(25e) High Throughput Genomic Analysis Using Maldi-Tof MS and Solid-Phase Capturable Dideoxynucleotides

We report here a high-throughput method for rapid, accurate and cost-effective genomic analysis. The method is based on SPC-SBE, an appoach utilizing solid phase capturable dideoxynucleotides and the matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) [Kim et al., Nucleic Acids Res., 2002]. We demonstrate the method allows for the efficient analysis of single nucleotide polymorphisms (SNPs) and gene expression levels.

SNPs, the most commonly occurring genetic variation in humans, are important genetic markers. For instance, SNPs from the human cytochrome P450 (CYP450) family of genes are responsible for inter-individual differences in metabolism of a large number of drugs and other xenobiotics including procarcinogens and hence play a key role in developing personalized medications. CYP450 SNPs Studies on CYP450 SNPs can be limited by the capacity and accuracy of the genotyping method, as a large number of SNPs have been reported in this family of genes. Previously, simultaneous genotyping of thirty mutations of the p53 gene has been reported, using the SPC-SBE approach [Kim et al., Genomics, 2004]. However analysis of thirty or more SNPs at a time has not been shown.

In this study, we show multiplex genotyping of fifty SNPs of the CYP2A13 gene. Briefly, a library of fifty primers are designed to have unique masses for each SNP anneal next to SNP locations is extended by one base at SNP sites with biotin-ddNTPs. The DNA extension fragments containing biotin moiety at their 3' end are readily isolated from reaction mixture by utilizing a streptavidin-coated surface. The isolated DNA molecules, free from salts, primers and other reaction components are then analyzed with MALDI-TOF MS. In this approach, one can remove irrelevant DNA fragments that can potentially interfere with the detection of target molecules (the extension products) in MS analysis, and therefore can increase the number of extension products that are analyzed simultaneously. The fifty-fold genotyping as shown in this study is the highest level of multiplexing by MALDI-TOF MS. Results of our experiments indicate that SPC-SBE is a robust, high throughput genotyping method, allowing rapid and accurate analysis of CYP450 SNPs.

Furthermore, we show our genotyping method can be effectively modified for genomic analysis of gene expression levels, adapting the real-competitive PCR (rcPCR) approach [Ding and Cantor, PNAS, 2003]. Gene expression patterns in complicated diseases, such as cancers, are believed to profile the activity of related gene functions, and can be used to illustrate the physiological characteristics of the disease at the molecular level. Therefore, efficient measurement of gene expression levels is critical for the understanding of disease development and for the diagnosis of the disease.

In our approach, cDNA molecules are first reversely transcribed from the sample mRNAs of interest and PCR-amplified with an internal standard that contains an artificial SNP. Then the PCR products of gene transcripts and internal standards serve as a template in SBE reaction where a library of primers, one for each cDNA amplicon, is extended using biotin-ddNTPs, solid phase capturable dideoxynucleotides. Extension products are then isolated by solid phase capture on streptavidin-coated surface and subsequently analyzed with MALDI-TOF MS. Ratios of mass spectral peak areas are used to determine the relative quantity of gene transcripts. Our method provides higher accuracy in quantitative analysis of DNA samples (thus the level of gene expression) due to the facilitated isolation of DNA fragments by the solid phase capture. Additionally, the approach increases the number of gene transcripts that can be analyzed simultaneously since it allows for the removal of irrelevant DNA fragments that may overlap with the extension products in the mass spectrum and prevent the accurate measurement of sample products. We verify our system with the analysis of gene expression patterns in breast cancer cells.

In summary, we present a method for genomic analysis, which employs solid phase capturable dideoxynucleotides and MALDI-TOF MS. Our approach enhances the accuracy and speed of MS-based genomic analysis, and therefore provides high throughput genomic analysis of genetic variations (such as SNPs) and gene transcripts.