Chemistry: analytical and immunological testing – Including sample preparation – Liberation or purification of sample or separation of...
Reexamination Certificate
2001-02-22
2004-11-09
Ludlow, Jan M. (Department: 1743)
Chemistry: analytical and immunological testing
Including sample preparation
Liberation or purification of sample or separation of...
C436S094000, C436S161000
Reexamination Certificate
active
06815215
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention relates to a method of recovering nucleic acids and an apparatus thereof, more particularly a method and apparatus fit to recover nucleic acids from some kinds of sample substances without reducing the concentrations of the nucleic acids.
As the molecular biology advances, many kinds of gene-related technologies have been developed and used to isolate and identify various infected genes. As the result, these molecular biological techniques have been employed by a wide variety of fields including medical, diagnostic, and testing fields, enabling various new diagnostic methods that had not been realized by conventional methods and dramatically shortening test periods.
This rapid progress is attributed mainly by a nucleic acid amplifying method, particularly by PCR (Polymerase Chain Reaction).
As PCR can peculiarly amplify nucleic segments of a specific structure in a solution, for example, PCR can be used to testify to the existence of an extremely small amount of viruses in blood indirectly by amplifying and detecting a nucleic acid which is the gene of the virus.
However, PCR has some problems when it is used for daily testing jobs in a clinical field. The main concern is extraction and refining of nucleic acids in preprocessing. These steps are greatly affected by the inhibiting factors which remain unremoved after refining. Hemoglobin in blood and surface-active agent used for extraction have been widely known as such inhibiting factors.
Further, the extraction process requires time- and labor-consuming complicated manual operations by experts, which mainly causes a hospital to hesitate to employ a new gene testing system. The automation of this process has been longed for.
In a blood center which must quickly detect HCVs (hepatitis C virus), HIV (human immunodeficiency virus), and so on in a great many blood specimens, a screening (sieving) method is sometimes employed to quicken the test. The screening method comprises steps of mixing some specimens into one and testing it. This is because such viruses (HCV, HIV, etc.) are rarely detected and most specimen mixtures are free from such viruses. Only when such viruses are detected in a specimen mixture, the specimens of the specimen mixture are individually tested.
However, in this screening method, the specimen mixture is diluted too much for example, one fiftieth when 50 specimens are combined into one or one five-hundredth when ten 50-specimen mixtures are combined into one and the concentrations of specimens may go below those required to detect nucleic acids which are the genes of such viruses. In extreme cases, the virus-positive specimens may be evaluated as virus-negative.
For extraction of nucleic acids, Japanese Non-examined Patent Publication H08-320274 (1996) discloses a method of isolating genes by means of a plurality of containers and tips for a single specimen. This method comprises the steps of mounting a first tip onto the pipette nozzle which is moved by a driving mechanism, sucking up a specimen into the first tip, fitting a filter which breaks blood corpuscles to the lower end of the first tip and discharging the specimen from the first tip to a first container through this filter.
The method further comprises the steps of demounting the filter and the first tip from the pipette nozzle, mounting a second tip to the lower end of the second tip, and sucking the specimen from the first container into the second tip.
The method furthermore comprises the steps of fitting a silica membrane filter to the lower end of the second tip to catch genes, and discharging the specimen from the second tip to the second container through the silica membrane filter. With this, genes are caught by the silica membrane and impurities are discharged to the second container.
Further, the method comprises the steps of moving the pipette nozzle to a third container which contains a washing liquid, demounting the silica membrane filter which has genes from the second tip, immersing the filter into the washing liquid in the third container, mounting the third tip to the pipette nozzle from which the second tip was demounted, fitting the washed silica membrane to the lower end of the third tip, sucking up a mixture of the washing liquid and the genes into the third tip, and discharging the mixture to the fourth container.
Japanese Non-examined Patent Publication H02-289596 (1990) discloses a method of using silica particles which can bind with nucleic acids in the presence of a chaotropic agent as a stationary phase for binding nucleic acids. This method comprises the steps of adding a specimen containing nucleic acids to a reaction container which contains a silica particle suspension and guanizithiocyanate buffer solution working as the chaotropic agent, mixing thereof, centrifugally separating a complex which binds nucleic acids to silica particles, and disposing of the supernatant solution.
This method further comprises the steps of adding a washing liquid to the complex residue, washing thereof by means of a vortex mixer, washing the complex precipitate with an aqueous solution of ethanol, washing the precipitate again with acetone, removing acetone, drying the precipitate, adding a buffer solution for elution to the dried complex, and recovering the eluted nucleic acids.
Further, Japanese Non-examined Patent Publication H11-266864 (1999) discloses a method comprising the steps of connecting a nucleic acid capturing tip which contains a silica stationary phase to a nozzle, sucking and discharging a mixture of a specimen which contains nucleic acids and a substance which accelerates the nucleic acids to be bound to the stationary phase, causing the nucleic acids to be bound to the stationary phase in the nucleic acid capturing tip, and washing this tip.
The technology in accordance with Japanese Non-examined Patent Publication H11-266864 (1999) enables automated extraction of nucleic acids.
However, as the technology in accordance with Japanese Non-examined Patent Publication H08-320274 (1996) is so constructed to capture genes when discharging the specimen from the second tip through the silica membrane, the time in which the specimen is in contact with the silica membrane is very short and the rate of capturing genes is low. This may cause a suspected virus-negative case.
Further, the technology in accordance with Japanese Non-examined Patent Publication H02-289596 (1990) requires a centrifugal separation process which is an obstacle to automation of the refining process and takes a lot of time for refining.
As already explained above, although the screening method can increase the test speed, the specimen is diluted down to about one hundredth and high-accuracy detection of viruses (HCV, HIV, etc.) cannot be expected.
Therefore, any other fast and high-precision testing method than the screening method has been longed for.
A comprehensive object of the present invention is to provide a method and apparatus which can automatically recover nucleic segments of a specific structure from a bio specimen.
SUMMARY OF THE INVENTION
The present invention has attained the aforesaid object by Claims, particularly by making a plurality of specimens in contact with a stationary phase for extracting nucleic acids, capturing nucleic acids from the specimens by a single stationary phase, and extracting thereof by an eluate.
A first embodiment of the present invention comprises the steps of making a plurality of specimens containing nucleic components in contact with a stationary phase which can bind with said nucleic components, letting said stationary phase absorb the nucleic acids from the specimens, separating other components which are left unabsorbed from said stationary phase, performing said two processes on each of the other specimens, letting said stationary phase absorb nucleic acids, making an eluate in contact with said stationary phase, discharging thereof, and eluting. This embodiment further comprises the steps of removing components that are not absorbed by the stationary phase
Igarashi Yoshiaki
Sakurai Toshinari
Yokobayashi Toshiaki
Hitachi , Ltd.
Ludlow Jan M.
Mattingly Stanger & Malur, P.C.
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