Source: http://www.google.com/patents/US6106777?dq=7350717
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Patent US6106777 - DNA analyzing method and device therefor - Google PatentsSearch Images Maps Play YouTube News Gmail Drive More »Sign inPatentsThe object of the present invention is to provide a method capable of analyzing the presence or absence of a target DNA sequence, the level and sequence characteristics thereof at a high sensitivity, and a device therefor, wherein the overall process from pretreatment to the recovery of DNA information...http://www.google.com/patents/US6106777?utm_source=gb-gplus-sharePatent US6106777 - DNA analyzing method and device thereforAdvanced Patent SearchTry the new Google Patents, with machine-classified Google Scholar results, and Japanese and South Korean patents.Publication numberUS6106777 APublication typeGrantApplication numberUS 08/552,496Publication dateAug 22, 2000Filing dateNov 9, 1995Priority dateNov 9, 1994Fee statusLapsedAlso published asDE69527125D1, DE69527125T2, EP0711840A2, EP0711840A3, EP0711840B1, US20010036637Publication number08552496, 552496, US 6106777 A, US 6106777A, US-A-6106777, US6106777 A, US6106777AInventorsTakeshi Fujita, Shin-ichiro UmemuraOriginal AssigneeHitachi, Ltd.Export CitationBiBTeX, EndNote, RefManPatent Citations (4), Non-Patent Citations (20), Referenced by (32), Classifications (23), Legal Events (7) External Links: USPTO, USPTO Assignment, EspacenetDNA analyzing method and device therefor
US 6106777 AAbstract
The object of the present invention is to provide a method capable of analyzing the presence or absence of a target DNA sequence, the level and sequence characteristics thereof at a high sensitivity, and a device therefor, wherein the overall process from pretreatment to the recovery of DNA information and the analysis thereof can be completed in a speedy fashion by the simple device structure and procedures.
1. A DNA analyzer wherein said signal processing means executes the comparison of the melting curve data of the sample single-stranded DNA fragment with known melting curve data, by comparing the measured melting curve data sets with each of the data sets of known melting curves preliminarily prepared or with each of the data sets of curves prepared by linearly binding a plurality of the known melting curve data sets in combination, and determining that the data of the known melting curve with the least statistical error or the combination of the data sets with the least statistical error as the sequence characteristics of the measured single-stranded DNA fragment.
11. A DNA analyzer, comprising:a holding means for holding a sample solution containing one sequence type of single-stranded DNA fragments or plural sequence types of single-stranded DNA fragments, which form conformation depending on the sequence type of single-stranded DNA in the solution and the condition of the sample solution; a spectroscopic means for measuring the UV absorbance of the sample solution held in said holding means; a denaturing means for denaturing the conformation formed by the single-stranded fragments in the sample solution held in said holding means under preset conditions; and a signal processing means for presetting the denaturing conditions and for obtaining and saving signals from the spectroscopic means and said denaturing means, wherein said processing means prepares a sample melting curve data of the single-stranded DNA fragment sample held in the holding means based on the saved signals, compares the sample melting curve data with each one of the template melting curve data set of the known sequence type of the single-stranded DNA fragments provided preliminarily in said processing means, chooses one of the template melting curve data set which most closely corresponds to the sample melting curve data based on a least square method, and subsequently displays a correspondence between the sample melting curve data and the chosen one of the template melting curve data set as those melting curve data most related to each other. 12. A DNA analyzer, comprising:a holding means for holding a sample solution containing one sequence type of single-stranded DNA fragments or plural sequence types of single-stranded DNA fragments, which form conformation depending on the sequence type of single-stranded DNA in the solution and the condition of the sample solution; a spectroscopic means for measuring the UV absorbance of the sample solution held in said holding means; a denaturing means for denaturing the conformation formed by the single-stranded fragments in the sample solution held in said holding means under preset conditions; and a temperature controller for presetting the denaturing conditions, and a signal processor for obtaining and saving signals from the spectroscopic means and said denaturing means, wherein said signal processor prepares a sample melting curve data of the single-stranded DNA fragment sample held in the holding means based on the saved signals, compares the sample melting curve data with each one of the template melting curve data set of the known sequence type of the single-stranded DNA fragments provided preliminarily in said processing means, chooses one of the template melting curve data set which most closely corresponds to the sample melting curve data based on a least square method, and subsequently displays a correspondence between the sample melting curve data and the chosen one of the template melting curve data set as those melting curve data most related to each other. 13. A DNA analyzer according to claim 12, wherein a temperature of said sample held in the holding means is controlled within a temperature range of about -20° C. to about 70° C.
