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Patent Issued for Microfluidic Device-Based Nucleic Acid Purification Method

May 28, 2014



By a News Reporter-Staff News Editor at Journal of Engineering -- Rheonix, Inc. (Ithaca, NY) has been issued patent number 8722329, according to news reporting originating out of Alexandria, Virginia, by VerticalNews editors.

The patent's inventors are Young, Lincoln C. (Ithaca, NY); Zhou, Peng (Newtown, PA); Spizz, Gwendolyn (Ithaca, NY); Yasmin, Rubina (Ithaca, NY).

This patent was filed on February 21, 2012 and was published online on May 13, 2014.

From the background information supplied by the inventors, news correspondents obtained the following quote: "Embodiments of the invention are directed generally to methods for preparing, purifying, amplifying, and detecting biological molecules of interest such as nucleic acids and, more particularly, to such methods performed using microfluidic devices.

"The use of molecular diagnostics has expanded greatly since its inception in the early 1980s, particularly as a means to permit the detection of slow growing or fastidious bacteria responsible for infectious diseases. The detection of viral pathogens, including viral load testing has also been significantly improved by molecular diagnostics. As more data have become available regarding the human genome, the use of molecular diagnostics in pharmacogenomics, companion diagnostics, and other personalized medicine applications continues to gain momentum.

"Nucleic acid amplification is a standard technique known in the art by which nucleic acids may be isolated and more accurately and efficiently manipulated for use in molecular diagnostics and other nucleic acid screening purposes. Various methods have been described in the literature for amplifying nucleic acids, such as deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), present in a sample. Among these, the most widely practiced is the polymerase chain reaction (PCR), described in U.S. Pat. No. 4,683,195 (Mullis et al., entitled 'Process for amplifying, detecting, and/or cloning nucleic acid sequences,' issued Jul. 28, 1987) and U.S. Pat. No. 4,683,202 (Mullis, entitled 'Process for amplifying nucleic acid sequences,' issued Jul. 28, 1987). Briefly, PCR consists of amplifying denatured, complementary strands of target nucleic acid by annealing each strand to a short oligonucleotide primer, wherein the primers are chosen so as to flank the sequence of interest. The primers are then mediated by a polymerase enzyme to yield extension products that are themselves complementary to the primers and hence serve as templates for synthesis of additional copies of the target sequence. Each successive cycle of denaturation, primer annealing, and primer extension essentially doubles the amount of target synthesized in the previous cycle, resulting in exponential accumulation of the target.

"PCR methodologies in general suffer from several limitations that are well-known in the art. One such limitation is owing to the poor fidelity of commonly used, thermostable polymerase enzymes, such as Taq. This results in nucleotide base misincorporations that are propagated from one cycle to the next. It is estimated that such misincorporations may occur as often as once per one thousand bases of incorporation. A second limitation is that different cDNAs are amplified with different efficiencies, resulting in underrepresentation of some cDNA sequences and overrepresentation of others in the amplified product. Even a small difference in efficiency may result in a several-thousand fold differential in the representation of these cDNAs in the product after only as few as 30 cycles of amplification.

"Another limitation is the presence of inhibitors of PCR in the starting material (e.g., hemoglobin in blood). These inhibitors are often carried through purification and either limit or completely impede amplification reactions performed with the nucleic acids derived from the purification. Therefore this provides another rationale for needing better upstream purification strategies (see e.g., J. Bessetti, Profiles in DNA, PCR Inhibition, An Introduction to PCR Inhibitors, Promega Corporation, March 2007).

"Also significant is that nucleic acid amplification is only as accurate as the starting sample of nucleic acid to be amplified. Nucleic acid (e.g., DNA or RNA) of low purity will yield amplification products that may not reflect the composition of the starting sample, and therefore, cannot be used (or relied on) for diagnostic purposes.

"It is also recognized that the conventional practice of biochemistry and molecular biology can require physical process resources on a scale that are frequently inversely proportional to the size of the subject being studied. For example, the apparatus and process chemistry associated with the preparation and purification of a biological sample such as a nucleic acid fragment for prospective analysis may easily require a full scale bio-laboratory with sterile facilities. Furthermore, an environmentally isolated facility of similar scale may typically be required to carry out the known nucleic acid amplification procedures such as polymerase chain reaction (PCR) for amplifying the nucleic acid fragment.

"There is therefore a need in the art for improved methods of nucleic acid purification for use in nucleic acid amplification techniques, so that nucleic acids may be more accurately and efficiently purified, identified and manipulated for use in molecular diagnostics and other nucleic acid screening purposes. There also exists a need for improved microfluidic systems for processing fluids for analysis of biological or chemical samples, and in particular, in the detection and analysis of biologically active macromolecules derived from such samples such as DNA, RNA, amino acids and proteins. It is beneficial and advantageous that the systems are mass producible, inexpensive, and preferably disposable; that the systems be simple to operate and that many or substantially all of the fluid processing steps be automated; that the systems be customizable, and be modular such that the system can be easily and rapidly reconfigured to suit various applications in which the detection of macromolecules is desired; and, that the systems be able to provide straightforward and meaningful assay results. A more thorough discussion of the challenges and shortcomings in the art, as well as exemplary solutions that may be utilized in conjunction with the teachings of the instant invention, are disclosed in applicant's copending U.S. application Ser. No. 13/033,165 (Pub. No. US2011/0275058) entitled 'Self-contained Biological Assay Apparatus, Methods, and Applications,' the subject matter of which is incorporated herein by reference in its entirety."

