Chemistry: electrical and wave energy – Apparatus – Electrophoretic or electro-osmotic apparatus
Reexamination Certificate
1999-08-30
2002-05-14
Tung, T. (Department: 1743)
Chemistry: electrical and wave energy
Apparatus
Electrophoretic or electro-osmotic apparatus
60
Reexamination Certificate
active
06387235
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention relates to an apparatus for the separation and fractionation of differentially expressed gene fragments. It relates more particularly to an apparatus and a process for the separation and fractionation of differentially expressed gene fragments (DNA fragments), which are suitable for searching a differentially expressed gene specific to a disease or a function.
Progress in life science and biotechnology has increased the need for the separation and fractionation of the DNA fragments. In particular, in progress in human genome and other genome projects, intensive attempts have been made to analyze the whole of expressed genes in an individual, to extract a differentially expressed gene specific to a certain organ or a gene involved with a specific function or disease, and to analyze the functions of the extracted gene. Accordingly, demands have been made to provide an apparatus which can analyze the whole of a multitude of samples and separate and sample differentially expressed gene (DNA) fragments with efficiency.
Of processes for detecting expression patterns of genes, some processes are based upon the size analysis of DNAs using electrophoresis. Such processes include, for example, the differential display method (hereinafter briefly referred to as “DD method”) (Nucleic Acids Research, Vol. 25, No. 12, pp.2541-2542(1997)), the fluorescence differential display method (hereinafter simply referred to as “FDD method”) (FEBS Lett., 351(2), 231-236, September 1994), the amplified fragments length polymorphism method (hereinafter simply referred to as “AFLP method”) (Nucleic Acids Research, Vol. 23, No. 21, pp. 4407-4414(1995)).
To be more specific, according to these methods, mRNAs are extracted from different biological tissues (e.g., from a normal cell and a cancerous cell), translated to cDNAs through reverse transcription, the resulting cDNAs are fragmented with a restriction enzyme treatment, the resulting fragments are then subjected to amplification by polymerase chain reaction (PCR) using an arbitrary primer (according to the DD method or FDD method) or a selected primer (according to the AFLP method), and the obtained PCR products are electrophoresed for the size separation to compare the obtained electropherograms. By way of illustration, if there is a DNA fragment which is specifically strongly observed in the cancerous cell, the DNA fragment is a candidate for a differentially expressed gene specific to the cancer, and is separated and sampled. According to a conventional procedure, the size separation of DNAs by electrophoresis, and the separation and fractionation of a differentially expressed gene are conducted in the following manner.
By way of illustration, when a slab gel about 0.3 mm thick is used as a separation medium, a mixture (a base length marker) of DNA fragments each having a known length is electrophoresed in some electrophoresis lanes, and a sample DNA to be analyzed, which has been labeled with a fluorophore, is electrophoresed in the other electrophoresis lanes. In general, if base lengths are analyzed using a slab gel, the total amount of the sample DNA is approximately 1 pmol (picomole; 10
−12
mole), and the volume of the sample DNA solution is several microliters (&mgr;L), each supplied to one electrophoresis lane. The sample is supplied to wells formed at an end of the slab gel, and then a voltage is applied to electrophorese the sample DNA.
After completion of the electrophoresis, an image formed on the gel is read out by an image reader. Such an image reader generally employs a technique of scanning laser light upon the gel surface and imaging the obtained fluorescent intensities to read out the positions of DNA bands in the slab gel.
In the electrophoresis on a slab gel, electrophoresis lanes often bend due to the effect of a distribution of temperature in the gel surface or the like to cause distortions in image. These distortions in image are corrected or calibrated in actual analysis and a base length pattern of the sample DNA is determined by a comparison between the positions of bands of the marker and those of the sample DNA. If a differential band is detected based on the comparison among base length patterns of different samples, the band is cut out from the gel to separate and collect the DNA fragment. The cut-out piece of gel is immersed in a buffer solution, allowed to stand for several hours or overnight to elute the DNA fragment from the gel into the buffer solution. The eluted DNA fragment is purified and then subjected to a sequencing reaction.
The conventional procedure requires a minute and precise technique to cut out a differential band in a precise size from the gel and thus requires a skilled and experienced person to handle. In addition, the extraction and purification of the DNA fragment from the cut-out gel require labor and a long period of time.
The gene expression profiling requires to analyze large amounts of samples, since comparisons between individual organs, between a normal tissue and a disease (e.g., cancer) tissue, or between a parent and a child, for example, are carried out using a combination of several tens of primers. Strong demands have therefore been made to improve working efficiency.
As examples of automatic apparatus for the separation and fractionation of differentially expressed gene fragments (DNA fragments), there may be mentioned one described in Japanese Patent Laid-open No. 7-181164 (hereinafter referred to as “the first conventional technique”), in which sample DNAs are electrophoresed using capillaries or slab gels each filled with a separation medium, and DNA fragments eluted into sheath flows of a buffer solution, transferred with the flow of the buffer solution by a transfer tube and sampled to sampling vessels. In this apparatus, the vessels are actuated according to detection signals of the DNA fragments eluted into sheath flows to collect a target DNA fragment automatically.
Separately, an apparatus is described in Japanese patent Laid-open No. 6-138037 (hereinafter referred to as “the second conventional technique”), in which first capillaries and second capillaries are respectively disposed in an optical cell face to face at specified gaps, which optical cell serves to detect DNAs labeled with fluorophores, and the DNA fragments eluted from the first capillaries into the optical cell are transferred to the second capillaries. In this apparatus, inner diameters of the second capillaries are greater than those of the first capillaries to introduce the DNA fragments to the second capillaries with ease and reliability.
SUMMARY OF THE INVENTION
Japanese Patent Laid-open No. 7-181164 (the first conventional technique) discloses fundamental constitutive elements of the apparatus, but fails to disclose a practical structure of the transfer tube, a concrete flow rate of the buffer solution and other parameters in a practical manner, which parameters are essential features for determining the performances of separation and fractionation of DNA fragments (in particular precision in separation of the DNA fragments, and required time to collect the DNA fragments). Japanese Patent Laid-open No. 6-138037 (the second conventional technique) lacks descriptions regarding the separation and fractionation of DNA fragments.
Accordingly, it is an object of the invention to improve the apparatus disclosed in Japanese Patent Laid-open No. 7-181164 (the first conventional technique) and thus to provide an apparatus for the separation and fractionation of differentially expressed gene fragments, which can shorten a required time for the separation and fractionation of DNA fragments and provide high separation.
The present inventors reviewed in detail the apparatus (fraction collector) disclosed in Japanese Patent Laid-open No. 7-181164 (the first conventional technique), and found that the performances of the separation and fractionation are significantly affected by a process for transferring DNA fragments through a transfer tube. To be more specifi
Hasegawa Hideki
Irie Takashi
Kambara Hideki
Ninomiya Ken
Antonelli Terry Stout & Kraus LLP
Hitachi , Ltd.
Noguerola Alex
Tung T.
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