U.S. patent application number 16/189170 was filed with the patent office on 2019-05-16 for tubes.
The applicant listed for this patent is Paul Francis Day. Invention is credited to Paul Francis Day.
Application Number | 20190143319 16/189170 |
Document ID | / |
Family ID | 60788364 |
Filed Date | 2019-05-16 |
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United States Patent
Application |
20190143319 |
Kind Code |
A1 |
Day; Paul Francis |
May 16, 2019 |
Tubes
Abstract
A thin-walled microplate suitable for use in the Polymerase
Chain Reaction (PCR) technique comprising a plurality of
thin-walled tubes or wells arranged in a fixed array, each well
having an upper portion with an open top and a lower, frustoconical
portion having a substantially flat bottom.
Inventors: |
Day; Paul Francis; (Wotton,
GB) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Day; Paul Francis |
Wotton |
|
GB |
|
|
Family ID: |
60788364 |
Appl. No.: |
16/189170 |
Filed: |
November 13, 2018 |
Current U.S.
Class: |
422/553 |
Current CPC
Class: |
B01L 2300/0829 20130101;
B01L 2300/168 20130101; B01L 3/5453 20130101; B01L 9/523 20130101;
B01L 3/50855 20130101; B01L 2300/021 20130101; B01L 2300/0851
20130101; B01L 3/5085 20130101; B29L 2031/756 20130101; B29C
33/0022 20130101; C12M 23/22 20130101; B01L 3/50851 20130101 |
International
Class: |
B01L 3/00 20060101
B01L003/00 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 10, 2017 |
GB |
1718615.6 |
Claims
1. A thin-walled microplate for use in the Polymerase Chain
Reaction (PCR) technique comprising a plurality of wells arranged
in an access array, each well having an upper portion with an open
top and a lower, frustoconical portion having a substantially flat
bottom.
2. The thin-walled microplate according to claim 1, wherein the
substantially flat bottom of each of the wells in the array has a
surface that extends in substantially the same horizontal plane
such that an inner bottom surface of each well is substantially
equidistant from the top of a microplate in order that items, such
as cells, retained along said inner bottom surface of said
plurality of wells in the array are in a focal plane that is
substantially the same when viewed from above through the open top
of the respective wells.
3. The thin-walled microplate according to claim 2, wherein a
distance from the open top to the inner bottom surface of each well
is the same to within +/-0.1 mm.
4. The thin-walled microplate according to claim 2, wherein a
distance from the open top to the inner bottom surface of each well
is the same to within +/-0.05 mm or better.
5. The thin-walled microplate according to claim 1, wherein the
flat bottom surface of each well has an external diameter in the
range 2.0 mm to 4.5 mm.
6. The thin-walled microplate according to claim 5, wherein the
external diameter is in the range 2.5 mm to 3.5 mm.
7. The thin-walled microplate according to claim 1, wherein the
substantially flat bottom surface of one or more wells in the array
incorporates a machine-readable code.
8. The thin-walled microplate according to claim 7, wherein the
machine-readable code is readable using an optical vision
system.
9. The thin-walled microplate according to claim 7, wherein the
machine-readable code is on the external surface of the
substantially flat bottom.
10. The thin-walled microplate according to claim 1, wherein an
external surface of the substantially flat bottom on each well
incorporates a protection ring or bead.
11. The thin-walled microplate according to claim 10, wherein the
protection ring or bead is located substantially on an outer
circumference of the substantially flat bottom.
12. The thin-walled microplate according to claim 1, wherein the
substantially flat bottom of each well is formed from substantially
clear material, such that each well has a flat, optically-clear
window.
13. The thin-walled microplate according to claim 1, wherein the
array of wells are held in a substantially rigid frame.
14. The thin-walled microplate according to claim 13, wherein the
substantially rigid frame is formed from a first plastics material
and the tubes or wells are formed from a second plastics material
that is suitable for PCR use.
15. The thin-walled microplate according to claim 13, wherein the
wells are not integrally fixed in the rigid frame but can be
individually removed from the frame and are therefore randomly
accessible.
16. The thin-walled microplate according to claim 13, wherein the
wells are substantially permanently fixed in the rigid frame and
are therefore not randomly accessible.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of priority to
GB1718615.6, filed on Nov. 10, 2017; the entirety of which is
hereby incorporated by reference herein.
BACKGROUND OF THE INVENTION
Field of the Invention
[0002] The present invention relates to thin-walled microplates for
use in Polymerase Chain Reaction (PCR) reactions, comprising an
array of thin-walled tubes or wells adapted for use in a thermal
cycler. It is particularly applicable to an array of thin-walled
tubes having open tops and lower, frustoconical portions having a
substantially flat bottom.
