Method of purifying nucleic acid using silver nanoparticles

Tag : guanidine thiocyanate


Abstract
Provided is a method of purifying a target substance using silvernanoparticles. The method includes: mixing a sample containingmolecules having a thiol group with the silver nanoparticles toobtain a complex of the molecules having the thiol group with thesilver nanoparticles; and isolating and removing the complex fromthe mixture. By using the purification method, PCR amplifiable DNAscan be rapidly purified, and thus, the method can be veryefficiently applied to lab-on-chip (LOC).
Description of the Invention
CROSS-REFERENCE TO RELATED PATENT APPLICATION

This application claims the benefit of Korean Patent ApplicationNo. 10-2004-0097595, filed on Nov. 25, 2004, in the KoreanIntellectual Property Office, the disclosure of which isincorporated herein in its entirety by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a method of purifying nucleicacids using silver nanoparticles.

2. Description of the Related Art

The production of high purity double-strand plasmid DNAs,single-strand phage DNAs, chromosomal DNAs, and agarosegel-purified DNA fragments is very important in molecular biology.Ideal methods of purifying DNAs should be simple and can beperformed rapidly and include little additional manipulation ofsamples. The DNAs obtained using such methods are ready for directtransformation, restriction enzyme analysis, ligation, orsequencing. Such methods are very attractive in the automatedproduction of DNA samples, which is favored in research anddiagnosis labs. Generally, the preparation of plasmid DNAs fromcrude alcohol precipitates is laborious. Plasmid DNAs are oftenproduced using a CsCl gradient, gel filtration, ion exchangechromatography, RNAase, proteinase K, and repeated alcoholprecipitation. These methods require considerable downstream samplepreparation to remove CsCl and other salts, EtBr, and alcohol, etc.Further, small negatively charged cellular components can beprecipitated together with DNAs. Thus, the DNAs may be contaminatedto an undesirable degree.

Methods of purifying nucleic acids using solid phase materials arewell known in the art. For example, U.S. Pat. No. 5,234,809describes a method of purifying a nucleic acid using a solid phasematerial which can bind to the nucleic acid. Specifically, themethod includes mixing a chaotropic material with a nucleic acidbinding solid phase, separating the solid phase material with thenucleic acid bound thereto from the liquid, and washing the solidphase material-nucleic acid complexes. Examples of the chaotropicmaterial include guanidinium thiocyanate (GuSCN), guanidinehydrochloride (GuHCl), sodium iodide (NaI), potassium iodide (KI),sodium thiocyanate (NaSCN), urea, and combinations thereof.Examples of the solid phase material include silica particles.

However, this method is considerably time-consuming, complicated,and unsuitable for lab-on-a-chip (LOC). Further, this method usesthe chaotropic material as an essential component. If thechaotropic material is not used, nucleic acid cannot bind to thesolid phase material. Since the chaotropic material is harmful to ahuman body, it must be carefully handled. In addition, thechaotropic material inhibits a subsequent process, such as PCR(polymerase chain reaction), and thus, it must be removed from thenucleic acid during or after purification of the nucleic acid.

Research has been conducted to develop a solid phase materialhaving a large surface area for efficient binding with a nucleicacid. However, a method of purifying a nucleic acid using the solidphase material still requires many treatment processes and the useof chaotropic salts and is time-consuming.

A reversible immobilization method using a solid phase material hasbeen described [Hawkins, et al., Nucleic Acids Res. 1995; 23:22].This method is a simple method and easily automated. However, theapplication of this method to the detection of pathogens has notbeen described and this method is not suitable for the productionof LOC.

There have been reported kits for producing single tube samples,which are available from GeneReleaser (manufactured byBioventures), ReleaseIT (manufactured by CPG Inc.), andLye-N-Go.TM. RCR Reagent (manufactured by Pierce). By using thekits, samples can be prepared in only two steps and PCR samples canbe prepared within 10-15 minutes. However, areal-time PCR cannot beperformed due to the use of a white polymer reagent and after celllysis, reagents and a PCR mixture must be added, which isinconvenient and results in possible contamination of the samples.

The present inventors conducted research on a method of purifying anucleic acid based on the general techniques and discovered thatsilver nanoparticles can bind to a molecule having a thiol group ina sample, and can then be removed from the sample using aSH-modified membrane.

SUMMARY OF THE INVENTION

The present invention provides a method of efficiently purifying anucleic acid using silver nanoparticles in a short time.

According to an aspect of the present invention, there is provideda method of purifying a target substance using silvernanoparticles, comprising: mixing a sample containing moleculeshaving a thiol group with the silver nanoparticles to obtain acomplex of the molecules having the thiol group with the silvernanoparticles; and isolating and removing the complex from themixture.

The sample may comprise a cell or a virus and the method mayfurther comprise lysing the cell or the virus.

The target substance may be a nucleic acid or a sugar.

