Patent Application
20140371083
This application is related to published United States Application 2009/0042737, filed Aug. 8, 2008, and published Feb. 12, 2009, entitled Methods and Devices for Correlated, Multi-Parameter Single Cell Measurements and Recovery of Remnant Biological Material, the entirety of which is incorporated herein by reference as if fully set forth here. No claim of priority is made to that application.
Systems, apparatus and methods are provided for biochemical analysis of a sample (e.g., a cell or nucleic acids). Samples are analyzed for molecular information and preferably remain accessible for subsequent analysis or testing. The systems, apparatus and methods provided are useful for performing quantitative and highly parallel biochemical reactions on biological samples in a high-throughput manner.
Reactions that are conducted in solution such as, for example, biochemical reactions, are frequently carried out within a chamber or other container. Such chambers, or reaction vessels, are commonly made of glass or plastic and include, for example, test tubes, microcentrifuge tubes, capillary tubes and microtiter plates. Reaction chambers currently in use are not amenable for use with volumes below one microliter, due to problems such as large head volumes in the reaction chamber leading to evaporative losses of the reaction solution, and difficulty in adding and removing reaction mixtures from the reaction chamber. Accordingly, available reaction chambers do not provide means for readily recovering reaction mixtures for subsequent testing or analysis.
Many types of biochemical reactions, for example, nucleic acid amplification, require temperature cycling. Many reaction chamber materials are poor thermal conductors, thus there are time lags associated with changing the temperature of the reaction vessel and equilibration of a temperature change throughout the sample volume. Such lags in temperature change and temperature equilibration lead to longer cycle times, non-uniform reaction conditions within a single reaction, and lack of reproducibility among multiple reactions, both simultaneous and sequential.
It is often necessary to carry out a series of experiments on a large number of identical samples. Usually this set of samples must be serially duplicated, either manually or by means of robotic liquid delivery systems. These processes can be slow, as they depend on the total number of samples to be duplicated and, if applicable, the speed of the robot. Additionally, is may be necessary to carry out multiple biochemical reactions on the same sample. Current systems do not permit serial reactions to be performed on samples.
Real-time polymerase chain reactions (qPCR) are a technique used to quantitatively measure DNA and RNA extracted from biological samples (e.g., cells or plasma). Most qPCR reactions are done in bulk reactions using the pooled genomic equivalent of 10,000 to 100,000 cells. Increasingly, researchers are interested in measuring the genetic contents of biological samples, including, for example, individual cells or free nucleic acids in plasma, but this effort is impeded by the high cost of reagents and the labor intensive manual approaches available today. Even state of the art robotics and 1536 micro-well plates use volumes in the range of 1-10 μL per well still become costly beyond a few hundred wells. In cases where rare events (e.g., rare alleles) that may occur in a small percentage of the genetic material of interest, it may be desirable to examine thousands of aliquots of the biological sample one-by-one. Current technologies cannot achieve this level of throughput without significant costs in time and money. Further, current technologies are end-point systems and do not readily allow for recovery of genetic material following biochemical reactions for subsequent testing or analysis, such as, for example, sequencing.
Thus, there is a need for systems, apparatuses, and methods suitable for performing biochemical reactions on microvolume biological samples. There is also a need for improved methods of recovering such reacted samples for subsequent analysis or testing.
The present invention provides systems, apparatus and methods for performing a biochemical reaction on a sample (e.g., a cell or nucleic acids). In certain embodiments, the invention provides an integrated system for performing a biochemical reaction on a sample preferably comprising: a reaction module, said reaction module comprising a device, said device comprising a plurality of discrete reaction volumes, said reaction volumes comprising a reagent and a sample, wherein said reaction volume is accessible to perform more than one biochemical reaction on said sample; a dispensing module for dispensing said sample in the device, wherein said dispensing module can be operably coupled with said device; a thermal module comprising a heating element and a thermal control element, wherein said thermal module can be operably coupled to said device; a detection module to detect a signal from the discrete reaction volumes; a recovery module operable to individually recover a portion of or substantially all reacted material from each of said plurality of discrete reaction volumes, wherein said recovery module can be operably coupled with the device. Optionally, the reaction module or device is adapted to receive an oil to reduce evaporation of other materials from the reaction volume. One or more oils may be utilized. The oils may be non-volatile, or have the same or differing volatility profiles.
