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  Concentration and cleanup of nucleic acid samplesUSPTO Application #: 20060157349Title: Concentration and cleanup of nucleic acid samples Abstract: Methods and devices are described for concentration and cleanup of samples containing bio-molecule analytes (e.g., polynucleotides, such as DNA, RNA, PNA). Various embodiments provide for pH-mediated sample concentration and cleanup of nucleic acid samples with channel devices (e.g., cross-T format, microchannel devices). (end of abstract) Agent: Mila Kasan, Patent Dept. Applied Biosystems - Foster City, CA, US Inventor: Karl O. Voss USPTO Applicaton #: 20060157349 - Class: 204453000 (USPTO) Related Patent Categories: Chemistry: Electrical And Wave Energy, Non-distilling Bottoms Treatment, Electrophoresis Or Electro-osmosis Processes And Electrolyte Compositions Therefor When Not Provided For Elsewhere, Capillary Electrophoresis, With Injection The Patent Description & Claims data below is from USPTO Patent Application 20060157349. Brief Patent Description - Full Patent Description - Patent Application Claims
CROSS REFERENCE TO RELATED APPLICATIONS [0001] This application is a divisional patent application of Ser. No. 10/237,537 filed Sep. 9, 2002, which claims priority under 35 U.S.C. .sctn. 119(e) to provisional application Ser. No. 60/318,269, filed Sep. 7, 2001, both of which are incorporated herein by reference. FIELD OF THE INVENTION [0002] The present invention relates to methods and devices for the concentration and cleanup of samples containing analytes. Aspects of the invention relate to pH-mediated sample concentration and cleanup of nucleic acid samples with channel devices. REFERENCES [0003] Backhouse et al., DNA sequencing in a monolithic microchannel device, Electrophoresis 2000, 21, 150-156. [0004] Dolnik et al., Capillary electrophoresis on microchip, Electrophoresis 2000, 21, 41-54. [0005] Grossman and Colburn, Capillary Electrophoresis Theory and Practice, Chapter 1, Academic Press (1992). [0006] Kambara et al., U.S. Pat. No. 5,192,142 (1993). [0007] Madabhushi et al., U.S. Pat. No. 5,552,028 (1996). [0008] Sambrook et al., eds., Molecular Cloning: A Laboratory Manual, Second Edition, Chapter 5, Cold Spring Harbor Laboratory Press (1989). [0009] Woolley et al., Ultra-high-speed DNA fragment separations using microfabricated capillary array electrophoresis chips, Proc. Natl. Acad. Sci., vol. 91, pp. 11348-11352, November 1994, Biophysics. [0010] Xiong et al., Base Stacking: pH-Mediated On-Column Sample Concentration for Capillary DNA Sequencing. Anal. Chem. 1998, 70, 3605-3611. BACKGROUND OF THE INVENTION [0011] In many techniques of molecular biology, it is important to have samples of high quality. Results are generally enhanced in PCR, sequencing, fragment analysis, and so forth, when the subject bio-molecule materials are separated from potentially interfering contaminants. Thus, it is often desirable to purify/concentrate the bio-molecules (e.g., polynucleotides, such as DNA, RNA, PNA, etc.) of interest in samples prior to analysis. [0012] In analyses utilizing laser-induced fluorescence (LIF) detection techniques, typical DNA samples often contain, in addition to dye-labeled DNA: salts, residual enzyme, DNA oligonucleotides, dNTP's, dye-labeled ddNTP's, and/or surfactants. It is generally desirable to remove all species except the subject dye-labeled DNA fragments. However, even partial purification can be useful. Thus, at a minimum, it is often desirable to remove at least some of the species that are present at higher concentration and that could interfere with the analysis. [0013] Sample concentration can be used to improve the detection limits of various analytical methods, such as electrophoresis. For example, the starting zone length of a sample injection can be reduced by utilization of a process termed "stacking." Stacking reduces the width of the sample zone before separation, which can result in improved sensitivity and increased peak efficiency. [0014] Xiong et al. describe a method for pH-mediated sample concentration of DNA sequencing samples on a capillary tube. While the technique of Xiong et al. might allow for sufficient signal from direct load on unpurified sequencing samples, it would not be expected to remove unincorporated dyes and contaminants that can obscure the sequencing data. Briefly, according to the method of Xiong et al., a capillary is filled with a nucleic acid DNA separation polymer. However, the polymer solution is buffered with a basic buffer that is charged at low pH but neutralized at high pH. Xiong et al. employed Tris buffer for this purpose. The first stage of the process involves a very long electrokinetic injection from unpurified sequencing reaction (e.g., right off a thermocycler with no following cleanup step). Because the sample is very salty at this point, the electrokinetic injection process is inefficient and a long injection time is needed to move enough DNA into the capillary to obtain sufficient signal. The injection time is so long that the peaks would be far too broad to achieve the necessary resolution for DNA sequencing. To re-focus the DNA starting band, Xiong et al. follow the DNA injection with a period of electrophoresis from a sodium hydroxide