Chemistry: molecular biology and microbiology – Apparatus – Including measuring or testing
Reexamination Certificate
2001-05-10
2003-01-28
Leary, Louise N. (Department: 1627)
Chemistry: molecular biology and microbiology
Apparatus
Including measuring or testing
C435S287100, C435S283100, C435S006120, C204S157740, C422S935000
Reexamination Certificate
active
06511843
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a glass capillary for DNA analysis and a manufacturing method thereof, and to a DNA analyzer that uses the glass capillary.
2. Prior Art
One of the methods of analyzing DNA is electrophoresis. Electrophoresis has the advantages that, since laser-excited fluorescence is detected in real time, both the sensitivity and the throughput are high.
In a conventional DNA analyzer that uses electrophoresis, a plurality of glass capillaries pass through an optical cell filled with a buffer solution, and DNA fragments that migrate through the glass capillaries are analyzed using a laser beam. To minimize background light due to scattering, the glass capillaries are lined up in a single plane in a horizontal direction along the horizontally irradiated laser beam.
However, with such a conventional DNA analyzer, the glass capillaries have a circular cross section, and therefore the laser beam is scattered at the surfaces of the first glass capillary, resulting in it being impossible to irradiate all of the glass capillaries uniformly, that is, without the intensity of the laser beam dropping for subsequent glass capillaries.
To combat this problem, either the refractive index of the buffer solution in the optical cell is made to be the same as the refractive index of the glass capillaries (about 1.5), so that scattering of the laser beam at the surfaces of the glass capillaries is eliminated and all of the migration paths of the DNA fragments are irradiated with the laser beam, or else the portion of the capillary array corresponding to the region irradiated with the laser beam is removed so as to form buffer solution sheath flows. A device disclosed in Anal. Chem., 1994, Vol. 66, pages 1021-1026 provides an example of the latter.
FIG. 1
is a schematic view showing the structure of the DNA analyzer disclosed in the above-mentioned document.
In
FIG. 1
, twenty vertically oriented glass capillaries
11
are lined up in a single plane in a horizontal direction at a pitch of 0.35 mm in a sealed optical cell
10
. Each of the glass capillaries
11
is comprised of a gel-filled capillary
12
that passes through a top wall of the optical cell
10
, and an open capillary
13
that passes through a bottom wall of the optical cell
10
. The bottom end of each gel-filled capillary
12
faces the top end of the corresponding open capillary
13
, with a
1
mm gap in-between. A buffer solution
15
is fed into the optical cell
10
from a buffer vessel
14
, and DNA fragments
16
are introduced into the gel-filled capillaries
12
.
Due to the pressure of the buffer solution
15
in the buffer vessel
14
, a sheath flow
17
of the buffer solution
15
is formed in the vicinity of the top end opening of each open capillary
13
. When this sheath flow
17
flows into the open capillary
13
, DNA fragments
16
are guided into the open capillary
13
. A laser beam is irradiated horizontally onto the glass capillaries
11
at the level of the sheath flows
17
, thus performing DNA analysis on the DNA fragments in the sheath flows
17
. The laser beam used is, for example, an Ar
+
488 nm laser beam or a YAG 532 nm laser beam.
Quartz glass elements are used as the gel-filled capillaries
12
in such a DNA analyzer. As shown in
FIG. 2
, each quartz glass element has a square cross section (D=300 &mgr;m), and has an internal hole having a square cross section (d=5 to 10 &mgr;m).
However, with the method in which the refractive index of the buffer solution
15
in the optical cell
10
is made to be the same as the refractive index of the glass capillaries
11
(about 1.5), and the method in which a portion of each glass capillary
11
corresponding to the region irradiated with the laser beam is removed so as to cause a sheath flow
17
of the buffer solution
15
to be formed in each place where a portion of a glass capillary
11
has been removed, the DNA detection efficiency of the electrophoretic DNA analysis can be increased, but there are drawbacks, such as a special buffer solution having to be prepared, or the structure of the analyzer becoming complicated, with the gel-filled capillaries and the open capillaries having to be aligned with one another.
SUMMARY OF THE INVENTION
It is an object of the present invention to provide a glass capillary for DNA analysis which allows the DNA detection efficiency of a DNA analyzer that uses electrophoresis (hereinafter referred to as an ‘electrophoretic DNA analyzer’) to be increased, and allows the DNA analyzer to have a simple structure, a method of manufacturing the glass capillary, and a DNA analyzer that uses the glass capillary.
The inventors of the present invention have discovered that, if glass capillaries for DNA analysis that each have an internal hole wherein both the glass capillary and the internal hole have a rectangular cross section are used in an electrophoretic DNA analyzer, then scattering of the laser beam at the surfaces of the glass capillaries can be prevented and therefore the transmittance of the laser beam can be increased. As a result, the DNA detection efficiency of the electrophoretic DNA analyzer can be increased, and moreover the analyzer can be given a simple structure. Note that throughout this specification (including the claims), the term ‘rectangular’ includes ‘square’; when ‘square’ is not to be included, the term ‘oblong’ is used.
Moreover, the inventors of the present invention have discovered that, by making the internal hole of each glass capillary have an oblong cross section and placing each glass capillary such that the longitudinal axis of the internal hole therein extends along the direction of laser beam irradiation, thin-walled portions are formed in each glass capillary along the direction of the short sides of the cross section of the internal hole and hence absorption loss of the laser beam can be reduced, and moreover thick-walled portions are formed in each glass capillary along the direction of the long sides of the cross section of the internal hole and hence the strength of the glass capillary can be increased; the inventors of the present invention have also discovered that, by offsetting the internal hole in the direction of the short sides of the cross section thereof, a thin-walled portion is formed in the glass capillary along the direction of a long side of the cross section of the internal hole and hence absorption loss in the glass of the fluorescence from the DNA fragments caused by the laser beam irradiation can be reduced.
Furthermore, the inventors of the present invention have discovered that, if a mother glass having a through hole wherein each of the mother glass and the through hole has a rectangular cross section is prepared, and this mother glass is drawn while heating the same, then a glass capillary for DNA analysis can be easily produced, wherein the cross section of the glass capillary is approximately similar to but smaller than the cross section of the mother glass, and the cross section of the internal hole of the glass capillary is approximately similar to but smaller than the cross section of the through hole of the mother glass; the inventors of the present invention have also discovered that, if the heating temperature of the mother glass is such that the viscosity of the mother glass becomes 10
5
to 10
9
poise, then the glass capillary for DNA analysis and the internal hole thereof can both be given a rectangular cross section reliably and with good reproducibility.
To attain the above object, the present invention provides a glass capillary for DNA analysis having an internal hole, and each of the glass capillary and the internal hole has a rectangular cross section.
According to this glass capillary, since the glass capillary and the internal hole each have a rectangular cross section, scattering of the laser beam at the surface of the glass capillary can be prevented and hence the transmittance of the laser beam can be increased. As a result, the detection efficie
Hattori Akihiko
Mizuno Toshiaki
Morishita Masahiro
Frishauf Holtz Goodman & Chick P.C.
Leary Louise N.
Nippon Sheet Glass Co. Ltd.
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