In one aspect, said device is a slide, a micro-well plate, or a compact-disc micro-well array. In other aspects, the volume of each said reaction volume is sufficiently small to enhance reaction kinetics compared to a bulk reaction. In other aspects, said reaction volume is less than 1 microliter. In yet other aspects, said sample is a cell, a nucleic acid, a whole genome, a crude cell lysate, a buccal swab, plasma, serum, whole blood, or urine. In yet other aspects, said reagent is a lysis buffer, a neutralization buffer, or an amplification mix. In another aspect, said device is a slide, a micro-well plate, or a micro-well disc. In other aspects, said reaction volume is contained in a reaction chamber, said reaction chamber being configured to receive said reagent and said sample. In other aspects, said reagent is sufficient to perform a polymerase chain reaction (PCR) or isothermal amplification. In other embodiments, the system further comprises a sequencer for sequencing said reacted material. In other aspects, said recovery module comprises a pin tool, a pipette, a slide, or an absorbent material. In yet another aspect, said thermal module further comprises a second heating element.
In yet other aspects, the present invention further provides methods for analyzing a sample, the method preferably comprising: dispensing said sample and a reagent into a plurality of discrete reaction volumes; performing a biochemical reaction on said discrete reaction volumes; detecting a signal from said discrete reaction volumes; and recovering at least a portion of or substantially all reacted material from said discrete reaction volumes. In one aspect, the method further comprises amplifying said sample in said discrete reaction volumes. In other aspects, the method further comprises sequencing said reacted material. In yet other aspects, the method further comprises performing array comparative genomic hybridization (a-CGH) on said reacted material. In other aspects, the method further comprises genotyping said reacted material. In another aspect, the method further comprises measuring gene expression in said reacted material. Optionally, one or more oils, either volatile or non-volatile, may be provided to the reaction volume to reduce evaporation.
In yet a further aspect of the invention, an apparatus is provided for receiving a device, the apparatus comprising: a base surface and a top surface and sidewalls including at least one opening for receiving said device; a pair of spaced guide rails for contacting said device to said base and preventing contact between said device and said top surface; said apparatus configured to further receive a fluid. In one aspect, said apparatus further comprises a sealing arrangement to seal said slide into said device. In another aspect, said fluid is oil. In other aspects, said oil is a mineral oil, a silicone oil, or a fluorocarbon oil. In yet other aspects, said device is a slide.
Before the present systems, apparatus and methods are described, it is to be understood that the invention is not limited to the particular methodologies, protocols, assays, and reagents described, as these may vary. It is also to be understood that the terminology used herein is intended to describe particular embodiments of the present invention, and is in no way intended to limit the scope of the present invention as set forth in the appended claims.
It must be noted that as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural references unless context clearly dictates otherwise. Thus, for example, a reference to “a genetic condition” may include a plurality of such conditions; a reference to a “fetal genetic variation” may be a reference to one or more fetal genetic variations, and so forth.
Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods, systems, and materials are now described. All publications cited herein are incorporated herein by reference in their entirety for the purpose of describing and disclosing the methodologies, reagents, and tools reported in the publications that might be used in connection with the invention. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.
