1. Field of the Invention
The present invention relates to a nucleic acid-bondable magnetic carrier containing a magnetic-responsible particle, utilized for extracting or purifying a nucleic acid from a biological material containing the nucleic acid, or for purifying an amplified product of a nucleic acid. The present invention also relates to a method for isolating a nucleic acid from a nucleic acid-containing biological material utilizing the magnetic carrier and a magnetic field, and a kit for utilizing the method.
2. Description of the Related Art
In conventional methods for isolating a nucleic acid using a nucleic acid-bondable magnetic carrier, it is known to utilize a magnetic-responsible particle having a superparamagnetic iron oxide core covered with a polymeric silane layer to which a biocompatible molecule (for example, a nucleic acid) is covalently bonded (Japanese Laid-Open Patent Publication No. 60-1564).
A method for determining a ligate concentration including the following steps is also known: (1) using a magnetic-responsible particle containing superparamagnetic iron oxide covered with a polymeric silane layer to which a biocompatible molecule is capable of being bonded; (2) reacting a sample solution containing a ligate, a known amount of a labeled ligate, and the magnetic-responsible particle to which a ligate-specific ligand is bonded, so as to form a ligand-ligate complex on the magnetic-responsible particle; (3) magnetically separating the magnetic-responsible particle from the reaction solution; (4) measuring the labeled ligate which is bonded to the magnetic-responsible particle or free labeled ligate in the reaction solution; and (5) applying the measurement of the label ligate to the standard curve so as to obtain the ligate concentration. This method is described in Japanese Patent Publication No. 7-6986.
In the above-mentioned Methods, in order to bond the nucleic acid to the magnetic-responsible particle, it is necessary to form a silane layer to which a biocompatible molecule (for example, a nucleic acid) is covalently bonded.
Furthermore, an analyzing method and apparatus are known utilizing a sequence of a nucleic acid which is bonded to a material sensitive to the magnetic field (WO86/05815). The method utilizes a magnetic or magnetizable particle covered with a material which is capable of being bonding to a single strand nucleic acid so as to separate and detect the single strand nucleic acid. More specifically, the surface of the magnetic particle is covered with nitrocellulose, which is a type of cellulose derivative, and nitrocellulose is specifically bonded to a single strand DNA or RNA. The single strand DNA or RNA collected by the method is utilized for sequencing.
In the method, it is necessary to specifically bond the single strand DNA or RNA to the magnetic carrier.
It is also known to utilize a poly-cationic substrate for purifying, separating and hybridizing a nucleic acid, especially for purifying and separating a nucleic acid containing contaminants (Japanese Laid-Open National Publication No. 1-502319). In the method, a sample solution containing contaminants is contacted with the poly-cationic solid support (magnetic-responsible particle) so as to non-covalently bond the nucleic acid to the support without excessively bonding contaminants contained in the sample solution to the support. The support to which the nucleic acid has been bonded is then separated from the solution. Examples of the support include metal oxide, glass and polyamide. Examples of the poly-cationic magnetic-responsible particle include a magnetic microsphere (typically, a magnetic amine microsphere). The bond between the nucleic acid and the support is considered to be based on an ionic bond between the magnetic amine microsphere having a positive charge and a sugar phosphate principal chain in the nucleic acid having a negative charge.
Furthermore, it is also known a method for isolating a substance of interest in biological material utilizing a magnetic particle consisting of a polymeric inner core particle and a magnetic-responsible metal oxide/polymer coating uniformly covering the core particle (Japanese Laid-Open National Patent Publication No. 2-501753). The method includes the steps of reacting the magnetic particle with a biological material so as to form a complex consisting of the magnetic particle and a substance from the biological material; separating the complex from the biological material; and removing the magnetic particle from the complex so as to obtain the substance.
In the method, a polymer such as polystyrene is used as an inner core particle, and metal oxide and a polymer such as polystyrene uniformly cover the inner core particle.
A superparamagnetic particle with a plurality of separated oligonucleotide sequences having monodispersibility (less than 5% of particle diameter distribution), and a method for producing a magnetic particle which covalently bonds or adsorbs the oligonucleotides to functional groups (for example, biotinyl groups) or molecules on the surface thereof is known. It is also known to utilize a particle to which oligonucleotide is covalently bonded or adsorbed as a probe of a nucleic acid (WO90/06045).
The object of the method is to specifically form covalently bonds or adsorb a probe of a nucleic acid utilized for hybridization to the particle. Therefore, the above-mentioned particle is not a carrier which non-specifically immobilizes (i.e., bonds or adsorbs) a large amount of nucleic acids.
As described above, in the methods utilizing a silane or polymeric layer on the surface of the magnetic particle carrier, a nucleic acid is, for example, covalently bonded to the silane or polymeric layer on the carrier surface. Such methods require providing functional groups on the carrier (magnetic particle) surface. Accordingly, while such methods are advantageous to the separation or the quantitation of nucleic acids utilizing the specific adsorption thereof, they are not suitable for a solid phase carrier which non-specifically adsorbs a large amount of nucleic acids so as to produce a high yield.
In the case of utilizing a surface-coated magnetic particle as a solid phase carrier for isolating a nucleic acid, a large particle (for example, having a diameter of more than 20 μm) is capable of responding to a weak magnetic field or a small magnetic field variation; however, it tends to rapidly precipitate and have insufficient dispersibility. Therefore, it is difficult for such a large particle to adsorb and immobilize a small nucleic acid such as plasmid DNA in the reaction which requires homogeneity such as a solid phase adsorption. Furthermore, a large particle has a smaller specific surface per weight than that of a small particle. As a result, a large particle is only capable of bonding a small amount of biological material thereto.
A small particle (for example, having a diameter of less than 0.1 μm) has outstanding specific surface and dispersibility; however, it has insufficient settling properties. Therefore, when the small particle is used in a separation utilizing the magnetic field, a larger and more expensive magnet having a larger magnetic charge is required, and it takes a longer time to separate the particle utilizing magnetic field.
Regarding a method utilizing a silica particle for purifying a nucleic acid, a column separation essentially utilizing high performance liquid chromatography (HPLC) apparatus has been conventionally used. A desired nucleic acid is adsorbed to the silica carrier surface by passing a synthesized nucleic acid or an amplified product through the column. An impurity can be washed away by flowing a washing buffer. The desired nucleic acid can be collected by flowing a buffer. The method has an advantage in that the column comprising silica carriers can be used repeatedly. However, the isolation of a nucleic acid from whole blood (which is a biological material) cannot be conducted because the column clogs. As a result, the method has disadvantages in that only a small amount of a nucleic acid can be collected. In addition, the apparatus utilized for the method is very expensive.
A method for separating a nucleic acid from a biological material (for example, whole blood, urine) utilizing a silica particle as a solid phase carrier is also known (Japanese Laid-Open Patent Publication No. 2-289596 and Japanese Patent Publication No. 7-13077).
However, in such a method that utilizes a silica particle as a carrier, complicated procedures are required (for example, centrifugation must be conducted many times). In addition, after adding the sample solution to the particle, the method requires a mixing operation by vigorously stirring with a vortex mixer in order to sufficiently mix the sample solution and the particle. Because of the vigorous stirring, a nucleic acid contained in the sample tends to be degraded, and as a result, a long-chain nucleic acid can hardly be separated.
As described above, there are various problems related to non-specifically immobilizing a large amount of nucleic acids.