Device and method for manipulating droplets using gel-state medium

An operation method and technology of an operation device are applied in the field of gene amplification and can solve the problem that genetic examination chips have not reached the level of popularization.

Active Publication Date: 2013-01-02
SHIMADZU SEISAKUSHO CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, among the microdevices for genetic testing based on the principle of one-time use, practical genetic testing chips have not yet reached the level of popularization due to the problem of the manufacturing cost of the device.

Method used

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  • Device and method for manipulating droplets using gel-state medium
  • Device and method for manipulating droplets using gel-state medium
  • Device and method for manipulating droplets using gel-state medium

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0287] As the nucleic acid-containing sample, a mixture of oral wiping liquid and cell lysis liquid was used. Oral wipes are prepared by using a cotton swab to scrape and probe oral mucosal cells, and suspend them in 1 mL of distilled water. By mixing 25 μL of oral wipes with a cell lysis solution containing guanidine thiocyanate at a final concentration of 2M, 50 μL of mixed solution was prepared.

[0288] The washing solution was prepared as a mixed solution of 200 mM potassium chloride and 50 mM Tris hydrochloric acid (pH 8.0).

[0289] For the PCR reaction solution, prepare a 0.125U TaqDNA polymerase (manufactured by Takara Bio), each 500nM concentration of β-actin detection primer, 500μM concentration of dNTP, 10mM concentration of magnesium chloride, 10mM concentration of Tris-HCl buffer (pH 9.2) and 0.2% (weight ratio) bovine serum albumin (manufactured by SIGMA). The sequences of the primers for β-actin detection are 5'-TGGCATCGTGATGGACTCCGGTGA-3' (SEQ ID NO: 1) and 5'-GC...

reference example 1

[0303] As the magnetic particles having a hydrophilic surface, Magnetic Beads (hereinafter simply referred to as magnetic silica beads) which are constituent reagents of the Plasmid DNA Purification Kit MagExtractor-Genome-kit sold by Toyobo were used. The magnetic silica beads in the above kit are washed by suspending the stock solution in 10 times the volume of pure water in advance, and repeating the operation of removing the supernatant by 500×g centrifugation for 1 minute for 5 times. Then, the magnetic silica beads are suspended in pure water, and the content of the magnetic silica beads in the pure water is adjusted so that the dry weight of the same silica beads is converted to 100 mg (dry) / mL.

[0304] The composition of the PCR reaction solution is 50 mM potassium chloride, 10 mM Tris hydrochloric acid buffer (pH 9.5), 5 mM magnesium chloride, 0.6 μM β-actin detection PCR primer (Forward) (manufactured by Applied Biosystems), 0.6 μM PCR primers for β-actin detection (Re...

reference example 2

[0310] Except for the case where the fluorescent pigments of SYBR-Green I, YO PRO-1, and SYTO-13 (all manufactured by Invitrogen) were used separately, and the droplet side concentration and the oil side concentration of the fluorescent pigment were changed to perform PCR, and The same operation as in Reference Example 1 above. Tables 1 to 3 show the difference between the fluorescence intensity of the droplets supplied before the start of PCR and the fluorescence intensity supplied after the start of PCR. Table 1 shows the results when SYBR-Green I was used, Table 2 shows the results when YO PRO-1 was used, and Table 3 shows the results when SYTO-13 was used.

[0311] In addition, the droplet volume is set to 3μL, and the composition of the reaction solution is 25mM Tris-HCl (pH8.3), 8mM MgCl 2 , 0.2% (w / V) bovine serum albumin, 0.125 U / μL Ex Taq DNA polymerase (manufactured by Takara Bio), 250 μM dNTPs, 1 μM each of primers for human β-actin gene detection.

[0312] The sequence...

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Abstract

A method for manipulating droplets is provided with which it is possible to manipulate droplets by means of a magnetic field only without requiring the use of a physical manipulation such as an electric-field manipulation. Also provided is a droplet manipulation device which renders such method possible. The droplet manipulation device is a device for transporting a droplet in a droplet confinement medium. The droplet manipulation device comprises a vessel (4) that holds the droplet confinement medium, droplets (12, 13, 14) each constituted of an aqueous liquid, a gel-state droplet confinement medium (31) that is insoluble or sparingly soluble in the aqueous liquid, particles (8) of a magnetic material that are contained in a droplet constituted of the aqueous liquid, and a magnetic-field application means (61) for generating a magnetic field and thereby transporting a droplet together with the particles of a magnetic material. The method comprises using the device to manipulate droplets in a droplet confinement medium held in the vessel.

Description

Technical field [0001] The invention relates to a droplet operation device and method using a gel-like medium. That is, the present invention relates to a micro device and a droplet operation method in the micro device. Specifically, it relates to a method capable of performing nucleic acid extraction, purification, and gene amplification in a microdevice. Background technique [0002] In the method of extracting and purifying nucleic acid from biological samples, the standard phenol-chloroform method has been used conventionally. However, because the operation is complicated and the reagents used are harmful, and the waste liquid treatment is costly, it is not often used in fields other than basic research. For example, in a nucleic acid purification method that uses genetic testing as the purpose, the ability of nucleic acid to specifically adsorb silicon can be used to easily extract and purify nucleic acid without using harmful reagents. In particular, the purification met...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N35/08G01N37/00C12Q1/68
CPCC12Q1/6806B01L2200/0673B01L2400/043B01L2300/069B01L2200/0647B01L3/502784B01L3/502761B01L2300/12B01L2300/087B01L7/54G01N35/08C12Q2525/131C12Q2531/113Y10T137/0391Y10T137/206
Inventor 大桥铁雄
Owner SHIMADZU SEISAKUSHO CO LTD
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