Background
[0003] A typical human cell consists of about 6 billion base pairs
of DNA and 600 million bases of mRNA. Usually a mix containing
millions of cells is used in sequencing the DNA or RNA using
traditional methods. However by using Next Generation Sequencing
(NGS) of DNA and RNA from a single cell, cellular functions can be
investigated. In order to carry out experiments on a single cell
the following steps are required: isolation of a single cell,
nucleic acid extraction and amplification, sequencing library
preparation, sequencing and bioinformatic data analysis. Needless
to say, it is much more challenging to perform single cell
sequencing in comparison with sequencing from cells in bulk. The
minimal amount of starting materials from a single cell means that
degradation, sample loss and/or contamination can cause pronounced
effects on the quality of sequencing data. Nonetheless, recent
technical improvements make single cell sequencing a promising tool
for approaching otherwise intractable problems.
[0004] There is currently no standardized technique for single-cell
isolation. Individual cells can be collected by micromanipulation,
for example by serial dilution or by using a patch pipette or
nanotube to harvest a single cell. Currently this separation step
is generally carried out in sample tubes in which the cell(s) can
be viewed, but which are inherently unsuitable for use in a PCR
thermal cycler. Only once the operator has determined that only the
desired single cell is present is this then transferred to a PCR
tube or well for PCR and sequencing. This is because known PCR
wells are not suitable for harvesting and viewing a single cell.
This transfer from the harvesting tube to a PCR well requires an
additional manipulation and can easily give rise to the problems of
degradation, sample loss and/or contamination referred to
above.
[0005] A further problem with current PCR wells is how to apply a
marking to a PCR well to give it a unique identity. A cap to the
PCR tube can be labelled, but caps can become detached, and this is
not the same as labelling the tube itself. The upper part of the
outside of a PCR well can be labelled but this involves producing a
label on a tightly curved surface, making the machine reading of
any label problematic. Such machine reading becomes impossible if
the PCR well in question is in the middle of an array of wells, for
example a 12 by 8 array of 96 tubes or wells.
[0006] It is an object of the present invention to overcome or
mitigate some or all of the disadvantages outlined above.
SUMMARY OF THE INVENTION
[0007] According to a first aspect of the present invention there
is provided a thin-walled microplate according to Claim 1. For
example, there is described a thin-walled microplate suitable for
use in the Polymerase Chain Reaction (PCR) technique comprising a
plurality of thin-walled tubes or wells arranged in a fixed or
random access array, each well having an upper portion with an open
top and a lower, frustoconical portion having a substantially flat
bottom. By providing a flat bottomed well in an array of wells
suitable for use in the PCR technique a wide variety of new
possibilities are created. It will be understood that the bottom to
the well will have an internal surface, inside the well, and an
external surface on the outside of the well. The bottom surface of
the well can be made substantially planar, and horizontal when
stood on a horizontal surface, such that a single cell in the tube
can be viewed through the top of the tube. If the flat bottom
surface of the well is made substantially transparent, i.e.
incorporates an optically clear window, then a single cell, or some
other feature such as fluorescence within the well, can be viewed
from below the well through this optically window, as well as from
above. Or alternatively the bottom surface, preferably the external
bottom surface, may carry some form of label, preferably in the
form of a unique machine readable code.
[0008] Preferably the bottom surface of each of the wells in the
array are in substantially the same horizontal plane such that
items such as a cell or cells in the bottom of different wells in
the array are in substantially the same focal plane when viewed
from above through the opening in the top of the respective wells,
or from below in the case of a well with a substantially
transparent flat bottom window. By `in substantially the same
plane` is meant that the distance to the bottom of each well is the
same to within +/-0.1 mm, and more preferably within +/-0.05 mm or
better.
[0009] Preferably the external diameter of the flat bottom surface
or portion of each well is in the range 2.0 mm to 4.5 mm, more
preferably in the range 2.5 mm to 3.5 mm, with a particularity
preferred diameter being 3 mm.
[0010] Preferably the substantially flat bottom surface of one or
more wells in a random access array includes a machine readable
code, and more preferably substantially all the wells in the
randomly accessible array carry such a code.
[0011] Preferably the machine readable code is readable using an
optical vision system, sometimes referred to as a Machine Vision
(MV) System.
[0012] Preferably the machine readable code is on the external
surface of the substantially flat bottom surface of the well.