The present invention relates to a method for analyzing DNA information in the fields of clinical diagnosis and life science, and a device therefor.
In order to overcome the above-mentioned problems and disadvantages, in accordance with the present invention, the melting curve of the conformation of a single-stranded DNA is directly detected, whereby a more precise and practical method of signal processing is provided along with a device therefor with a simplified structure.
FIG. 1 is a block diagram of the structure of a detector of a first example in accordance with the present invention;
The present invention will now be illustrated hereinbelow in one example.
FIG. 1 is a block diagram of the fundamental structure of the DNA analyzer in accordance with the present invention. An ultraviolet ray (of a wave length of 260 nm) from light source 1 is divided into two beams at an optical system 2, which beams are then individually incident into sample part 4 and control part 5, both being placed in cell holder 6. Thereafter, the individual beams collimated with optical systems (not shown) are detected with photomultiplier 7, and are then passed through amplifier 9 and processed with analytical signal processing device 10. The cell holder 6 can control the temperatures of the sample part 4 and the control part 5, following the temperature profiles programmed optionally with temperature controller 8. Temperature control can be done at an optional rate of temperature increase or decrease. The temperature in the cell holder 6 is measured with a temperature sensor (not shown), and input to the feedback temperature controller 8 and the signal processing device 10 simultaneously. Because it is required that the temperature of some sample should be controlled within the range from -20° C. to 100° C., the cell holder 6 has such a structure in which dry air flows from the bottom of each sample holding cell 4 and 5 to the top thereof so as to prevent the occurrence of bedewing on the surface of the both cells.
By the standard procedure, a DNA sample solution with the extracted genomic DNA, a PCR buffer solution containing a final 10 mM concentration of Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 0.02% gelatin, and 200 μM of each of deoxyribonucleotide triphosphates (dATP, dCTP, dGTP, and dTTP), 2.5 U Taq polymerase, 20 pmol of each of the two types of primers GH 26 and GH 27 corresponding to the HLA-DQA1 region to be analyzed (Ulf B. Gyllensten and Henry A. Erlich; Proceedings of the National Academy of Sciences of USA, Vol. 85, pp. 7652-7656, October 1988) were mixed together in a test tube, followed by overlaying mineral oil on the mixture. The PCR cycling condition was 27 cycles of 94° C. (1 minute), 57° C. (1.2 minute) and 72° C. (1 minute) in this order.
Using 1/100 of the reaction product, asymmetric PCR was done. The asymmetric PCR solution was almost the same as described for PCR, except that the amounts of the primers were modified such that GH 26 was 10 pmol and GH 27 was 1 pmol. The PCR cycling condition was 15 cycles of 94° C. (1 minute), 57° C. (1.2 minute) and 72° C. (1 minute) in this order.
The reaction product was desalted and concentrated with a microfilter (Microcon™ 30, manufactured by Grace Japan), which was then dissolved in the TNE buffer (10 mM Tris-HCl, 1 mM EDTA (pH 8.3), 30 mM NaCl). The resulting product was defined as a sample solution.
Placing the sample solution at the sample part 4 while placing the TNE buffer as the control solution at the control part 5, the sample temperature was once decreased to 0° C. Subsequently, the temperature was elevated to 60° C. at an elevation rate of 1° C./min.
In the present Example, a satisfactorily accurate melting curve can be generated in a practical sense, at a temperature elevation rate of 5° C./min at maximum. In this case, the analysis time is about 10 minutes per sample, achieving speeding up by 20 fold or more compared with the conventional DNA sequencing and SSCP method (4 hours or more).
By substantially uniformly regulating the temperature of samples at a high speed, the dynamic response of the conformation of a single-stranded DNA fragment of some sample to the temperature change was identified in a range above 10° C./min of temperature elevation or decrease. More specifically, it was identified that during the denaturing and forming of the conformation (during temperature elevation and decrease, respectively), the melting curve drew a hysteresis curve (1→2→3→4→5→6→2→3→4→5→ . . . ) as shown in FIG. 11. Specific to the difference in sequence such as the substitution, depletion or insertion of bases, the hysteresis curve varies depending on the sample. This indicates that the method is not only applicable to the change in the absorbance but also to the hysteresis curve of the absorbance, wherein more accurate determination of such type at a higher speed can be done by comparing a measured hysteresis curve with the template hysteresis curves in the same manner as in the case of the signal processing method.
