News Column

Patent Application Titled "Microfluidic Device for Nucleic Acid Extraction and Fractionation" Published Online

July 15, 2014



By a News Reporter-Staff News Editor at Life Science Weekly -- According to news reporting originating from Washington, D.C., by NewsRx journalists, a patent application by the inventors O'Halloran, Jonathan James (Uckfield, GB); Warburton, Elaine Harrington (Purley, GB); Solomon, Matthew Daniel (Hughesdale, VIC, AU); McCormack, John Edward (North Warrandyte, VIC, AU); Schuenemann, Matthias (Brunswick, VIC, AU); Briggs, David James (Lancefield, VIC, AU); Andre, Mindy Lee (Croydon, VIC, AU), filed on November 30, 2011, was made available online on July 3, 2014 (see also Quantumdx Group Limited).

The assignee for this patent application is Quantumdx Group Limited.

Reporters obtained the following quote from the background information supplied by the inventors: "Various embodiments of the present disclosure generally relate to molecular biological protocols, equipment and reagents for the extraction and fractionation of DNA molecules, from whole or lysed samples, in a single flow-through device.

"DNA is a long polymer consisting of units called nucleotides. The DNA polymers are long chains of single units, which together form molecules called nucleic acids. Nucleotides can be one of four subunits (adenine (A), cytosine (C), guanine (G) & thymine (T)) and, when in a polymer, they may carry the genetic information in the cell. DNA comprises two long chains of nucleotides comprising the four different nucleotides bases (e.g. AGTCATCGTAGCT . . . etc) with a backbone of sugars and phosphate groups joined by ester bonds, twisted into a double helix and joined by hydrogen bonds between the complementary nucleotides (A hydrogen bonds to T and C to G in the opposite strand). The sequence of nucleotide bases along the backbone may determine individual hereditary characteristics, or other acquired diseases, such as cancer.

"The central dogma of molecular biology generally describes the normal flow of biological information: DNA can be replicated to DNA, the genetic information in DNA can be 'transcribed' into mRNA, and proteins can be translated from the information in mRNA, in a process called translation, in which protein subunits (amino acids) are brought close enough to bond, in order (as dictated by the sequence of the mRNA & therefore the DNA) by the binding of tRNA (each tRNA carries a specific amino acid dependant on its sequence) to the mRNA.

"To study, or analyze the sequence and biology of DNA or RNA from a sample it is usually necessary to extract, or isolate, the nucleic acids from the rest of the clinical or biological sample (i.e. other cellular components such as lipids, carbohydrates, proteins, etc.). This is presently performed by a number of methods by those familiar with the art. These methods are described briefly below.

"The standard methodology consists of a protocol with different variations depending upon application and sample type, begins with cell disruption or cell lysis, to release the DNA. This is commonly achieved by mechanical lysis (such as grinding, or grinding tissue in liquid nitrogen), sonicating, enymatically or chemically (such as adding a chaotropic salts (e.g. guanidinium thiocyanate) to the sample). The cells lipid membranes and other lipids, are usually removed by adding a detergent and the proteins usually removed by adding a protease (such as Protinase K, optional but almost always done). Water-saturated phenol, chloroform allows for phase separation by centrifugation of a mix of the aqueous sample and a solution, containing resulting in an upper aqueous phase and a lower organic phase (mainly chloroform). Nucleic acid is found in the aqueous phase, while proteins are found in organic phase. In a last step, RNA is recovered from the aqueous phase by precipitation with ice cold 2-propanol or ethanol. DNA will be located in the aqueous phase in the absence of guanidinium thiocyanate. Since DNA is insoluble in these alcohols, it will precipitate and aggregate, giving a pellet upon centrifugation. This step also removes alcohol-soluble salt. Adding a chelating agent to sequester divalent cations such as Mg2+ and Ca2+ prevents dnase enzymes from degrading the DNA. Cellular and histone proteins bound to the DNA can be removed either by adding a protease or by having precipitated the proteins with sodium or ammonium acetate, or extracted them with a phenol-chloroform mixture prior to the DNA-precipitation.

