Scientific Presentations Summer 2009 - Dana-Farber/Harvard ...

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Massively Parallel Amplification of cDNAs Ugo Nduaguba Mentors: David E. Hill, PhD and Marc Vidal, PhD Scientific Advisors: Lila Ghamsari, Elizabeth Mello, Kourosh Salehi-Ashtiani Dana-Farber Cancer Institute Primer, cloning, and sequencing costs are major obstacles in carrying out genome wide amplification and cloning of an organism’s protein coding regions. Although the advent of “next generation” sequencing technology platforms has led to decreased cost and increased throughput of DNA sequencing, the power of these methodologies cannot be fully utilized due to the lack of PCR technologies that match the throughput at which these new sequencing platforms operate cost effectively. In situ microarray synthesis of primers provides an avenue for inexpensive synthesis of primers. However, the use of such array-synthesized primers in PCR, typically generated as a complex mixture of several thousand primers, remains problematic. One of the challenges in carrying out such complex amplification reactions is that because primers are used in pools, the concentration with respect to each specific primer pair can be considerably lower than what they would be in a conventional PCR, altering the primer hybridization kinetics significantly. In order to develop a PCR protocol capable of generating hundreds of amplicons simultaneously, we have used a set of 500 primer pairs, targeting metabolic protein coding regions of Chlamydomonas reinhardtii, to determine the required amplification parameters. To compensate for the lower primer concentration, we increase the primer pool concentration by tenfold and extend the annealing time by ~15 to 30 fold (~1,000 - 2,000 seconds) for each PCR cycle. Using these modified parameters along with reverse transcribed Chlamydomonas RNA as template; we obtain gene-specific amplification products in reactions containing the 500 primer pairs. Cloning and sequencing of the obtained products verifies correct amplification of the intended targets.
Identifying Potential HLA-A*0201-restricted, Immunogenic Epitopes from Myosin VI Osamede Obanor Mentor: Simo Arredouani, PhD Scientific Advisors: Wen Yue, MD; Bin Lu, PhD; and Martin G. Sanda, MD Beth Israel Deaconess Medical Center Background: Myosin VI previously found to be overexpressed in prostate tumors and play a role in tumorigenesis, could potentially be a target for immunotherapy geared towards prostate cancers. Hypothesis: By analyzing and testing Myosin VI’s HLA-A2.1 epitopes, novel epitopes will be identified that can elicit a cytotoxic lymphocyte immune response to prostate tumors. Preliminary data: Comprehensive in-silico analysis was conducted that identified 57 novel, human prostate cancer-restricted, tumor-associated antigens (TAA). Fifteen of these TAA, including Myosin VI, were ascertained as showing prostate cancer-associated expression by Q-RTPCR of human specimens. Aims & Methods: in-silico analysis using multiple prediction algorithms was performed in order to identify novel peptides of Myosin VI that bind to human HLA-A2.1 receptors. Potential binders were then run through an in vitro HLA-A2.1 stabilization assay to determine whether they bind to the HLA-A2.1 receptor. HLA-A2.1 transgenic (HHD) mice were injected with the 5 peptides that showed significant binding, along with a constant helper peptide to test for their immunogenicity. Spleens were harvested from the immunized mice 12 days later; splenocytes were prepared and subjected to an ELISPOT testing that determined if an immune response was elicited. Results: 10 peptides from Myosin VI were chosen to go through HLA-A2.1 stabilization assay, and 5 of the 10 showed significant binding to HLA-A2.1 receptors. When the 5 peptides were injected in transgenic mice, 2 showed the ability to elicit an immune response. Conclusions: 2 Myosin VI peptides were able to bind to HLA-A2.1 receptors and elicit an immune response, making them potential targets for immunotherapy.
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