The inventions provide integrated systems, apparatus and methods for biochemical analysis of a sample (e.g., a cell or free nucleic acids). Samples are analyzed for genetic information and preferably remain accessible for subsequent analysis or testing. The methods and systems provided are useful for performing quantitative and highly parallel biochemical reactions on biological samples in a high throughput manner. In some embodiments, described more fully herein, one or more oils, either non-volatile oil or volatile oils, or a combination of multiple oils, provide an oil strategy to facilitate access to a sample following a biochemical reaction. As shown in
The present invention incorporates the use of methods and devices for dispensing portions of a sample onto or into a device 30 to form a plurality of discrete reaction volumes upon which biochemical reactions, such as, for example, PCR amplification can be performed. The methods and devices used to dispense the sample and any test reagents onto or into the device are not critical to present invention, and one of skill in the art will appreciate that many methods and devices may be used to generate such discrete reaction volumes and dispense such samples and reagents. Accordingly, the present invention is not intended to be limited to a particular method or device for generating discrete reaction volumes or conducting biochemical reactions on or in a device.
The dispensing module 20 may comprise any one of a number of commercially available reagent dispensers, piezoelectric dispensers, solenoid valve dispensers, and the like. Those of skill in the art will appreciate that other types of dispensers and valve actuation devices exist and may be used efficaciously. Examples of dispensers that may be used in the present invention include, for example, air brush dispensers, piezoelectric dispensers, fluid impulse dispensers, heat actuated dispensers, and the like. Suitable dispensers for use in the systems of the present invention are described in U.S. Patent Application Pub. No. 2010/0273680 and U.S. Pat. No. 6,576,295, both of which are incorporated by reference in their entirety.
In certain embodiments, a positive displacement syringe pump is hydraulically coupled to the dispenser. Alternatively, the pump may be any one of several varieties of commercially available pumping devices for metering precise quantities of liquid. A wide variety of other direct current fluid sources may be used, however. These may include, without limitation, rotary pumps, peristaltic pumps, squash-plate pumps, and the like, or an electronically regulated fluid current source.
In certain aspects, the sample and/or reagent is dispensed in a volume between about 1 nL to about 500 nL. Discrete reaction volumes generated using the dispensing module 20 described herein may be formed with dispensed sample and/or reagent volumes as small as about 1 nL, 5 nL, 10, nL, 25 nL, or 50 nL. Typically, reaction volumes may range from about 1 nL to about 250 nL, although particular reaction volumes range from about 10 nL to about 250 nL; more particular reaction volumes range from between about 50 nL to about 250 nL. This small reaction volume allows multiple discrete reaction volumes to be applied to a single device 30, such as, for example, a microscope slide, so that between about 1-5, 5-10, 10-100, 500-1000 or more, discrete samples, including a combination of control and test samples, may be formed on or in the device 30. The small sample volume also significantly reduces the amount of test reagent that is necessary for the biochemical reaction, thereby further decreasing the overall cost of conducting the biochemical reaction while at the same time increasing the fidelity of the results of the biochemical reaction because the test and control samples can be processed under identical conditions. As compared to the volume of a prior art bulk reaction, the preferred reaction volumes of the invention is from about 1:20 to 1:100,000 times smaller. Further, since reaction volumes contained in or on a device of the present invention remain accessible, multiple biochemical reactions may be carried out with the subsequent dispensing of additional sample or reagent(s).
Several suitable syringe pumps are commercially available to one of skill in the art. For example, the Biodot CV1000 Syringe Pump Dispenser, available from Biodot, Inc. (Irvine, Calif.) incorporates an electronically controlled stepper motor for providing precision liquid handling using a variety of syringe sizes. Such syringe pumps may have anywhere from 3,000-24,000 steps, although higher resolution pumps having 48,000-768,000 steps may be used in the integrated systems of the present invention. Higher resolution pumps, such as, for example, piezoelectric pumps may also be used to provide even finer resolutions, if desired. Multiple syringe pumps may be employed in parallel, for example, to deliver varying concentrations of reagents and/or other liquids to the dispenser or to alternate dispensing operations between two or more reagents as may be desired when conducting the biochemical reactions contemplated by the present invention.
