US-12618801-B2 - System and method for presenting large DNA molecules for analysis

US12618801B2US 12618801 B2US12618801 B2US 12618801B2US-12618801-B2

Abstract

Systems and methods for presenting nucleic acid molecules for analysis are provided. The nucleic acid molecules have a central portion that is contained within a nanoslit. The nanoslit contains an ionic buffer. The nucleic acid molecule has a contour length that is greater than a nanoslit length of the nanoslit. An ionic strength of the ionic buffer and electrostatic or hydrodynamic properties of the nanoslit and the nucleic acid molecule combining to provide a summed Debye length that is greater than or equal to the smallest physical dimension of the nanoslit.

Inventors

David C. Schwartz
Kristy Kounovsky-Shafer
Juan Hernandez-Ortiz
Theo Odijk
Juan DePablo
Kyubong Jo

Assignees

WISCONSIN ALUMNI RESEARCH FOUNDATION
UNIVERSITEIT LEIDEN
UNIVERSITY OF CHICAGO

Dates

Publication Date: 20260505
Application Date: 20210503

Claims (18)

1 . A micro-fluidic device comprising: a first microchannel; a second microchannel; a nanoslit extending between the first and second microchannels, the nanoslit providing a fluid path between the first and the second microchannels, the nanoslit having nanoslit electrostatic or hydrodynamic properties; a nucleic acid molecule having a first end portion, a second end portion, and a central portion positioned between the first end portion and the second end portion, the nucleic acid molecule having nucleic acid molecule electrostatic or hydrodynamic properties; and an ionic buffer within the nanoslit and the first and second microchannels, the ionic buffer having an ionic strength and a buffer temperature, producing a Debye length; the first microchannel including a first cluster region adjacent to a first end of the nanoslit and the second microchannel including a second cluster region adjacent to a second end of the nanoslit, the nucleic acid molecule in a dumbbell configuration such that the first cluster region contains the first end portion, the second cluster region contains the second end portion, and the nanoslit contains the central portion, the nucleic acid molecule having a contour length that is greater than a nanoslit length of the nanoslit, and the ionic strength and the buffer temperature of the ionic buffer providing a summed Debye length that is greater than or equal to a nanoslit height or a nanoslit width, wherein the nanoslit height or nanoslit width is the smallest physical dimension of the nanoslit, wherein the summed Debye length, is a function of the ionic strength and the buffer temperature of the ionic buffer, the nanoslit electrostatic or hydrodynamic properties and the nucleic acid molecule electrostatic or hydrodynamic properties, and is four times the Debye length at the given ionic strength and the buffer temperature, wherein central portion of the nucleic acid molecule is fully stretched within the nanoslit by virtue of the summed Debye length being greater than or equal to the nanoslit height or the nanoslit width, the nucleic acid molecule occupying the dumbbell configuration, the nanoslit electrostatic or hydrodynamic properties, and the nucleic acid molecule electrostatic or hydrodynamic properties, wherein the ionic strength is between 0.11 mM to 1.0 mM, wherein the nanoslit width is between 200 nm and 10 μm, and wherein the nanoslit length is less than or equal to half a contour length of the nucleic acid molecule.
2 . The micro-fluidic device of claim 1 , the nanoslit height is less than or equal to 100 nm.
3 . The micro-fluidic device of claim 1 , wherein at least one of the microchannels has one or more of the following: a microchannel width of about 20 um, a microchannel length of about 10 mm, and a microchannel height of about 1.66 μm.
4 . The micro-fluidic device of claim 1 , the device further comprising a temperature adjustment module.
5 . The micro-fluidic device of claim 1 , wherein the buffer temperature is less than or equal to 20° C.
6 . The micro-fluidic device of claim 1 , wherein the ionic buffer further comprises a viscosity modifier.
7 . The micro-fluidic device of claim 1 , wherein the nucleic acid molecule has a relaxation time of at least about 30 seconds.
8 . The micro-fluidic device of claim 1 , wherein the nucleic acid molecule is a DNA molecule.
9 . A method of stretching a nucleic acid molecule in an ionic buffer, the method comprising: positioning the nucleic acid molecule in a dumbbell configuration such that a central portion of the nucleic acid molecule occupies a nanoslit, a first end portion of the nucleic acid molecule occupies a first cluster region adjacent to a first end of the nanoslit, and a second end portion of the nucleic acid molecule occupies a second cluster region adjacent to a second end of the nanoslit, the nanoslit, the first cluster region, and the second cluster region including the ionic buffer, the nucleic acid molecule having a contour length that is greater than a nanoslit length of the nanoslit, and the ionic buffer having an ionic strength and a buffer temperature, producing a Debye length, the nanoslit having nanoslit electrostatic or hydrodynamic properties and the nucleic acid molecule having nucleic acid molecule electrostatic or hydrodynamic properties, the ionic strength and the buffer temperature providing a summed Debye length that is greater than or equal to a nanoslit height or a nanoslit width, wherein the nanoslit height or nanoslit width is the smallest physical dimension of the nanoslit, wherein the summed Debye length, is a function of the ionic strength and the buffer temperature of the ionic buffer, the nanoslit electrostatic or hydrodynamic properties and the nucleic acid molecule electrostatic or hydrodynamic properties, and is four times the Debye length at the given ionic strength and the buffer temperature, wherein the nucleic acid molecule is fully stretched within the nanoslit by virtue of the summed Debye length being greater than or equal to the nanoslit height or the nanoslit width, the molecule occupying the dumbbell configuration, the nanoslit electrostatic or hydrodynamic properties, and the molecule electrostatic or hydrodynamic properties, wherein the ionic strength is between 0.11 mM to 1.0 mM, and wherein the nanoslit width is between 200 nm and 10 μm, and wherein the nanoslit length is less than or equal to half a contour length of the nucleic acid molecule.
