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Abstract
Grant Number: 1R43GM069068-01 PI Name: EGHOLM, MICHAEL PI Email: michaele@molecularstaging.com PI Title: Project Title: Isothermal cDNA/mRNA Amplification Technology Abstract: DESCRIPTION (provided by applicant): High throughput mRNA expression profiling studies are useful to evaluate changes in global gene expression under different physiological environments, e.g. diseased vs. normal tissue, drug-treated vs. untreated tissues, carcinogen/toxin-exposed vs. unexposed tissues. Often small quantity of total RNA samples, such as those obtained from cells acquired by aspiration needle biopsies, are available for these studies. Thus, high throughput expression-profiling studies need to be carried out using amplified cDNA samples. Current cDNA amplification methods are based on RT-PCR technology and suffer from the same defects of general PCR: e.g. successive high temperature denaturing steps leading to template damage; use of low fidelity, low processivity Taq DNA polymerase; primer-dimer amplification. Molecular Staging Inc. (MSI) is a life science company developing a portfolio of advanced nucleic acid amplification technologies. MSI's DNA amplification technology, Multiple Displacement Amplification (MDA), being marketed as Repli-g, amplifies whole genomic DNA under isothermal conditions. Our proposed Isothermal Total Transcript Amplification (ITTA) technology will also utilizes key components of MDA. 1st strand cDNA is performed using a 5'-phosphorylated primer. Single-stranded (ss) cDNA molecules are then ligated using a universal bridge oligonucleotide. The 3'-end of the bridge oligonucleotide contains six random nucleotides and hybridizes to the 3'-end of the cDNAs whereas the 5'-end is complementary to the cDNA primer sequences. Ligated cDNA molecules are amplified using 1229 DNA polymerase and exonuclease-resistant hexamer primers to give long multimeric double-stranded (ds) DNA products. Multimer amplified products are converted to unit length amplified cDNA molecules upon digestion with Not I or other rare cutter restriction endonuclease, whose site is incorporated in 1st strand cDNA primer, for expression profiling or other applications. The objective of the Phase-1 segment of this project is to establish scientific and technical feasibility of this cDNA/mRNA amplification technology, referred to as ITTA (patent pending). Specifically, we propose to: 1. Establish and optimize protocols for various steps of ITTA procedure to obtain efficient amplification of all transcripts. We will also determine the sensitivity of ITTA procedure regarding minimum RNA input, maximum fold amplification, and size-dependency on transcript amplification. 2. Demonstrate non-biased amplification of at least 40 different transcripts from more than two RNA samples using Taqman quantitative-PCR technology. 3. Document all assay protocols as standard operating procedures (SOP) for future use. The successful development of ITTA technology will transform global amplification of mRNAs/cDNAs. Isothermal amplification will provide a cost effective and simple technology that will not require expensive equipment or reagents. Given the fact that whole genome amplification using our isothermal amplification technology generates insignificant amplification bias compare to PCR based technologies (as determined by allele representation studies using amplified gDNA), we anticipate that isothermal cDNA amplification technology will also produce little amplification bias of the RNA population.
Thesaurus Terms:
nucleic acid amplification technique, technology /technique development
complementary DNA, genetic transcription, messenger RNA, thermostability
polymerase chain reaction
Institution: MOLECULAR STAGING, INC. 300 GEORGE ST, 7TH FL NEW HAVEN, CT 06511 Fiscal Year: 2003 Department: Project Start: 01-JUL-2003 Project End: 31-DEC-2004 ICD: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES IRG: ZRG1