Typing Highly Degraded DNA Using Circularized Molecules and Target Enrichment

As submitted by the proposer: Due to the expansion of biological evidence presented to forensic laboratories, DNA samples analyzed today can be highly degraded, limited in quantity and/or present as complex mixtures. Although there is substantial evidence that falls into this category of challenged samples, there are few if any options for the analyst to extract genetic information from such evidence. Yet the demands to rely on DNA analyses are increasing. With complex mixtures, a portion of profiles are being deemed inconclusive or have low statistical strength. There is a need to develop systems that can analyze severely compromised samples and if possible make mixtures less complicated. This proposal seeks to employ molecular biology tools and novel procedures associated with the advances of massively parallel sequencing (MPS) to enable analysis of degraded DNA. The concept is that genetic markers within highly degraded DNA fragments can be selected away from non-target DNA by a capture technique. These enriched fragments then can be converted into small circles that are ideal for serving as templates to be copied many times by rolling circle amplification (RCA). RCA is a highly robust process for amplifying DNA, but until now could not be used on highly degraded linear DNA fragments. Circularization of degraded DNA changes everything as a circle essentially is an infinite linear molecule ideal for RCA. The methodology workflow would be capture target markers, circularize the highly degraded fragments, copy the circles carrying human identity markers, and sequence the amplified products by MPS. The procedure also would be tested by switching the order with circularization first and then capture. Another approach is use of molecular inversion probes (MIP) that are highly specific for target markers and form circles if they bind to the target marker template. Contained within the MIP are universal PCR primers that provide standard and robust PCR amplification. These two methods should enable analysis of highly degraded DNA. The MIP method holds promise of being highly specific and sensitive. The circle/RCA method holds promise for sensitivity of detection but more importantly may preferentially detect degraded (minor contributors) in complex mixtures, a capability not yet possible by any other means. These methods will enable analyses of the most challenging samples, enhancing chances of successful typing of touch DNA, cold case evidence, low level male components from rape cases, and human remains from mass disasters, missing persons cases, and acts of terrorism. Note: This project contains a research and/or development component, as defined in applicable law. ca/ncf