"A second method isolates DNA from a lysate (regardless of what method of lysis is used) by virtue of its ability to bind to silica in the presence of high concentrations of chaotropic salts (Chen and Thomas, 1980; Marko et al. 1982; Boom et al. 1990). The DNA can bind to any silica surface, whether this is pillars with microfluidics cassettes, silica coated paramagnetic beads, a silica filter within a spin column, or other silica surface. The chaotropic salts are then removed with an alcohol-based wash and the DNA eluted in a low-ionic-strength solution such as TE buffer (a buffer consisting of tris hydroxymethylaminomethane ('Tris') and Ethylenediaminetetraacetic acid ('EDTA')) or water. DNA binds to silica because of dehydration and hydrogen bond formation, which competes against weak electrostatic repulsion (Melzak et al. 1996). Hence, a high concentration of salt will help drive DNA adsorption onto silica, and a low concentration will release the DNA. As the DNA is bound to the silica surface the rest of the cellular and other debris is simply washed away with wash buffers prior to eluting the DNA bound to the silica in either H.sub.2O or TE buffer.

"The ChargeSwitch.RTM. (Invitrogen) methodology sees negatively charged DNA (through their negatively charged phosphate backbone) in a lysate bind to a special ligand that acquires a positive charge at low pH values (8.5) and the positive charge is neutralized.

"The Nexttec DNA isolation system, allows purifying DNA with a single centrifugation step within four minutes following cell lysis. It is up to five times faster than currently used DNA isolation systems. This is possible through a proprietary sorbent matrix, which, in a reversal of silica based methods, retains inhibiting substances, such as proteins and low molecular weight substances and lets pass the pure DNA within a lysed sample. One limitation of this method is that it relies on a long enzymatic lysis step, at 60.degree. C.

"The methodologies presently used by those skilled in the art are deployed in tubes, spin-columns or plates and require substantial hands on operator time and multiple steps and thus present a bottle-neck for the analysis of DNA. The use of liquid handling machines and deployment of the technologies presented above in multi-well plates, utilizing vacuums to draw the samples and reaction solutions in to the active matrix for each technology, has been used for higher through put use, however these require batching of samples to make them cost effective and still cause a bottle-neck. For many applications, such as molecular analysis of clinically relevant DNA at the point of care, which is fast becoming recognized as the only methodology to control the emerging drug resistance problems in infectious diseases, especially in developing and third world nations, these methodologies are unsuitable and can not be readily deployed in point of care devices.

"Some methodologies, such as coating micro-pillars or other features and/or structures in microfluidic channels, or paramagnetic beads with silica surfaces have been translated and deployed in microfluidics devices, however the requirement for multiple wash steps and buffers means the fluidic programming and the microfluidic device design itself is complex, thus making the devices expensive."

In addition to obtaining background information on this patent application, NewsRx editors also obtained the inventors' summary information for this patent application: "In one embodiment a device is disclosed for simultaneously extracting and fractionating DNA from a lysate or a whole sample, the device comprising a single flow-through microfluidic channel, the channel comprising buffer and reagent chambers and a sorbent filter.

"The sorbent filter may comprise a support at least partially covered by a polymeric coating comprising polyaniline or derivatives thereof.

"The device may comprise a glass material.

"The device may comprise a PDMS material.

"In an embodiment of the device, the single flow-through microfluidic channel comprises a bulbous structure at an inlet thereof and wherein the bulbous structure tapers to a thinner structure at an outlet thereof.

"In some embodiments, the sorbent filter comprises a series of solid or hollow microstructures fabricated into the walls of the microfluidc channel.

"In some embodiments, the sorbent filter is lose and packed within the microfluidic channel

"In another embodiment, the sorbent filter is a matrix configured to bind cellular and other clinical/biological sample material other than nucleic acids.

"A method is disclosed in accordance with some embodiment of the invention for fabricating a microfluidics device to extract and fractionate DNA from a sample. The method comprises: forming at least two blank layers having a channel; forming an adhesive layer with ports cut through the layer corresponding to an inlet and an outlet port of the channel; vacuum packing a portion of the channel with a sorbent material; and aligning the blank layers with the adhesive layer therebetween and bonding the layers together under pressure.

"A method for simultaneously extracting and fractionating DNA from a lysate or a whole sample is disclosed in another embodiment. The method comprises: providing a device comprising a single flow-through microfluidic channel, the channel comprising buffer and reagent chambers and a sorbent filter; applying the sample to an inlet of the single flow-through microfluidic channel; activating the sorbent filter with a buffer; flowing the sample into the portion of the channel containing the sorbent filter; flowing the sample through the channel and looping it though the portion of the channel containing the sorbent filter from between one and ten times; and flowing the extracted and fractionated DNA out of the device.