Supplementing the background information on this patent, VerticalNews reporters also obtained the inventors' summary information for this patent: "An embodiment of the instant invention is a microfluidic device-based method for purifying nucleic acid (e.g., DNA or RNA) from a lysed liquid sample solution. The method includes the steps of providing a suitable microfluidic device; adding an organic solvent to the lysed solution; adding magnetic beads to the lysed solution; binding the nucleic acid to the beads; separating the nucleic acid-bound magnetic beads from the solution; washing the nucleic acid-bound magnetic beads in a wash solution; separating the washed beads from the wash solution; eluting the nucleic acid from the washed beads; reestablishing an appropriate molecular charge on the eluted nucleic acid; recapturing the nucleic acid from the solution, washing the recaptured nucleic acid with a wash solution; and eluting the recaptured nucleic acid to obtain a purified nucleic acid. As used herein and in the claims, a suitable microfluidic device means a microfluidic device or system (a system generally includes a companion instrument that interfaces with the microfluidic device to control the process steps performed by the microfluidic device) having the capability to support and carry out the disclosed processes and claimed process steps. An exemplary suitable microfluidic device and companion instrument is one such as is disclosed in U.S. application Ser. No. 13/033,165 (id.). Other examples of suitable microfluidic devices and systems are known in the art. For example, the device may be composed of any materials commonly used for the production of microfluidic devices such as but not limited to silicon, glass, quartz, elastomeric materials (e.g. PDMS) and polymers (e.g. polystyrene, acrylics, COC's and others). The microfluidic devices may be produced by any known methods for producing microfluidic systems such as photolithography, embossing, laser etching, machining, injection molding; and the devices may incorporate various materials or be constructed from a single material. The device may incorporate discrete assembled parts or be composed of various layers designed to provide the functions required to perform the method. The microfluidic device may be operated manually or robotically, where all or at least some of the reagents are added to the device during the performance of the purification or, the device may include all of the reagents required for the purification placed in reservoirs and operate fully automatically. Generally the microfluidic device will be associated with an instrument capable of implementing the functions designed into the microfluidic device. According to various non-limiting, exemplary aspects, the method may further include the following steps, features, or characteristics: the step of reestablishing an appropriate molecular charge on the nucleic acid further comprises the steps of adding a suitable buffer to the eluted nucleic acid and adding an organic solvent to the solution, so that the nucleic acid is capable of binding to a substrate during purification; manually or robotically adding at least some of the reagents to the microfluidic device to carry out the purification process; providing the microfluidic device containing all of the necessary reagents to automatically carry out the purification process; estimating, analyzing, and/or amplifying the purified nucleic acid; repeating the steps of washing the nucleic acid-bound magnetic beads in a wash solution, and separating the washed beads from the wash solution a desired number of times; mixing the solution for a time sufficient to bind the nucleic acid to the beads; performing an RNAse treatment after the step of eluting the nucleic acid from the washed beads to remove RNA and only purify DNA; performing a DNAse treatment after the step of eluting the nucleic acid from the washed beads to remove DNA and only purify RNA.

"Another embodiment of the invention is a microfluidic device-based method for purifying nucleic acid (e.g., DNA or RNA) from a source containing nucleic acids. The method includes the steps of obtaining a liquid sample of the source; lysing the liquid sample to create a liquid sample solution; providing a suitable microfluidic device; adding an organic solvent to the lysed solution; adding magnetic beads to the lysed solution; agitating the solution for a time sufficient to bind the nucleic acid to the beads; separating the nucleic acid-bound magnetic beads from the solution; washing the nucleic acid-bound magnetic beads in a wash solution; separating the washed beads from the wash solution; eluting the nucleic acid from the washed beads; reestablishing an appropriate molecular charge on the eluted nucleic acid; capturing the nucleic acid from the solution; and eluting the captured nucleic acid to obtain a purified nucleic acid. According to various non-limiting, exemplary aspects, the method may further include the following steps, features, or characteristics: the step of reestablishing an appropriate molecular charge on the nucleic acid further comprises the steps of adding a suitable buffer to the eluted nucleic acid and adding an organic solvent to the solution, so that the nucleic acid is capable of binding to a substrate during purification; manually or robotically adding at least some of the reagents to the microfluidic device to carry out the purification process; providing the microfluidic device containing all of the necessary reagents to automatically carry out the purification process; estimating, analyzing, and/or amplifying the purified nucleic acid; repeating the steps of washing the nucleic acid-bound magnetic beads in a wash solution, and separating the washed beads from the wash solution a desired number of times; performing an RNAse treatment after the step of eluting the nucleic acid from the washed beads to remove RNA and only purify DNA; performing a DNAse treatment after the step of eluting the nucleic acid from the washed beads to remove DNA and only purify RNA."

For the URL and additional information on this patent, see: Young, Lincoln C.; Zhou, Peng; Spizz, Gwendolyn; Yasmin, Rubina. Microfluidic Device-Based Nucleic Acid Purification Method. U.S. Patent Number 8722329, filed February 21, 2012, and published online on May 13, 2014. Patent URL: http://patft.uspto.gov/netacgi/nph-Parser?Sect1=PTO2&Sect2=HITOFF&p=93&u=%2Fnetahtml%2FPTO%2Fsearch-bool.html&r=4604&f=G&l=50&co1=AND&d=PTXT&s1=20140513.PD.&OS=ISD/20140513&RS=ISD/20140513

Keywords for this news article include: Polymerase, Rheonix Inc, DNA Research, Enzymes and Coenzymes.

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Source: Journal of Engineering


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