[0013] In a particularly preferred embodiment the flat bottom
surface or window of each well incorporates a protection ring or
bead, and preferably the protection ring/bead is located
substantially on the outer bottom peripheral circumference of the
flat bottom surface or window. This bead, ring or downstand serves
to protect the flat bottom surface of the well from accidental
damage caused by, for example, solvents or abrasion. Contact with
solvents for example might degrade any code or other label on the
bottom of the well. Any abrasion would detract from optical
measurements made through an otherwise substantially optically
transparent window, or interfere with the 2D code reading. Keeping
the flat bottom exterior surface of the well distanced away from
potential contaminants is an important feature of the
invention.
[0014] Preferably the substantially flat bottom surface of each
well is formed from substantially clear material, such that each
well has an optically-clear window. This feature, if present,
provides the advantages set out above.
[0015] Preferably the array of thin-walled tubes or wells are held
in a substantially rigid frame, and this frame may be formed from a
different plastics materials to the wells, such that the
substantially rigid frame is formed from a first plastics material
and the tubes or wells are formed from a second plastics material
that is suitable for PCR use. The wells may be permanently fixed in
the frame or may be individually removable from the frame and
therefore randomly accessible.
[0016] The summary of the invention is provided as a general
introduction to some of the embodiments of the invention, and is
not intended to be limiting. Additional example embodiments
including variations and alternative configurations of the
invention are provided herein.
BRIEF DESCRIPTION OF SEVERAL VIEWS OF THE DRAWINGS
[0017] The accompanying drawings are included to provide a further
understanding of the invention and are incorporated in and
constitute a part of this specification, illustrate embodiments of
the invention, and together with the description serve to explain
the principles of the invention.
[0018] Preferred embodiments of the present invention will now be
described by way of example only with reference to the accompanying
Figures wherein:
[0019] FIGS. 1A to 1D show side elevation, cross-sectional, bottom
elevation and perspective views respectively of a single
thin-walled PCR well:
[0020] FIG. 2 shows a perspective view of a single thin-walled PCR
well as shown in FIG. 1 with a 2D machine readable code on the
bottom of the well;
[0021] FIG. 3A to 3 H show top elevation, side cross-section along
C-C, side cross-section along B-B, an end elevation, a side
elevation, a partial view from underneath, detail F from FIG. 3B,
and a perspective view from above to one side of a 96 well random
access array of the type of wells shown in FIG. 1 in a frame.
[0022] Corresponding reference characters indicate corresponding
parts throughout the several views of the figures. The figures
represent an illustration of some of the embodiments of the present
invention and are not to be construed as limiting the scope of the
invention in any manner. Further, the figures are not necessarily
to scale, some features may be exaggerated to show details of
particular components. Therefore, specific structural and
functional details disclosed herein are not to be interpreted as
limiting, but merely as a representative basis for teaching one
skilled in the art to variously employ the present invention.
DETAILED DESCRIPTION OF THE ILLUSTRATED EMBODIMENTS
[0023] As used herein, the terms "comprises," "comprising,"
"includes," "including," "has," "having" or any other variation
thereof, are intended to cover a non-exclusive inclusion. For
example, a process, method, article, or apparatus that comprises a
list of elements is not necessarily limited to only those elements
but may include other elements not expressly listed or inherent to
such process, method, article, or apparatus. Also, use of "a" or
"an" are employed to describe elements and components described
herein. This is done merely for convenience and to give a general
sense of the scope of the invention. This description should be
read to include one or at least one and the singular also includes
the plural unless it is obvious that it is meant otherwise.
[0024] Certain exemplary embodiments of the present invention are
described herein and are illustrated in the accompanying figures.
The embodiments described are only for purposes of illustrating the
present invention and should not be interpreted as limiting the
scope of the invention. Other embodiments of the invention, and
certain modifications, combinations and improvements of the
described embodiments, will occur to those skilled in the art and
all such alternate embodiments, combinations, modifications,
improvements are within the scope of the present invention.
[0025] FIG. 3 illustrates various views of one embodiment of a
thin-walled microplate according to the present invention, in this
example a microplate containing a 12.times.8 array of 96 wells. A
12.times.8 array is given here by way of example only, and other
arrays having different configurations and numbers of wells are
possible. FIG. 1A to 1D show various views of one of the individual
thin-walled wells used in the array of wells shown in FIG. 3. The
thin-walled reaction wells shown in more detail in FIG. 1 are
designed specifically for use in performing Polymerase Chain
Reaction (PCR) experiments in a thermal cycler. Each well comprises
a hollow tube 10 having an opening 11 at a first end, a short
substantially straight sided upper section 18 adjacent to the open
end, and a frustoconical section 12 which is closed in a
fluid-tight fashion by a substantially flat bottom portion 13,
having internal and external bottom surfaces. The wells are formed
from a plastics material with good heat transfer properties and
which is suitable for PCR, such as polypropylene.
