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High Throughput Determination of Gene Expression Levels Utilizing Mass Spectrometry and Solid-Phase Captureable Dideoxynucleotides

Daniel S. Duffield1, Ashish Misra1, Li Cai2, and Sobin Kim2. (1) Chemical and Biochemical Engineering, Rutgers University, 98 Brett Rd, Piscataway, NJ 08854, (2) Biomedical Engineering, Rutgers University, 98 Brett Rd, Piscataway, NJ 08854

Here we present a rapid, precise and cost-effective method for determining gene expression levels. Current tools for expression analysis include DNA microarrays, Serial Analysis of Gene Expression (SAGE), and RT-PCR based methods. These offer efficient analysis of gene expression with a level of accuracy and throughput. However, there are notable limitations in these methods, such as long and laborious procedures (microarray and SAGE), and low throughput (RT-PCR). Thus new approaches that will allow more accurate and cost-effective analysis with multiplexing capability are highly desirable. To this end, we have developed a new method utilizing MALDI-TOF MS (matrix assisted laser desorption/ionization time-of-flight mass spectrometry) and biotinylated dideoxynucleotide triphosphate, the solid phase capturable terminators.

MALDI-TOF MS has been extensively studied for DNA analysis, i.e., DNA sequencing, SNP genotyping, and allele frequency determination, since it is accurate, suitable for quantification and easy for automation. Previously SPC-SBE, a multiplex genotyping method using solid phase capturable (SPC) dideoxynucleotides (ddNTPs) and single base extension (SBE) reaction, has been developed for MALDI-TOF based genetic variation detection. The method involves generating DNA fragments extended by a single base corresponding to the SNP of interest with SPC-ddNTPs such as biotinylated ddNTPs (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 are then analyzed with MALDI-TOF MS, allowing accurate DNA analysis for MALDI-TOF MS. The presented method further explores the use of biotin-ddNTPs and MALDI-TOF MS for gene expression analysis. Briefly, cDNAs are reversely transcribed from mRNAs isolated from breast tissue (normal and cancerous) and the cDNAs of interest and a known amount of internal standard (internal standard 1, IS-1) are PCR-amplified. A library of primers, one for each cDNA amplicon, are extended using biotin-ddNTPs with another internal standard (IS-2). Extension products are isolated by SPC on streptavidin-surface and analyzed with MALDI-TOF. Ratios between mass spectral peak areas are used to determine the relative quantity of each transcript. The system can enhance the accuracy in quantitative analysis of RNA samples, thus the level of gene expression. Additionally, our approach will increase the number of gene transcripts that can be analyzed simultaneously.

We have demonstrated the method in the analysis of gene expression in the mouse retinal tissue. Expression levels of five genes including, Rho, Lhx2, Hprt1, Sfrp2, and Nrl were determined utilitizing the method. The results strongly indicate that the method can provide fast processing time, great accuracy, and can be expanded to include high multiplexing capability.

As the proposed system utilizes biotin-ddNTPs and affinity capture of biotinylated DNA fragments, rapid capture, efficient washing and simple recovery of extension fragments is indispensable to further enhance the throughput provided by SPC in our gene expression analysis approach. Thus, we are constructing a novel microbead tip device for DNA isolation, which utilizes microbeads coated with biotin-binding proteins and a pipette tip. Together with the microbead tip system, our method will make a highly effective tool for the analysis of gene expression level changes.