Development of a high throughput methodology to measure recombination in male meiosis

Recombination is not uniform across the human genome but occurs in small regions of 1-2 kilobases known as recombination hotspots. The position of recombination hotspots is not constant over time and varies between closely related species such as humans and chimpanzees or even between different human populations and among individuals of the same ethnicity. My long term goal is characterizing the inter- and intra- population variation in recombination hotspot position and activity. Studying recombination hotspots is quite relevant since not much is know about what controls the activity of hotspots or how this activity changes over time. We now know that hotspots are very common, but we know very little about the molecular nature of meiotic hotspots. Currently, the techniques that accurately measure recombination in single individuals are very laborious and costly. Therefore, the focus of this proposal is to develop a high throughput methodology that will allow the characterization of many hotspots in a large sample size of individuals to further understand recombination hotspots. A new technology based on the amplification of single molecules on magnetic beads contained in micro- droplets suspended in an emulsion will be used for this purpose. On a broader perspective, adapting this new technology to study recombination hotspots will have also the benefit of perfecting and extending a technology which, given its high throughput, has fostered new approaches to analyzing biological problems. My specific aims are to: 1. Adapt the emulsion-bead technology for measuring recombination events. This assay will allow "counting" crossover events in human male meioses (sperm DNA). Specifically, a reaction where two distinct genetic markers can be simultaneously amplified on a single magnetic bead in the emulsion needs to be developed. 2. Find sensitive and specific assay conditions that allow distinguishing the rare recombinants from the much more numerous non-recombinants. 3. Improve the throughput of the emulsion-bead technology by testing different detection biochemistries, as well as optimizing the optical, mechanic, and fluidic components of the microscope that allows a fast analysis of the arrayed beads.