[0026] It is important that the bottom of each well is
substantially flat and substantially horizontal when the microplate
is positioned on a horizontal surface. It is also important that
the inner bottom surface of each well in an array is substantially
equidistant from the top of the microplate. By substantially
equidistance it is meant that the distance to the bottom of each
well is the same within +/-0.1 mm, and preferably +/-0.05 mm or
better. This tolerance arrangement means that an operator can view
an item, such as a single cell, resting on the flat bottom surface
of any of the wells through an optical imaging system, without
having to alter the focal plane of the instrument. This
significantly speeds up the process of checking the content of each
well.
[0027] The flat bottom surface may advantageously be made
substantially optically clear or transparent, thus creating a
substantially clear optical window in the bottom of the well. One
method of achieving this high transparency is to highly polish the
opposing surfaces of the moulding tool that form the bottom of each
well. In this way visual observations may also be made from
underneath the microplate as well as from above. Observation of
other effects such as colour changes or fluorescence from the
contents of a well may also be made from above or below the
microplate. This is the first time this has been possible in an
array of PCR wells in a microplate.
[0028] In a further embodiment, rather than having an optically
clear window on the bottom of a well, the flat-bottom surface or
portion can carry some form of label or machine readable code. One
example of this is shown in FIG. 2 which shows a 2D code 117 on the
bottom of a well. There are a wide variety of known ways to apply
machine readable data to the bottom of a test tube, but not a PCR
well. For example, U.S. Pat. No. 6,372,293B (Matrix Technologies
Corp) describes a method of applying a multi-layer coating onto a
planar exterior surface of a test tube and removing portions of the
outer layer. U.S. Pat. No. 6,270,728B (Micronic B.V) describes a
process of laser burning a code onto the base of a test tube. A
further method is to use a treatment, for example a corona
treatment, to change the properties of the exterior of the
flat-bottom surface of each well. A machine readable code can then
be applied directly onto the bottom of each treated well. This is a
particularly cost effective method of applying a code to the bottom
of each well.
[0029] The provision of PCR tubes with unique machine readable
codes represents a real advance for experimenters, particularly
when those tubes are in a random access array as illustrated in
FIG. 3. In this example the individual tubes 210 are not integrally
(permanently) fixed in the rigid frame 201, 202 but can be
individually removed from the frame and are therefore randomly
accessible. That is to say, an operator can push any one tube
upwards out of the frame in order to process that particular tube
without disturbing the other 95 tubes that are still held captive
in the frame.
[0030] It will be appreciated that in an alternative embodiment the
tubes may be integrally (permanently) fixed into a rigid frame, if
that is the format preferred by the experimenter. Methods of making
integrally (permanently) fixed arrays of wells are known, such as
those described in U.S. Pat. No. 6,340,589B1 (Eppendorf).
[0031] Whether the outer surface of the bottom of each well is
optically clear or carries a machine readable code, it is important
that the surface is protected in some way. One method of giving a
degree of protection is to incorporate a protection ring or bead
14, around part of or around the entire outer circumference of the
bottom surface, as shown in FIGS. 1B, 1D and 2. This protection
ring can be formed as a downstand around some of or substantially
the entire outer perimeter of the bottom of the well in the
moulding process.
[0032] The protection ring need not be present around the entire
circumference of the bottom of the well, and may include one or
more gaps 15 as shown in FIGS. 1D and 2. Furthermore, it need not
take the form of a conventional `ring`, but instead could take the
form of a plurality of individual protrusions set around the
perimeter of the bottom portion. The protection ring, or its
equivalent, serves to keep the outer surface of the flat-bottom
away from any surface that it might otherwise rest on. This keeps
contaminants, such as solvents, away from the machine readable code
and also prevents the surface from getting scratched or abraded.
Such abrasion would impair the quality of imaging if the bottom of
the well is optically clear, as well as potentially destroy the
integrity and readability of any code.
[0033] The diameter of the bottom surface of the frustoconical
portion of the wells is an important feature of this invention. The
diameter, which is measured to the inside edge of any protection
ring, is preferable 2.0 to 4.5 mm and more preferable 2.5 to 3.5
mm. A particularly preferred diameter for the window in the bottom
surface is 3 mm.+-.10%.