Pin tools may be used to dispense sample and reagent(s) in the systems of the present invention. Pin tools refer to devices that are capable of transferring substantially all or a portion of fluid from a fluid source to a fluid destination without drawing the aliquots from the fluid source under an applied pressure. An exemplary pin tool useful in the methods and systems of the present invention is shown in
The devices used in the methods and systems of the present invention are configured to receive a plurality of discrete reaction volumes. In some embodiments, the discrete reaction volumes remain accessible while in or on the device. For example, following a biochemical reaction on a plurality of discrete reaction volumes in a micro-well plate, the discrete reaction volumes remain accessible for recovery or for addition of reagent for performing another biochemical reaction.
Devices useful in the methods and systems of the present invention include planar surfaces, such as, for example, glass or plastic (e.g., polymer) slides.
As shown in
In some embodiments, the surface of the slide is homogenous. In other embodiments, the surface of the slide is pretreated or coated with a hydrophobic or hydrophilic material. In one aspect, the hydrophobic material is PDMS. In other aspects, the hydrophilic material is PDMS with subsequent oxygen or air plasma treatment, plasma treatment with a mask (i.e., selective treatment or patterning), or silanization. Once the sample and reagent(s) have been dispensed onto the planar surface (e.g., slide surface), oil or any other suitable encapsulation media is applied to isolate the discrete reaction volumes arrayed on the planar surface (See, e.g.,
The contact angle and wetting between the reaction volume and planar surface are selected to prevent movement of the discrete reaction volumes on a slide. (See, e.g.,
In certain embodiments, the device used in the systems and methods of the present invention is a microwell plate such as, for example, a nanowell array plate.
In other embodiments, the device used in the systems and methods of the present invention is a compact disc (CD) micro-well array. An exemplary embodiment of a CD micro-well array is shown in
In certain aspects, the present invention provides apparatus for receiving a device used in the methods and systems of the present invention.
In a biochemical reaction, for example, in a PCR thermal cycling reaction, pouch 300 can be arranged and in contact with a first heating and cooling unit positioned on a first side 320, and a second heating and cooling unit comprising a heat-transfer surface in contact with side 330 of pouch 300. In some embodiments, the heating and cooling units can each comprise a Peltier unit
Any effective temperature that will support the desired biochemical reaction may be employed in the isothermal biochemical reactions of this invention. Accordingly, the isothermal reactions may be conducted at any substantially constant and effective temperature, including at about 20° C., 21° C., 22° C., 23° C., 24° C., 25° C., 26° C., 27° C., 28° C., 29° C., 30° C., 31° C., 32° C., 33° C., 34° C., 35° C., 36° C., 37° C., 38° C., 39° C., 40° C., 41° C., 42° C., 43° C., 44° C., 45° C., 46° C., 47° C., 48° C., 49° C., 50° C., 51° C., 52° C., 53° C., 54° C., 55° C., 56° C., 57° C., 58° C., 59° C., 60° C., 61° C., 62° C., 63° C., 64° C., 65° C., 66° C., 67° C., 68° C., 69° C., 69° C., 70° C., 71° C., 72° C., 73° C., 74° C., 75° C., and the like.
In other embodiments, thermal cycling conditions are provided by a thermal module of the present invention. For example, in embodiments performing biochemical reactions such as DNA amplification via PCR, the temperature is cycled to produce suitable temperatures for the desired number of PCR cycles. This may be accomplished with a standard thermal cycler using a heat block or Peltier device, or it may be accomplished with alternative technologies such as an oven, hot and cold air, flowing a heated liquid with good thermal conductivity, transferring the device between instrument components held at different temperatures or any other suitable heating elements known in the art.
In some embodiments, the systems and methods of the present invention comprise a detection module, the detection module comprising one or more optical, electrical, or chemical detectors. In certain embodiments the optical detector is a scanning detector or alternatively a stationary detector. In other embodiments, the optical detector is a photomultiplier tube, a CCD camera, photodiodes or photodiode arrays. In some aspects, the detector measures the intensity of the fluorescence signal from a discrete volume reaction.
