Method of DNA analysis using micro/nanochannel
Inventors
Schwartz, David C. • Jo, Kyubong • Dhingra, Dalia M.
Assignees
Wisconsin Alumni Research Foundation • National Institutes of Health NIH • US Department of Health and Human Services
Publication Number
US-7960105-B2
Publication Date
2011-06-14
Expiration Date
2026-11-29
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Abstract
Methods are provided for tagging, characterizing and sorting double-stranded biomolecules while maintaining the integrity of the biomolecules.
Core Innovation
The invention provides methods for tagging, characterizing, and sorting double-stranded polymeric nucleic acid molecules while maintaining their integrity. It involves introducing a sequence-specific single-strand break (nick) using a nicking endonuclease without cleaving the entire molecule, labeling the nicked sites, elongating the molecule, and analyzing or sorting the labeled nucleic acid molecules in a nanochannel or micro/nanochannel device. This approach allows the molecule to be prepared and chemically manipulated in solution without attachment to a surface, thereby avoiding surface interference and preserving the molecule for subsequent uses.
The background describes problems with existing DNA analysis methods, where DNA is either fixed on surfaces that interfere with chemical reactions or suspended inside nanochannels that are difficult and costly to fabricate, hard to load with DNA and reagents, and difficult to clear of reaction by-products. Nanochannels also often do not generate sufficient tension in DNA for effective detection of enzyme cleavage. The invention addresses these issues by performing biochemical processing, including sequence-specific nicking and labeling, on DNA molecules in solution before their introduction into channels, and by employing buffer conditions to increase DNA stiffness for improved handling in channels that can be larger and easier to fabricate.
The invention also introduces the use of low ionic strength buffers to increase the stiffness (persistence length) of DNA molecules, which facilitates their elongation and proper alignment in channels having a nanometer scale in height but micrometer scale in width. This allows the use of channels that are simpler to manufacture and more practical, including disposable devices. Additionally, the invention enables high-throughput identification and sorting of nucleic acid molecules using characteristic fluorescent barcodes generated by site-specific labeling, followed by capture on addressable charge-based electrodes or sorting microchannels controlled by computer systems to selectively collect molecules of interest for further analysis or use.
Claims Coverage
The patent includes three independent claims covering methods for DNA analysis, sequence structure determination, and sorting of nucleic acid molecules.
Sequence-specific nicking and labeling of intact DNA molecules
Introducing single-strand sequence-specific nicks into double-stranded DNA molecules using nicking endonucleases that maintain the molecule's integrity, followed by labeling the nicked sites with a first label and the rest of the DNA with a second label, thus creating a pattern characteristic of the DNA molecule.
Elongation and channel-based analysis of labeled DNA
Elongating the DNA molecule and passing it into nanochannels or micro/nanochannels that hold the molecule in an elongated state to enable optical detection of the labeled sites revealing the DNA's sequence structure.
Sorting nucleic acid molecules based on labeled pattern recognition
Correlating the pattern of labeled nucleic acid molecules with known sequences, passing molecules from the channel, and capturing them selectively using activated charge-based capture pads or electrodes controlled by a controller system based on user-defined sorting criteria.
The claims cover the inventive features of sequence-specific nicking without cleavage, dual labeling for pattern detection, elongation of DNA in nano/microchannels for analysis, and controlled sorting and capture of nucleic acid molecules based on their fluorescence-labeled sequence-specific patterns.
Stated Advantages
Maintains the integrity of DNA molecules during nicking and labeling, allowing further analysis and use after characterization.
Avoids surface interference by manipulating DNA in solution and suspending DNA in nanochannels without attachment to channel walls.
Increases DNA stiffness by using low ionic strength buffers, enabling elongation and alignment in larger, easier to fabricate channels.
Simplifies channel construction and loading, making disposable channels practical and reducing costs.
Enables high-throughput analysis and sorting of nucleic acid molecules by combining site-specific fluorescent barcoding with addressable capture electrodes controlled by a user interface.
Documented Applications
Tagging, characterizing, and sorting double-stranded polymeric nucleic acid molecules for high-throughput genetic analysis.
Analyzing sequence structure of genomic DNA molecules by fluorescently labeling sequence-specific nick sites and detecting patterns in nanochannel devices.
Sorting molecules of interest based on fluorescent barcodes for subsequent use, including collection in arrays or preparation for nucleotide-level sequencing.
Use of nanochannel and micro/nanochannel devices to elongate and stabilize DNA molecules for optical mapping and further biochemical analysis.
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