Patent Description:
A typical human cell consists of about <NUM> billion base pairs of DNA and <NUM> 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 cut 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 challeng ng 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, recert technical improvements make single cell sequencing a promising tool for approaching otherwise intractable problems.

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 unsu table 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 anc 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.

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 <NUM> by <NUM> array of <NUM> tubes or wells.

It is an object of the present invention to overcome or mitigate some or all of the disadvantages outlined above.

<CIT> discloses a collection/extraction container for collecting solid forensic samples and/or for extracting biological material from these solid forensic samples and its use.

<CIT> discloses a method and procedure for colour coding cuvettes used in laboratory tests.

<CIT> discloses a cell screening method having: a step in which a target cell is sorted from a plurality of cells into a first tray having an array share wherein a plurality of containers are arrayed; a step in which the cell sorted into the container is imaged; and a step in which on the basis of the image captured in the imaging step, the contained into which the cell has been sorted is separated from the first tray and relocated to a second tray.

<CIT> discloses a two-dimensional code anti-theft sample container.

<CIT> discloses a system and method for storage and transportation of process tubes in a carrier tray. The process tube includes a securement region having an annular ledge, a neck, and a protrusion.

According to a first aspect of the present invention there is provided a thin-walled microplate according to Claim <NUM>, being a thin-walled microplate suitable for use 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 is 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.

The bottom surface of each of the welts 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 +/- <NUM> and more preferably with n +/- <NUM> or better.

The external diameter of the flat bottom surface on portion of each well is in the range <NUM> to <NUM>, more preferably in the range <NUM> to <NUM>, with a particularly preferred diameter being <NUM>.

Preferably the substantially lat 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 cede.

Preferably the machine readable code is readable using an optical vision system sometimes referred to as a Machine Vision (MV) System.

Preferably the machine readable code is on the external surface of the substantially flat bottom surface of the well.

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 cr 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.

Preferably the substantia ly flat bottom surface cf 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.

Preferably the array of thin-walled tubes or wells are he d 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.

Preferred embodiments of the present invention will now be described by way of example only with reference to the accompanying Figures wherein:-.

<FIG> illustrates various views of one embodiment of a thin-walled microplate according to the present invention, in this example a microplate containing a 12X8 array of <NUM> wells. A 12X8 array is given here by way of example only, and other arrays having different configurations and numbers cf wells are possible. <FIG> show various views of one of the individual thin-walled wells used in the array of wells shown in <FIG>. The thin-walled reaction wells shown in more detai' in <FIG> are designed specifically for use in performing Polymerase Chain Reaction (PCR) experiments in a thermal cycler. Each well comprises a hollow tube <NUM> having an opening <NUM> at a first end, a short substantially straight sided upper section <NUM> adjacent to the open end, and a frustoconical section <NUM> which is closed in a fluid-tight fashion by a substantially flat bottom portion <NUM>, 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.

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 substant ally 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 +/- <NUM>, and preferably +/- <NUM> 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.

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 m croplate 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 fast tine this has been possible in an array of PCR wells n a microplate.

In a further embodiment, rather than having an optically clear window or the bottom of a well, the flat-bottom surface or portion can carry some form of abel, or machine readable code. One example of this is shown in <FIG> which shows a 2D cede <NUM> on the bottom of a well. There are a wide variety of known ways to apply machine readable data :o the bottom of a test tube, but not a PCR well. For example, <CIT> 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. <CIT> describes a process of aser burning a code onto the base of a test :ube. 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.

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>. In this example the individual tubes <NUM> are not integrally (permanently) fixed in the rigid frame <NUM>, <NUM> 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 <NUM> tubes that are still held captive in the frame.

t 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 <CIT>).

Whether the outer surface of the bottom of each well is optically clear or carries a machine readable code, it is important that the surace is protected in some way. One method of giving a degree of protection is to incorporate a protection ring or bead <NUM>, <NUM> around part of or around the entire outer circumference of the bottom surface, as shown in <FIG>. This protection ring is formed as a downstand around some of or substantially the entire outer perimeter of the bottom of the well in the moulding process.

The protection ring need not be present around the entire circumference of the bottom cf the well, and may include one or more gaps <NUM> as shown in <FIG> Furthermore, it need not take the form of a conventional 'ring', but instead could take the form of a plurality of individual protrusions se: 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 cf any code.

The diameter of the bottom surface of the frustocorical 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 <NUM> to <NUM> and more preferable <NUM> to <NUM>. A particularly preferred diameter for the window in the bottom surface is <NUM> ± <NUM>%.

The wall thickness of the side wall of each well in the frostoconical portion is <NUM> ± <NUM>, 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 cycer, and a typical thickness for the bottom of the well is <NUM>-n. The angle of the side wall of each well in the frustoconical section must correspond to the well angle found in commercially availab'e thermal cyclers. Typically this angle is <NUM> degrees. The full well volume of a typical well is in the region of <NUM> microlitres. 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.

As referred to above, <FIG> illustrates a thin-walled microplate <NUM> of the invention, containing an <NUM> by <NUM> array of <NUM> wells of the type illustrated in <FIG>. As explained above the individual wells <NUM> are removably held captive in a substantially rigid frame made up of a skirt portion <NUM> and a deck portion <NUM>. in the example illustrated in <FIG>, the wells are held ir a randomly accessible fashion in the deck portion by a collar <NUM> and a substantially planar region <NUM> 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 wil 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.

In an example, not forming part 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 12X8 array of <NUM> 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 reacer as a pre-screen in applications like single cell PCR procedures.

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 n 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.

Wells incorporating optical windows in the 12X8 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 <NUM> tubes that are still held captive in the frame.

Claim 1:
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 random access array in a substantially rigid frame, each well having an upper portion with an open top; a lower, frustoconical portion of thickness of <NUM>±<NUM>, and a substantially flat bottom, the substantially flat bottom of each of the wells in the array having an external diameter in the range <NUM> to <NUM> and an internal flat bottom surface, viewable through the open top of the well, that extends on a substantially horizontal plane such that the internal flat bottom surface of each well is substantially equidistant from the top of the microplate in order that items retained along the internal flat bottom surfaces of the 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, and an external surface of the substantially flat bottom on each well incorporating a protection ring or bead axially down from the external surface to protect the external surface, wherein the wells are not integrally fixed in the rigid frame but can be individually removed from the frame and therefore randomly accessible and wherein the substantially flat bottom surface of each well in the array incorporates a <NUM>-D machine-readable code that is readable using an optical vision system.