are highly abundant, are binary in

nature, can be anchored in intragenic regions, and are relatively easy to analyze. A wide variety of methods using diverse detection devices has been presented for all scales of genotyping studies, each having its advantages and pitfalls (see References 5 and 6, and Chapter 10 by Kalim U. Mir and Jiannis Ragoussis). MALDI mass spectrometry as a detection device has been successfully applied for SNP genotyping using allele-specific hybridization, ligation of allele-specific oligonucleotides, allele-specific cleavage of oligonucleotides, and primer extension to generate allelespecific products.7 In principle, MALDI mass spectrometry is ideally suited for high-throughput genotyping studies due to the rapid and accurate nature of its data accumulation. Mass, a physical property of the product rather than a signal deriving from a tag of the product, is read out. The multichannel detecting capabilities of mass spectrometers allow multiplexing.8 In the beginning, analyzing nucleic acids with mass spectrometers proved to be more difficult than anticipated. Signal intensity is about 100 times lower than for peptides of the same mass. Cations, especially of sodium and potassium, form adducts with DNA resulting in diffuse signals and can only be removed by stringent purification protocols. However, purification contributes largely to the genotyping costs and makes automation more cumbersome. The GOOD assay circumvents purification with the help of a chemical modification strategy.9 Negative charges from the backbone are neutralized and a single positive or negative charge is introduced chemically, rendering the oligonucleotide insensitive to the formation of adducts and thereby increasing the detection sensitivity about 100-fold, equaling that of peptides. This strategy was implemented in the GOOD assay (Figure 12.1).10,11 The regular GOOD assay consists of five steps: (1) a stretch of DNA including he SNPs of interest is amplified by PCR, (2) remaining dNTPs are removed by a Shrimp Alkaline Phosphatase (SAP) digest, (3) the SNPs are queried using a primer extension reaction, (4) extension primers are reduced to the core-sequence by phosphodiesterase II digestion, and (5) charges from the backbone of the remaining extension primer are alkylated using iodomethane. All reaction steps are simple additions of reagents into the same tube or wells of a microtitre plate followed by incubations. Finally, samples are transferred onto the MALDI target without any purification whatsoever.