In this state, PCR is performed while regulating the temperature of the PCR cell 600 in hot air or cold air. The PCR cycling condition is 25 cycles of 94° C. for 30 minutes, 55° C. for 1 minute 72° C. for 30 seconds and in this order. The total reaction time was 50 minutes. The time period required for the PCR is possibly shortened as short as about 20 minutes, by making the cell shape into a thinner form and enlarging the surface/volume ratio. By closing the valves 724, 728, the vaporization of the reaction solution at higher temperatures could be reduced to substantially zero.
The spectroscopic micro-cell 300 in the present Example is made of quartz glass and black quartz glass; the window of the light path is made of quartz (transparent) glass, and the remaining parts of the cell box in a rectangular parallelepiped with the upper top open are made of black quartz glass. On the upper top are internally arranged spacers of black quartz glass, with the flow gates 303 on both sides. Therefore, a sample solution holding part of a square shape, with a light path of a 10 mm length and a cross section of a 1.4 mm×1.4 mm-square shape, is formed below the spacers. A temperature sensor 301 is slightly projected toward the sample solution holding part at the central part of the spacers. As shown (inserted) with the broad arrow in the figure, the spectroscopic micro-cell 300 is placed internally inside temperature controller 302 to regulate the temperature of a sample solution.
In the present embodiment, the cell wall is made of black quartz glass with a thickness of about 1 mm, because the glass has far less reflection stray light with a relatively high thermal conductivity and a substantially great strength; for the cell material, a material with less reflection stray light and an excellent thermal conductivity is suitable. Another example is aluminum alloy coated with platinum black and TiN (titanium nitride). In the cell of the present Example, temperature sensor 301 is embedded in temperature contoller 302 to measure the sample temperature at the central location of the light path. Therefore, the cell can regulate the temperature of samples at a high efficiency. Additionally, the cell can measure the temperature at a high precision. Furthermore, compared with general commercially-available spectroscopic cells, the cell of the present Example has such a larger surface/volume ratio of 2800 that the cell can realize the temperature increase or decrease at the rate of from 0.1° C./min to 5° C./sec.
FIG. 15 depicts the block diagram of an example of a detector structure to detect the fluorescence from the DNA and the intercalating agent. An ultra-violet ray (of a wave length of 260 nm) from light source 801 passes through a filter and optical system 802, such as a lens, to be incident into sample 804 placed in sample holder 805. Via the presence of ethidium bromide intercalated with the sample DNA, fluorescence of 590 nm is emitted, which is then collimated with the optical system 807 followed by detection with photoelectric converter 808. The signal is thereafter processed through amplifier 805 at an analytical signal processor 810. The sample holder 805 can regulate the sample temperature following the temperature profile optionally programmed with temperature controller 806. Temperature regulation can be preset optionally at a rate of temperature increase or decrease from 0.1° C./min to 2° C./sec. Temperature can be regulated within the range of -20° C. to 100° C. by an electronic heating-cooling apparatus using Peltier effect.
