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DEVICE FOR RAPID DNA EXTRACTION, PURIFICATION, AND QUANTIFICATION

The separation, purification, concentration, quantification, and extraction of charged analytes, such as biomolecules and deoxyribonucleic acid (DNA) from crude samples remains a technical and practical challenge. For example, crude samples may contain environmental contaminants, such as soil, blood, bacteria, particulate material, cell detritus, ionic species, and biomolecule inhibitors, which complicate analysis of the sample. Additionally, conventional apparatus and techniques for analyzing crude samples are labor intensive, time consuming, and require access to a laboratory, skilled technicians, and specialized equipment. Moreover, such apparatus and techniques typically deliver the purified analytes (e.g., DNA) in small fluid volumes, (about 50 μL) which limits further analysis. Further, conventional apparatus and methods for separation of charged analytes from crude samples may require pre-separation and post-separation sample preparation steps (e.g., filtration, centrifugation, and precipitation). Such further sample preparation steps may reduce the quantity of charged analytes delivered from the sample and lower the final concentration of charged analytes. The reduction of both quantity and concentration of delivered charged analytes can negatively impact the likelihood of further post-separation analyses, such as, e.g., in the case of DNA, short tandem repeat (STR) analysis for human identification. Apparatus for separating, purifying, concentrating, quantifying, and extracting charged analytes and methods are desired.

This invention is the combination of the components that allows for extraction, purification, concentration, and quantification of DNA from crude samples, all in one rapid step. The components comprising the device are: A fused silica capillary or microfluidic channel (typically ~ 8 cm long and 75 um i.d.), having two ends, a buffer end and a sample end; a run buffer reservoir connected to one end (the buffer end) of the capillary/microchannel; a fluorescence detector, interfaced with the capillary to detect fluorescent species in the capillary; a contactless conductivity detector, interfaced with the capillary to detect the conductivity at a point along the capillary; a high voltage power supply with a high voltage electrode connected to the fluid in the run buffer reservoir; a pressure controller connected to control the pressure of the head space of the run buffer reservoir; a device for measuring the current through the capillary or microchannel; a computer or other controller to record measurements of the current through the capillary, the voltage applied, the pressure, the fluorescence detector signal and the conductivity detector signal as a function of time and to control the sequence of applied voltage and pressure required to implement the extraction, purification , concentration, and quantification of DNA.

The invention is superior to current practice because it uses a different mechanism (electrophoresis) to discriminate between the DNA that is desired from a sample and other molecules in a crude sample that may be polymerase chain reaction (PCR) inhibiting. It also can be used with crude samples such as soil without pre-filtering the particulates from the sample solution; this is beneficial for field-portable applications. It is also faster than current methods for DNA extraction and purification (5 minutes as compared to 20-60 minutes). Finally, it combines DNA quantification into the extraction/purification process, which reduces the time and complexity of the overall DNA analysis process.

The figure below is a schematic illustration of a system for performing gradient elution isotachophoresis, wherein double headed arrows in between the processor and the storage medium of the controller and in between the isotachophoretic apparatus and the controller represent a communicative coupled relationship.

Abstract: 

Gradient elution isotachophoretic apparatus and systems for performing gradient elution isotachophoresis to separate, purify, concentrate, quantify, and/or extract charged analytes from a sample is described. The isotachophoretic apparatus include an electrophoretic assembly, a sampling assembly connected to the electrophoretic assembly, and/or a support structure connected to the electrophoretic assembly and/or to the sampling assembly. The system includes an isotachophoretic apparatus, and a controller communicatively coupled to the isotachophoretic apparatus. The controller includes a storage medium and a processor for executing computer readable and executable instructions. The combination of the components that allows for extraction, purification, concentration, and quantification of DNA from crude samples are all in one rapid step.

Benefits: 

This invention is the combination of the components that allows for extraction, purification, concentration, and quantification of DNA from crude samples, all in one rapid step

Inventors: 

Alyssa Henry, Christopher Konek, David Ross, Elizabeth Strychalski

Patent Number: 
10067088
Technology Type(s): 
Advance Manufacturing Processes, Analytical Chemistry, Manufacturing, Material Removal Processes, Materials, Materials Physics and Chemistry, Health Care, Biochemical Science, Chemical Sciences, Physical and Chemical Properties, Precision Measurement,
Internal Laboratory Ref #: 
15-022US
Patent Issue Date: 
September 4, 2018
Lab Representatives
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