The lysing of the cell or the virus may be performed using a methodselected from the group consisting of mechanical grinding, a methodusing a chemical reaction, a method using an electrochemicalreaction, a method using a biochemical substance, a method usingultrasonic waves, a method using sound waves, a method usingmicrowaves, heating, a method using a laser, a method usingelectric field, and electrolysis.

The silver nanoparticle complex may be removed by passing themixture through a structure having SH groups.

The structure having SH groups may be a glass fiber membranemodified with 3-(mercaptopropyl)trimethoxysilane.

The silver nanoparticles may have a size of 1-100 nm.

The concentration of the silver nanoparticles may be 10-1000 ppm,preferably 100-1000 ppm.

The silver nanoparticles may be directly added to the sample orgenerated by electrolysis using a silver electrode.

DETAILED DESCRIPTION OF THE INVENTION

Hereinafter, the present invention will be described in more detailstep by step.

According to an embodiment of the present invention, there isprovided a method of purifying a target substance using silvernanoparticles, comprising:

mixing a sample containing molecules having a thiol group with thesilver nanoparticles to obtain a complex of the molecules havingthe thiol group with the silver nanoparticles; and

isolating and removing the complex from the mixture.

FIG. 1 (see Original Patent) is a flow diagram illustrating amethod of purifying a nucleic acid using silver nanoparticlesaccording to an embodiment of the present invention. Referring toFIG. 1, first, cells and silver nanoparticles are mixed in acontainer. Then, cells are lysed using boiling or microwavetreatment, or a similar method. Once the cell are lysed, the silvernanoparticles bind to proteins. The proteins are modified duringthe cell lysis, thereby exposing the thiol groups, which allows thesilver nanoparticles to bind to the thiol groups of the proteins.It is assumed that bonds between the silver nanoparticles and theproteins are formed via thiol groups in cysteine and methionine ofthe proteins. The bonds between the silver nanoparticles and thethiol groups are very strong, irreversible, and covalent. Next, theobtained protein-silver nanoparticle complexes are filtered througha SH-modified membrane. At this time, most proteins are removedfrom the cell lysate and DNAs remain in the solution. Thus, a PCRamplification can be facilitated using the resultant DNA solution.

According to an embodiment of the present invention, the targetsubstance is a nucleic acid or a sugar. The target substance may beany substance not having a thiol group, and preferably a nucleicacid for realizing LOC.

According to an embodiment of the present invention, the lysing ofa cell or a virus may be performed using a method selected from thegroup consisting of mechanical grinding, a method using a chemicalreaction, a method using an electrochemical reaction, a methodusing a biochemical substance such as enzyme, a method usingultrasonic waves, a method using sound waves, a method usingmicrowaves, heating, a method using a laser, a method usingelectric field, and electrolysis. The cell lysis method using thecells to which the silver nanoparticles are added barely affectsthe PCR results.

According to an embodiment of the present invention, theprotein-silver nanoparticle complex is isolated from the samplethrough a SH-modified membrane. The SH-modified membrane may be anymaterial modified with a thiol group, and is preferably glass fibermembrane modified with 3-(mercaptopropyl)trimethoxysilane.

According to an embodiment of the present invention, the silvernanoparticles may have a size of 1-100 nm. If the size of thesilver nanoparticles is greater than 100 nm, the efficiency of thebinding of silver nanoparticles to proteins can be low. If the sizeof the silver nanoparticles is less than 1 nm, the silvernanoparticles cannot be easily manufactured.

According to an embodiment of the present invention, theconcentration of the silver nanoparticles may be 10-1000 ppm,preferably 100-1000 ppm. If the concentration of the silvernanoparticles is less than 100 ppm, the proteins cannot be removedfrom the sample. If the concentration of the silver nanoparticle isgreater than 1000 ppm, the PCR amplification is not wellfacilitated, even after the SH filtration. That is, when theconcentration of the silver nanoparticles is too high, the PCRamplification cannot be performed although a large amount of themodified protein can be removed from the sample. If theconcentration of the silver nanoparticles is 100 ppm or less, thePCR is not inhibited regardless of whether the SH filtration hasbeen performed. Further, the results of the PCR are not affected ifthe concentration of the silver nanoparticles is 10 ppm or less. Byusing a plurality of membranes or increasing the surface area ofthe membrane in the SH filtration apparatus, the maximumconcentration of the silver nanoparticles at which PCRamplification is not affected can be increased.

According to an embodiment of the present invention, the silvernanoparticles are directly added to the sample or generated byelectrolysis using a silver electrode.

Claim 1 of 9 Claims
1. A method of purifying a target substance comprising: mixingsilver nanoparticles with a sample comprising the target substanceand molecules having a thiol groups, wherein the target substanceis a nucleic acid or a sugar, and the target substance does nothave a thiol group; forming a complex of the molecules having thethiol group and the silver nanoparticles; removing the complex fromthe mixture; and obtaining a solution comprising the purifiedtarget substance.

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