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Du Pont De Nemours And CompanyFluorometer with low heat-generating light sourceUS7670832 *Aug 15, 2005Mar 2, 2010University Of Utah Research FoundationSystem for fluorescence monitoringUS7700275May 24, 2002Apr 20, 2010The Secretary Of State Of DefenseDetection systemUS7785776May 8, 2003Aug 31, 2010Idaho Technology, Inc.Genotyping by amplicon melting curve analysisUS7968856May 6, 2009Jun 28, 2011E.I. Du Pont De Nemours And CompanyFluorometer with low heat-generating light sourceUS8053213Sep 7, 2010Nov 8, 2011Roche Diagnostics Operations, Inc.Detection of PCR products in gel electrophoresisUS8271205Apr 29, 2009Sep 18, 2012Corbett Research Pty LtdMethod and system for analysis of melt curves, particularly dsDNA and protein melt curvesUS8343754 *May 7, 2012Jan 1, 2013University Of Utah Research FoundationAnnealing curve analysis in PCRUS9393566Jun 23, 2008Jul 19, 2016Canon U.S. Life Sciences, Inc.System and method for temperature referencing for melt curve data collectionUS20030224434 *May 8, 2003Dec 4, 2003Wittwer Carl T.Genotyping by amplicon melting curve analysisUS20040241679 *May 24, 2002Dec 2, 2004Lee Marin AlanDetection systemUS20050112647 *Oct 6, 2004May 26, 2005The Secretary Of State For DefenceDetection systemUS20060029965 *Aug 15, 2005Feb 9, 2006Wittwer Carl TSystem for fluorescence monitoringUS20060127906 *Oct 10, 2003Jun 15, 2006Lee Martin ADetection systemUS20070194246 *Aug 7, 2006Aug 23, 2007Lee Jerald DFluorometer with low heat-generating light sourceUS20080212090 *Mar 20, 2008Sep 4, 2008Lee Jerald DFluorometer with low heat-generating light sourceUS20090065471 *Feb 10, 2004Mar 12, 2009Faris Sadeg MMicro-nozzle, nano-nozzle, manufacturing methods therefor, applications thereforUS20090230323 *May 6, 2009Sep 17, 2009Lee Jerald DFluorometer with low heat-generating light sourceUS20090258414 *Oct 29, 2007Oct 15, 2009Wittwer Carl TSystem for fluorescence monitoringUS20090318306 *Jun 23, 2008Dec 24, 2009Canon U.S. Life Sciences, Inc.System and method for temperature referencing for melt curve data collectionUS20100076690 *Apr 29, 2009Mar 25, 2010Corbett Research Pty LtdMETHOD AND SYSTEM FOR ANALYSIS OF MELT CURVES, PARTICULARLY dsDNA AND PROTEIN MELT CURVESUS20100227326 *Feb 18, 2010Sep 9, 2010The Secretary Of State For DefenceDetection SystemUS20100330579 *Sep 7, 2010Dec 30, 2010Christian BirknerDetection of pcr products in gel electrophoresisUS20110207142 *May 6, 2011Aug 25, 2011Lee Jerald DFluorometer with low heat-generating light sourceEP2482056A1Feb 25, 2003Aug 1, 2012Waters Technologies CorporationLight-guiding flowcellsWO2009124665A1Mar 27, 2009Oct 15, 2009Roche Diagnostics GmbhDetection of pcr products in gel electrophoresisWO2010123625A1Mar 2, 2010Oct 28, 2010University Of Southern CaliforniaCd133 polymorphisms predict clinical outcome in patients with cancerWO2010123626A1Mar 2, 2010Oct 28, 2010University Of Southern CaliforniaCd133 polymorphisms and expression predict clinical outcome in patients with cancerWO2011031234A1 *Sep 8, 2009Mar 17, 2011Agency For Science, Technology And ResearchMethod and system for thermal melt analysisWO2011084757A1Dec 20, 2010Jul 14, 2011University Of Southern CaliforniaGermline polymorphisms in the sparc gene associated with clinical outcome in gastric cancerWO2011085334A1Jan 10, 2011Jul 14, 2011University Of Southern CaliforniaCd44 polymorphisms predict clinical outcome in patients with gastric cancer* Cited by examinerClassifications U.S. Classification422/50, 536/24.3, 436/800, 436/807, 422/82.08, 536/23.1, 359/350, 536/24.33, 435/287.2, 422/82.05, 435/91.1, 436/808, 435/6.12, 435/6.13International ClassificationC12Q1/68Cooperative ClassificationY10T436/143333, Y10S436/80, Y10S436/807, Y10S436/808, C12Q1/6816European ClassificationC12Q1/68B, C12Q1/68, C12Q1/68B6Legal EventsDateCodeEventDescriptionNov 9, 1995ASAssignmentOwner name: HITACHI, LTD., JAPANFree format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:FUJITA, TAKESHI;UMEMURA, SHIN-ICHIRO;REEL/FRAME:008169/0260Effective date: 19951027Mar 4, 1997ASAssignmentOwner name: HITACHI, LTD., JAPANFree format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:KIYAMA, MASAHARA;REEL/FRAME:008384/0099Effective date: 19970218Jan 29, 2004FPAYFee paymentYear of fee payment: 4Jan 18, 2008FPAYFee paymentYear of fee payment: 8Apr 2, 2012REMIMaintenance fee reminder mailedAug 22, 2012LAPSLapse for failure to pay maintenance feesOct 9, 2012FPExpired due to failure to pay maintenance feeEffective date: 20120822RotateOriginal ImageGoogle Home - Sitemap - USPTO Bulk Downloads - Privacy Policy - Terms of Service - About Google Patents - Send FeedbackData provided by IFI CLAIMS Patent Services