In some embodiments, recovery modules are used to recover a portion or substantially all of an individual reaction volume for subsequent analysis and/or further biochemical reactions. Material recovery following one or more biochemical reactions may be achieved using any of the dispensing devices described herein. Material recovered from the discrete reaction volumes may optionally be used in subsequent assays or additional biochemical reactions. In some embodiments, capillary action may be used to recover a liquid in the systems and methods of the present invention. For example, a capillary tube can brought into contact with a target volume reaction. When the capillary tube contacts the volume reaction, the target volume reaction is recovered by capillary action without active control, such as a vacuum. Hydrophilic materials such as PMMA or glass can be used to enhance capillary action. In this aspect, the tube is transparent and thus the volume of the recovered material can be quantified. In other embodiments, recovered reaction volumes can be dispensed to a second device for subsequent assays or biochemical reactions by, for example, applying pressure on the tubing using a pipette or syringe.
In other embodiments, the pin tools described above are used to recover material following one or more biochemical reactions. (See, e.g.,
In yet other embodiments, an absorbent (e.g., porous) material is used in the systems and methods of the present invention to recover material following one or more biochemical reactions.
In other embodiments, recovery or splitting of a plurality of reaction volumes is achieved by bringing a first slide with a plurality of reaction volumes into contact with a second slide. As shown in
In other embodiments, the dispensers described above are used in the systems and methods of the present invention to recover material following one or more biochemical reactions.
The present invention provides methods for analyzing a sample, the method comprising: dispensing said sample and a reagent into a plurality of discrete reaction volumes; performing a biochemical reaction on said discrete reaction volumes; detecting a signal from said discrete reaction volumes; and recovering a portion of or substantially all reacted material from said discrete reaction volumes. In some embodiments, the discrete reaction volumes remain accessible for subsequent analysis or testing. In other embodiments, the discrete reaction volumes are not sealed.
The present invention further provides methods for performing serial or multiple biochemical reactions on a sample or a discrete reaction volume. As shown in Example 2 below, a plurality of discrete reaction volumes were subjected to a first whole genome amplification reaction and a second whole genome amplification. In some embodiments, the present invention provides methods for splitting, aliquoting, or recovering a sample to or from a device. For example, a pin tool as described above may be used to recover a portion or substantially all of a reaction volume following a biochemical reaction.
In some embodiments, the discrete reaction volumes are encapsulated by applying a hydrophobic encapsulation media to the discrete reaction volume. In some aspects, the oil is a volatile oil. Use of volatile oils in the systems and methods of the present invention provide access to the reaction volumes for further analysis or the introduction of additional reagents. For example, the evaporation of a volatile oil overlay can be controlled to allow for the addition of multiple reagents to reaction volumes or recovery of reaction volumes following a biochemical reaction. In some aspects, different molecular weight oils are used to provide a variety of vapor pressures, or different evaporation rates. For example, 0.65 cSt silicone oil evaporates faster than 5 cSt silicone oil. Accordingly, 0.65 cSt silicone oil can be used for low temperature incubations or high temperature incubations for a short period of time. For biochemical reactions requiring longer high temperature incubation times, 5 cSt silicone oil or high molecular weight silicone oil may be used (instead of 0.65 cSt silicone oil which has a higher vapor pressure) at high temperature. Following incubation the 5 cSt silicone oil or other high molecular weight oil can be removed by introducing 0.65 cSt silicone oil. The 0.65 cSt silicone oil is subsequently removed via evaporation following exposure to air which provides access to the reaction volumes and allows for the dispensing of additional reagents or recovery of the reaction volumes. The methods and techniques for use of one or more oils described in this methods section are applicable to the systems and apparatus described in this application.