"In one variation, the sample is flowed into the portion of the channel containing the sorbent filter and then incubated therein for between 15 seconds and 15 minutes, before being flowed out the device. In another variation, the sample is flowed into the portion of the channel containing the sorbent filter and then oscillated back and forth within the sorbent filter channel, before flowing the sample out of the device.

"In some embodiments, the sorbent filter is activated with a buffer, and lysed sample is flowed into the channel containing the filter and the sample flowed through the channel to the end, resulting in a pure, or near pure DNA solution.

"The resultant eluate is sufficiently pure and concentrated to be detected in an agarose gel and can be used in PCR, RT-PCR, DNA sequencing, hybridization experiments and can be detected in nanobiosensors such as nanopores, carbon nanotubes and nanowire biosensors.

"In some embodiments, the buffer(s) and other reagents are stored off cassette and delivered via the fluidics of an external device.

BRIEF DESCRIPTION OF THE DRAWINGS

"FIG. 1 depicts an exploded schematic view of a microfluidics device for DNA extraction and fractionation.

"FIG. 2 depicts a top view of a microfluidic device.

"FIG. 3 depicts a bottom view of a microfluidic device.

"FIG. 4 illustrates a polycarbonate insert of a microfluidic device.

"FIG. 5 illustrates a polycarbonate shell of a microfluidic device.

"FIG. 6 illustrates a laser-cut double-sided tape layer which may be used to bond the insert (FIG. 4) and shell (FIG. 5) together, thereby forming the microfluidic channels of the microfluidic device.

"FIG. 7 illustrates a shell and insert laminated to cap fluid reservoirs.

"FIG. 8 depicts the filters placed into the cartridge insert at the inlet and outlet of the sorbent packed chamber to ensure that the sorbent material remains in place.

"FIG. 9 shows a double-sided tape layer being applied to the insert to hold the cassette halves (insert and shell) together and to create the microfluidics channels.

"FIG. 10 illustrates a sorbent chamber filled under vacuum.

"FIG. 11 illustrates the top view of an assembled nucleic acid extraction microfluidics device.

"FIG. 12 illustrates the bottom view of an assembled nucleic acid extraction microfluidics device.

"FIG. 13 shows the results from running 80 .mu.l of 11.0 .mu.g/ml salmon sperm through an extraction experiment using the extraction cassette and bench marking it with Nexttec clean column DNA extraction.

"FIG. 14 shows PCR from eluate fractions from lysed human blood passed through the extraction microfluidics device. FIG. 14a shows the mass ladder in lane 1 and eluates 1 through 11 in lanes 2 to 12. FIG. 14b shows a mass ladder in lane 1 with lanes 2-10 containing eluate fractions 13 to 21.

"FIG. 15 illustrates a gel image from a BioAnalyzer analysis of eluate fractions that separate DNA based upon size.

"FIG. 16 shows an alternative embodiment of a DNA extraction and fractionating device. A.

"FIG. 17 is a perspective view showing a point of care device and microfluidics cassette that includes the DNA extraction and fractionation device within the cassette.

"FIG. 18 is a schematic view showing the components of a microfluidics cassette designed for handheld diagnostics in accordance with some embodiments.

"FIG. 19 illustrates a microfluidics cassette design designed for handheld sequencing in some embodiments."

For more information, see this patent application: O'Halloran, Jonathan James; Warburton, Elaine Harrington; Solomon, Matthew Daniel; McCormack, John Edward; Schuenemann, Matthias; Briggs, David James; Andre, Mindy Lee. Microfluidic Device for Nucleic Acid Extraction and Fractionation. Filed November 30, 2011 and posted July 3, 2014. Patent URL: http://appft.uspto.gov/netacgi/nph-Parser?Sect1=PTO2&Sect2=HITOFF&u=%2Fnetahtml%2FPTO%2Fsearch-adv.html&r=2129&p=43&f=G&l=50&d=PG01&S1=20140626.PD.&OS=PD/20140626&RS=PD/20140626

Keywords for this news article include: Genetics, Peptides, Protease, Proteins, Amino Acids, DNA Research, Enzymes and Coenzymes, Quantumdx Group Limited.

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Source: Life Science Weekly


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