LATE-PCR and Its Allied Technologies maximum Information from a Single Tube Application to Mitochondria

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SELECTBIO

Lawrence Wangh, Professor, Brandeis University

Abstract
Novel diagnostic technologies developed at Brandeis University, including LATE-PCR, ThermalightTm Probes, and PrimeSafeTm have been used to construct a closed-tube triplex assay for simultaneous analysis of sequence variation in mitochondrial genes in cultured cells, before and after selection for hypoxia resistance. Each gene target is several hundred nucleotides long and displays a characteristic “fluorescent signature” in one color. This signature undergoes distinguishable changes in response to each nucleotide alteration in the target sequence. For this reason we call this approach Virtual Sequencing. Virtual sequencing is first used to analyze all of the mitochondria from many cells to establish the fluorescent signatures of the complex population. Next, the fluorescent signatures of individual cells are analyzed to establish whether there are high-frequency mitochondrial DNA mutations in cells before and after selection. Finally, individual mitochondrial molecules within single cell are analyzed to reveal the extent of heteroplasmy in a single cell. This is the first thorough, cost effective approach to analyzing mitochondrial genome heteroplasmy. It is suitable for analysis of mitochondrial genome changes due to pharmaceuticals, chemicals, high levels of glucose, aging and many other factors thought to damage or alter mitochondrial genes over time.