The present invention provides methods for performing a biochemical reaction on a plurality of reaction volumes. In some embodiments, the invention further provides methods for sequencing said reaction volume. For example, following one or more biochemical reactions individual reaction volumes may be sequenced in a microreactor. Sims et al. describe techniques for fluorogenic DNA sequencing in microreactors. (Sims et al. (2011) Nature Methods 8:575-80.) In other embodiments, the invention further provides methods for amplifying said sample in said discrete reaction volumes. In yet other embodiments, the invention provides methods for performing array comparative genomic hybridization (a-CGH) on said reacted material. In other embodiments, the invention provides methods for genotyping said reacted material. In some embodiments, the invention further provides methods for measuring gene expression in said reacted material.
The systems and methods described herein can be employed in a wide variety of applications. In some embodiments, the systems and methods of the present invention are used to detect alleles found in a fetal genome. Currently known prenatal diagnostic methods typically involve invasive techniques such as amniocentesis, the removal chorionic villi and the removal of fetal blood or tissue biopsies. Non-invasive methods based on enriching maternal blood samples for fetal cells and analyzing the population of cells in the sample to identify fetal cells have been described. (See International Publication Nos. WO 2008/048931 and WO 2010/075459.) U.S. Pat. No. 6,258,540 discloses a method of performing a prenatal diagnosis on a maternal blood sample, which method comprises obtaining a non-cellular fraction of the blood sample, amplifying a paternally inherited nucleic acid from the non-cellular fraction and performing nucleic acid analysis on the amplified nucleic acid to detect paternally inherited fetal nucleic acid. The methods described herein, in particular embodiments, may be used for the biochemical analysis of fetal genomic DNA in biological samples, such as, for example, maternal blood or plasma.
The present systems and methods can be combined with methods known in the art for determining fetal gene variants. For example, Oliphant, International Publication No. WO 2010/075459, the contents of which are incorporated by reference herein in its entirety, discloses a method in which a maternal sample is genotyped, a mixture of maternal and fetal cells is obtained, and the sample is concentrated for fetal cells and divided into subsamples. A panel of at least one target locus at which the maternal sample is homozygous is selected for screening or genotyping of the subsamples. Each of the subsamples is individually screened or genotyped at at least one of these loci, with detection of a heterozygous genotype indicating the presence of a non-maternal allele in the subsample. Alternatively, a panel of at least one target locus at which the maternal sample is heterozygous is selected for screening or genotyping of the subsamples. Each of the subsamples is individually screened or genotyped at at least one of these loci, with detection of a homozygous genotype indicating the presence of a non-maternal allele in the subsample. The systems and methods for biochemical analysis described herein are particularly suited for use in that and other methods described in Oliphant, and provide a way of detecting and analyzing fetal DNA in maternal samples, particularly by amplification and detection of fetal genomic material.
A biological sample was analyzed for the presence of a rare allele (e.g., a fetal allele). Twenty microliters of an admixture of maternal and fetal DNA (0.5 ng/uL) was mixed with 35 uL of 2× Genotyping Master Mix (Part No. 4371353, Life Technologies, Carlsbad, Calif.), 1.75 uL of Taqman SNP genotyping pre-mix (40×, dbSNP:rs4144457) and 13.25 uL of DEPC H2O. The resulting volume was dispensed with a dispenser (Biodot, Irvine, Calif.) onto a COP slide (Zeon Chemicals, Louisville, Ky.) in a 20×40 array to form 800 discrete reaction volumes of 30 nL. After dispensing, the slide was inserted into a plastic sleeve of the present invention containing 1 mL of FC40 oil (3M, St. Paul, Minn.). A plastic cap was used to enclose the slide in the sleeve. The oil encapsulation prevents reaction volume evaporation and cross-contamination. Guide rails in the sleeve prevented contact of the discrete reaction volumes with the top surface of the sleeve. The sleeve was then inserted into a thermal cycler and subjected to the following heating cycles: 1 cycle of 50° C. for 2 minutes, 1 cycle of 95° C. for 10 minutes, and 46 cycles of 95° C. for 15 seconds and 60° C. for 1 minute. Following thermal cycling, the slide was removed from the sleeve and exposed to air to evaporate the oil, and placed in an imaging module to detect fluorescent signals (VIC and FAM) from the discrete reaction volumes contained on the surface of the slide.
