The goal of this project was to use 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 defraded DNA fragments can be selected away from the non-target DNA by a capture technique. These enriched fragments can then be converted into small circules that are ideal for serving as templates to be copied many times by rolling circle amplification (RCA). RCA is a robust process for simplifying DNA, but until now could not be used on highly degraded linear DNA fragments. Circularization of degraded DNA changes everything, since a circle essentially is an infinite liner molecule ideal for RCA. The workflow methodology would be to capture target markers, circularize the highly degraded fragments, copy the circles carrying human identity markers, and sequence the amplified products by massively parallel sequencing (MPS). The procedures should also be tested by switching the order, with circularization first and then capture. Another approach is to use 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.
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