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Quantifying Random Genomic Mutations in CLL. Anna Freed, Thomas Kipps and Bradley Messmer. Moores Cancer Center, University of California, San Diego, La Jolla, CA, United States. OBJECTIVES. APPROACH-METHODOLOGY.

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PROGRESS

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  1. Quantifying Random Genomic Mutations in CLL Anna Freed, Thomas Kipps and Bradley Messmer. Moores Cancer Center, University of California, San Diego, La Jolla, CA, United States. OBJECTIVES APPROACH-METHODOLOGY • To explain the large heterogeneity in tumor cells, we can propose a mutator phenotype, which occurs due to mutations in the genes that maintain the genetic stability of a normal cell. • A procedure (the random mutation capture method or RMC) to quantify random genomic mutations, which would be indicative of a mutator phenotype, was recently developed. • We will use the RMC in order to see whether there is an increase in mutation rate in CLL. • Genomic DNA is digested with several restriction enzymes (that do not cut within the mutational target, the TaqI recognition sequence). • DNA is hybridized with an excess of probe that contains U’s instead of T’s and a biotinylated nucleotide at the 5’ terminus. • This probe is complementary to the mutational target. • Hybridized target is isolated by magnetic separation after it is complexed with streptavidin superparamagnetic beads. • Mutations within the target are ascertained by digesting with TaqI. TaqI cleaves the wild-type sequence, but will not digest DNA is a mutation has occurred. • Probe is disabled by digestion with UDG • DNA is diluted then amplified by QPCR. Bielas, J.H. & Loeb, L.A. Quantification of random genomic mutations. Nature Methods (2005). PROGRESS Synthesis of Probe Digestion of probe with Taq I and UDG Hybridization of genomic DNA to bead complex Figure 3. A) Gel demonstrating the digestion of the probe with TaqI restriction enzyme. B) Digestion using UDG. Figure 2. QPCR with bead/probe complex hybridized to genomic DNA yielded no products. This step is in progress. Figure 1. Gel showing successful synthesis of probe using biotinylated reverse primer. Probe should be 909 bp. QPCR for detection of mutations B A Blue: Step is in progress Red: Step is successful • Figure 4. A) Successful QPCR standard curve, with digested genomic DNA as template. B) Gel indicating that product is the correct size.

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