The results are shown in
Fresh human cells (9802 cells) were resuspended in PBS (0.1 cell/nL) and dispensed in 10 nL aliquots into each well of a 32×32 multi-well plate using a dispenser (Biodot, Irvine, Calif.) to deliver, on average, a single cell to each well in the plate to form discrete reaction volumes. Next, 20 nL of a lysis buffer (200 mM KOH and 83 mM DTT) was dispensed into each well. The plate was inserted into a pouch of the present invention containing 5.0 cSt silicone oil, thereby encapsulating the reaction volume in each well. The oil encapsulation prevents reaction volume evaporation and cross-contamination. The pouch was heat sealed and the reaction volumes were subjected to a first biochemical reaction (i.e., cell lysis) at 65° C. for 10 minutes. Following incubation, the plate was removed from the pouch and introduced into another pouch with 0.65 cSt silicone oil and gently agitated for 5 minutes to dilute the 5 cSt silicone oil present on the plate. The plate was removed from the second pouch and the 0.65 cSt silicone oil on the plate was evaporated to expose the reaction volume in each well. Next, 20 nL of neutralization buffer (900 mM Tris and 200 mM HCl) was dispensed into each well followed by dispensing of 150 nL of a WGA reaction mix consisting of phi29 polymerase, random primers, 25 mM dNTPs, and a salt mix. Following the dispensing, the plate was introduced into a pouch with 0.65 cSt silicone oil, the pouch was heat sealed, and the reaction volumes were subjected to a second biochemical reaction (whole genome amplification, WGA) at 30° C. for 4 hours followed by 65° C. for 10 minutes to inactivate phi29 polymerase (see
For genomic analysis, PCR was carried out on the reaction volumes following WGA. Briefly, the plate was removed from the pouch and exposed to air to remove the 0.65 silicone oil. A 32×32 pin array was introduced into the 32×32 well plate, removed, and dipped into a second 32×32 well plate that was prefilled with 150 nL of duplex PCR master mix containing primers and probes for the SRY gene (Y chromosome) and the X2 gene (X chromosome). The transfer process was repeated for a replicate plate to validate the whole genome amplification reaction. The reaction volumes in the two PCR plates were encapsulated with a 5 cSt oil overlay and incubated at 95° C. for 10 minutes followed by 50 thermal cycles of 15 seconds at 95° C. and 60 seconds at 60° C. Following PCR, FAM and VIC fluorescence images of the plates were taken and locations of the 9802 cells were identified by double positive signals of FAM and VIC (see
These results showed that the systems and methods in embodiments of the present invention are useful for performing serial biochemical reaction on cells and subsequent genomic analysis. These results also showed that the systems and methods in embodiments of the present invention are useful for performing quantitative and highly parallel biochemical reactions on biological samples in a high throughput manner.
These and other embodiments of the present invention will readily occur to those of ordinary skill in the art in view of the disclosure herein. The present invention is not limited in scope by the exemplified embodiments, which are intended as illustrations of single aspects of the invention only. Any apparatus or methods that are functionally equivalent are within the scope of the invention. Various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications fall within the scope of the appended claims.
All the references referred to herein are incorporated by reference in their entirety for the subject matter discussed. The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention.
This is an international filing claiming priority to and benefit of U.S. Provisional Patent Application No. 61/507,966, filed Jul. 14, 2011, entitled “Systems, Apparatus and Methods For Biochemical Analysis” (Ref. 144,717-025), which is hereby incorporated by reference in its entirety.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US12/46776 | 7/13/2012 | WO | 00 | 7/28/2014 |
| Number | Date | Country | |
|---|---|---|---|
| 61507966 | Jul 2011 | US |
