NCJ Number
242180
Date Published
October 2011
Length
45 pages
Annotation
This study developed two microfluidic components for the analysis of low copy number (LCN) samples: a post-PCR clean-up component and an optimized electrophoretic injection component.
Abstract
Microfluidic biochip clean-up produced a six- to eight-fold increase in signal strength compared with untreated samples. This improvement enabled additional alleles to be called from LCN samples. In addition, these results indicated that NetBio's DNA purification, thermal cycling, and separation and detection modules are effective with LCN templates and that the microfluidic UF process is compatible with these modules. For the PCR clean-up methodologies, two filter membrane materials over a range of filter pore sizes were evaluated, and single sample biochips for microfluidic clean-up were designed and fabricated. The optimized injection protocol developed demonstrated its potential for analyzing LCN samples; optimal injection conditions showed that it can detect PCR samples amplified with 0.006 ng of DNA template (a single human genome equivalent) and of greater than 10 ng of DNA template without saturating. Optimized injection protocols (the optimal amount of cleaned PCR sample for separation and detection and the optimal electrophoretic conditions) were developed, and experiments demonstrated that injection efficiency increased as the amount of purified PCR product applied to microfluidic separation and detection was increased. An increase in injection efficiency of two- to three-fold was achieved by allowing the entire PCR product to be loaded for microfluidic separation and detection. An injection time of 90 seconds and an injection voltage of 175 V were optimal. Testing involved the analysis of LCN blood, saliva, and buccal cell samples (0.025 ng of DNA template). The availability of an STR typing instrument that combines DNA extraction and purification, amplification, and separation into a single instrument would be a substantial advance in forensic DNA analysis. 22 figures
Date Published: October 1, 2011