10 . The method of claim 9 , wherein positioning the nucleic acid molecule includes threading the nucleic acid molecule through the nanoslit.
11 . The method of claim 9 , wherein positioning the nucleic acid molecule includes electrokinetically driving the central portion of the nucleic acid molecule into the nanoslit.
12 . The method of claim 9 , wherein the nanoslit height is less than or equal to 100 nm.
13 . The method of claim 9 , wherein at least one of the microchannels has one or more of the following: a microchannel width of about 20 μm, a microchannel length of about 10 mm, and a microchannel height of about 1.66 μm.
14 . The method of claim 9 , wherein the buffer temperature is less than or equal to 20° C.
15 . The method of claim 9 , wherein the ionic buffer further comprises a viscosity modifier.
16 . The method of claim 9 , wherein the nucleic acid molecule has a relaxation time of at least about 30 seconds.
17 . The method of claim 9 , wherein the nucleic acid molecule is a DNA molecule.
18 . The method of claim 9 , the method further comprising imaging at least a portion of the central portion.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS This application is a continuation of U.S. application Ser. No. 14/485,119, filed Sep. 12, 2014, which application claims the benefit of U.S. Provisional Patent Application Ser. No. 61/877,570 filed on Sep. 13, 2013, the disclosure of which is incorporated by reference herein as if set forth in its entirety. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH This invention was made with government support under HG000225 awarded by the National Institutes of Health and 0832760 awarded by the National Science Foundation. The government has certain rights in the invention. BACKGROUND OF THE INVENTION The field of the invention is nucleic acid molecule manipulation. More particularly, the invention relates to stretching nucleic acid molecules in order to better present portions of the nucleic acid molecules for inspection by various techniques. Much of the human genome is comprised of DNA sequences that are present in multiple copies. Although such elements play an important role in biological regulation and evolution, their presence troubles current DNA sequencing approaches. Accordingly, serious issues arise when trying to complete the sequencing of human, or cancer genomes because short analyte molecules, currently used by major sequencing platforms, often present redundant sequence data. Like trying to assemble a jigsaw puzzle with pieces bearing no uniquely discernible features, such sequence data make it difficult to assemble the sequence of an entire genome. Furthermore, our ability to assess genomic alterations within populations as mutations, or polymorphisms is also limited. To meet this challenge, genomewide analysis1-3 systems are now featuring modalities that present large, genomic DNA analytes3,4 for revealing genomic alterations through bioinformatic pipelines. Achieving utility for genome analysis using nanoconfinement approaches requires integration of system components that are synergistically poised for dealing with large data sets. Such components include sample preparation, molecular labeling, presentation of confined DNA molecules, and detection, complemented by algorithms incorporating statistical considerations of experimental error processes for data analysis.5-8 While a number of approaches to confine DNA molecules have been examined and implemented in the past few years,5,19-15 few elongate DNA molecules close to their contour length. Kim et al.,9 for example, elongated λ-DNA within poly(dimethylsiloxane) (PDMS) replicated nanochannels (250 nm×400 nm) and achieved a stretch of 0.88 using ultralow ionic strength conditions (0.06 mM). To our knowledge, it was the longest stretch reported for DNA molecules within nanochannels, using low ionic strength buffers. In different work, Reisner et al.11 used 50 nm fused silica nanochannels with higher ionic strength conditions (˜5 mM) to elongate DNA molecules up to 0.83. Although the stretch with these two approaches was higher than 0.8, both techniques exhibit limitations. The approach of Reisner et al. is demanding in that it requires fabrication of extreme nanoconfinement devices, smaller than the molecular persistence length,16 to elongate DNA molecules close to the molecular contour length, thereby increasing the complexity of the molecular loading process. Accordingly, a need exists for an approach to stretching a nucleic acid molecule that overcomes the aforementioned drawbacks. SUMMARY OF THE INVENTION The present invention overcomes the aforementioned drawbacks by providing a microfluidic device and a method of stretching a nucleic acid molecule. In accordance with the present disclosure, the micro-fluidic device can include a first microchannel, a second microchannel, a nanoslit, a nucleic acid molecule, and an ionic buffer. The nanoslit can extend between the first and second microchannels. The nanoslit can provide a fluid path between the first and second microchannels. The nucleic acid molecule can include a first end portion, a second end portion, and a central portion positioned between the first end portion and the second end portion. The ionic buffer can be within the nanoslit and the first and second microchannel. The first microchannel can include a first cluster region adjacent to a first end of the nanoslit and the second microchannel can include a second cluster region adjacent to the second end of the nanoslit. The first cluster region can contain the first end portion. The second cluster region can contain the second end portion. The nanoslit can contain the central portion. The nucleic acid molecule can have a contour length that is greater than a nanoslit length of the nanoslit. An ionic strength of the ionic buffer and electrostatic or hydrodynamic properties of the nanoslit and the nucleic acid molecule can combine to provide a summed Debye length that is greater than or equal to a nanoslit height or a nanoslit width. The nanoslit height or nanoslit width can be the smallest phys