[0034] By way of example only of some other typical dimensions, the
wall thickness of the side wall of each well in the frustoconical
portion is 0.25 mm.+-.0.025 mm, which is a typical wall thickness
for carrying out PCR reactions. The thickness of the bottom portion
or surface is not critical, as it is not in direct contact with the
thermal cycler, and a typical thickness for the bottom of the well
is 0.5 mm. The angle of the side wall of each well in the
frustoconical section must correspond to the well angle found in
commercially available thermal cyclers. Typically, this angle is 17
degrees. The full well volume of a typical well is in the region of
200 microliters. The top of each well could incorporate a raised
rim or chimney if desired (not shown), to facilitate sealing the
wells with some form of sealing strip or film, but this is not
essential.
[0035] As referred to above, FIG. 3 illustrates a thin-walled
microplate 200 of the invention, containing a 12 by 8 array of 96
wells of the type illustrated in FIG. 1 or FIG. 2. As explained
above, the individual wells 210 are either fixed or removably held
captive in a substantially rigid frame made up of a skirt portion
201 and a deck portion 202. In the example illustrated in FIG. 3,
the wells are held in a randomly accessible fashion in the deck
portion by a collar 16 and a substantially planar region 18 close
to the top of each well. The substantially planar portion of each
well may incorporate a slight indentation in order that the wells
are held more tightly in the substantially rigid frame, and are
therefore less prone to being dislodged accidently during handling.
The rigid frame can be made of a first plastics material such as a
polycarbonate, a nylon or Acrylonitrile Butadiene Styrene (ABS),
and the wells formed from a second plastics material that is
suitable for PCR such as polypropylene. It will be understood that
new materials do become available over time and the optimum
materials for both components will be selected by a materials
expert as required.
[0036] In summary, in a first version of the present invention
there is provided an array of tubes with highly polished flat
optical bases, or optical windows, where the wells are
substantially permanently fixed in a rigid frame in, for example, a
12.times.8 array of 96 wells. This arrangement is of interest to
scientists who want to make optical measurements of a tube's
contents from either above or below the plate, i.e. by using a
laboratory plate reader as a pre-screen in applications like single
cell PCR procedures.
[0037] There is a growing trend in the market for scientists to
want to do single cell PCR whereby they populate a screening plate
that is not a PCR plate but in which each individual tube in the
plate has an optical base and then after correct identification of
the desired target cell transfer that cell to a PCR well for lysis
and amplification. A plate with an array of PCR wells with flat
optical windows in the bottom of each well enables scientists for
the first time to screen a plate for single cell morphology and to
confirm there is only one cell per well and then use the same plate
for performing the PCR. This offers a huge advantage in that the
scientist does not have to capture and transfer a single cell from
one plate or well to another, with the inherent possibility of
degradation, sample loss and/or contamination.
[0038] In a second version, wells incorporating optical windows in
the 12.times.8 array of wells are not integrally (permanently)
fixed in the rigid frame but can be individually removed from the
frame and are therefore randomly accessible. That is to say, an
operator can push any one tube out of the frame to process that
particular tube without disturbing the other 95 tubes that are
still held captive in the frame.
[0039] In a third version of the present invention, a flat bottom
PCR tube we can be used to carry a 2D code on the flat bottom
surface. This 2D coded PCR tube can then be tracked through the
laboratory work flow, which is becoming an important feature for
DNA storage and for the Molecular Diagnostic market. Thus, flat
bottomed PCR tubes with a modified treated bottom surface that has
been labelled with a 2D code are held in a randomly accessible
array in a substantially rigid frame, such as a 12.times.8 array of
labelled tubes, where the tubes are not integrally (permanently)
fixed in the rigid frame but can be individually removed from the
frame and are therefore randomly accessible. The key point to
labelling all 96 tubes on the underside of the flat bottom surface
is that these tubes can be randomly accessed and removed from the
array with their code intact. These random accessible 96 tubes may
be processed in a 96 well format but can then be accessed
individually in downstream processing procedures/experiments. The
flat bottom PCR tube with machine readable code on the flat bottom
surface can have the code positioned on the flat bottom surface in
such a way as to still allow the flat bottom to serve as a flat
optical window for readings.
[0040] It will be apparent to those skilled in the art that various
modifications, combinations and variations can be made in the
present invention without departing from the scope of the
invention. Specific embodiments, features and elements described
herein may be modified, and/or combined in any suitable manner.
Thus, it is intended that the present invention cover the
modifications, combinations and variations of this invention
provided they come within the scope of the appended claims and
their equivalents.
* * * * *