solution. The hydroxide migrates into the capillary, neutralizing the Tris buffer as it enters. In the area where the Tris is neutralized the conductivity becomes very low and therefore the electric field increases. The increased electric field at the injection end of the capillary allows the DNA at the back of the injection plug to travel faster than the DNA at the front of the injection plug. This refocuses the injection plug and allows for reasonable resolution to be obtained. When this technique is used with standard capillary electrophoresis, the contaminating dye labeled terminators, which are present in much higher concentration than the DNA, also migrate into the separation capillary. The large concentration of dye can migrate with the DNA and may negatively impact some section of the sequencing data. SUMMARY OF THE INVENTION [0015] Aspects of the present invention relate to sample concentration and cleanup; e.g., cleanup of a DNA sample to reduce or eliminate unincorporated dyes. Among other things, the present invention provides for direct loading of unpurified sequencing reactions on microfabricated separation devices. By way of the present methods and devices, the need to purify sequencing reactions (e.g., after themocycling, which typically involves centrifugation) can be reduced or eliminated. [0016] Aspects of the present invention relate to a channel device, various embodiments of which include (i) an injection channel and a separation channel, each channel having a first end and a second end, with the separation channel intersecting the injection channel at a region between the ends of the injection channel; (ii) a first reservoir disposed for fluid communication with one of the ends of the injection channel; (iii) a first separation medium held within the injection channel; and (iv) a second separation medium held within the separation channel; wherein the second separation medium differs from the first separation medium; [0017] Aspects of the present invention relate to a sample-manipulation method using such a channel device. For example, various embodiments of methods herein include (a) introducing a sample, including a polynucleotide-analyte component and one or more contaminants, into the injection channel; (b) introducing a pH-modulating composition into the injection channel; (c) stacking the polynucleotide-analyte component at a stacking region of the device defined by the intersection of the channels, and locating the one or more contaminants of the sample at a region between the stacking region and the second end of the injection channel; and (d) electrophoresing the polynucleotide-analyte component down at least a portion of the separation channel, with a substantial amount (e.g., a majority) of the one or more contaminants remaining in the injection channel. [0018] Aspects of the present invention relate to a sample-manipulation method, various embodiments of which include (a) providing a channel device, the device including (i) an elongate injection channel and an elongate separation channel, each channel having a first end and a second end, with the separation channel intersecting the injection channel at a region between the ends of the injection channel (e.g., in a cross-T format), and (ii) a loading region disposed for fluid communication with the first end of the injection channel; (b) placing a sample containing a polynucleotide analyte (e.g., DNA) and one or more contaminants into the loading region; (c) applying a first driving force (e.g., electric field) sufficient to cause at least some of sample to move from the loading region into the injection channel; (d) placing a basic solution (e.g., NaOH) into the loading region; (e) applying a second driving force (e.g., electric field) sufficient (i) to cause at least some of the basic solution to move from the loading region into the injection channel, thereby causing the polynucleotide analyte to stack in the region of the intersection of the channels, and (ii) to cause at least a portion of the one or more contaminants to move to a region between the intersection of the channels and the second end of the injection channel; and (f) applying a third driving force (e.g., electric field) sufficient to cause at least a portion of the stacked DNA to move into and along at least a portion of the separation channel, leaving behind in the injection channel at least a substantial portion (e.g., most) of the contaminants. [0019] According to various embodiments, a separation medium is placed in each of the injection and separation channels. The separation medium can be the same in each of the channels, or it can differ in one or more respects (e.g., concentration and/or composition). Continue reading... Full patent description for Concentration and cleanup of nucleic acid samples Brief Patent Description - Full Patent Description - Patent Application Claims Click on the above for other options relating to this Concentration and cleanup of nucleic acid samples patent application. ###  How KEYWORD MONITOR works... a FREE service from FreshPatents1. Sign up (takes 30 seconds). 2. Fill in the keywords to be monitored. 3. Each week you receive an email with patent applications related to your keywords. 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