UNHEATED EXTRACTION OF GENOMIC DNA IN AN AUTOMATED LABORATORY SYSTEM

Abstract

A method for analyzing genomic DNA includes introducing a plurality of samples comprising human cells into individual vessels in each of a plurality of multi-vessel well plates. At least a subset of the human cells in the plurality of samples is lysed without the use of heat. DNA in the at least a subset of lysed human cells is isolated with the use of a plurality of paramagnetic beads. The isolated DNA is analyzed to identify one or more single nucleotide polymorphisms (SNPs), wherein the lysing, isolating, and analyzing steps are performed substantially in parallel for each of the plurality of samples.

Claims

1. A method for analyzing genomic DNA, the method comprising: introducing a plurality of samples comprising human cells into individual vessels in each of a plurality of multi-vessel well plates; lysing at least a subset of the human cells in the plurality of samples without the use of heat; isolating DNA in the at least a subset of lysed human cells with the use of a plurality of paramagnetic beads; and analyzing the isolated DNA to identify one or more single nucleotide polymorphisms (SNPs), wherein the lysing, isolating, and analyzing steps are performed substantially in parallel for each of the plurality of samples.

2. The method of claim 1, wherein the lysing further comprises introducing one or more chemical reagents to the plurality of samples.

3. The method of claim 2, wherein the one or more chemical reagents comprises a chaotropic salt solution.

4. The method of claim 2, wherein the one or more chemical reagents comprises a protease enzyme.

5. The method of claim 4, wherein the protease enzyme is Proteinase K.

6. The method of claim 1, further comprising labeling each of the plurality of multi-vessel well plates with a barcode.

7. The method of claim 6, further comprising scanning the barcodes and associating the barcodes with a unique patient sample identifier in a computing device.

8. The method of claim 7, further comprising tracking each of the plurality of multi-vessel well plates with the computing device as the plurality of multi-vessel well plates are processed by an automated liquid handling system.

9. The method of claim 1, further comprising performing the introducing, lysing, isolating, and analyzing steps for at least 4000 samples in a 24 hour period.

10. The method of claim 1, further comprising analyzing the one or more SNPs to assess cardiovascular health, effectiveness of a cardiovascular disease treatment, or a risk of developing cardiovascular disease for a subject.

11. The method of claim 1, further comprising analyzing the one or more SNPs to assess diabetes, effectiveness of a diabetes treatment, or a risk of developing diabetes for a subject.

12. The method of claim 1, further comprising analyzing the one or more SNPs to assess fatty liver health, effectiveness of a fatty liver disease treatment, or a risk of developing fatty liver disease for a subject.

13. The method of claim 1, wherein the one or more SNPs are selected from the group consisting of APOE 112, APOE 158, MTHFR C677T, FII, FVL, CYP2C19*2, CYP2C19*3, CYP2C19*17, CYP2C9*2, CYP2C9*3 and VKORC1.

14. The method of claim 1, wherein the plurality of samples are selected from the group consisting of body fluids, body wastes, body excretions, and blood.

15. The method of claim 1, wherein the plurality of samples comprise blood.

16. The method of claim 1, wherein the introducing, lysing, isolating, and analyzing steps arc performed at room temperature.

17. The method of claim 1, wherein one or more of the introducing, lysing, isolating, and analyzing steps are performed in a single liquid sample handling instrument.

18. The method of claim 1, wherein each of a plurality of multi-vessel well plates comprise 96-sample multi-vessel well plates.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0021] FIG. 1 is a flowchart of an exemplary method for unheated extraction of genomic DNA;

[0022] FIG. 2 is an exemplary liquid handling system with an exemplary two-dimensional scanner;

[0023] FIG. 3 is an exemplary liquid handling system with exemplary magnetic devices;

[0024] FIG. 4 is an exemplary liquid handling system with exemplary source carriers; and

[0025] FIG. 5 is an exemplary liquid handling system with exemplary shaker devices.

DETAILED DESCRIPTION

[0026] Referring to FIG. 1, a flowchart of an exemplary method for unheated extraction of genomic DNA is illustrated. The steps of the method described and illustrated below with reference to FIG. 1 are performed at room temperature, without the introduction of heat from an external source. Additionally, at least steps 102-106 of the method described and illustrated with reference to FIG. 1 are performed in a single liquid sample handling instrument, such as a MicroLab Star platform liquid handling system available from Hamilton Robotics, Inc. of Reno, Nev., although other liquid handling systems can also be used. The method of the present invention is particularly useful for providing rapid extraction of DNA from human clinical samples for use in a high throughput screening assay as, for example, an assay to detect SNPs in the genome of a patient.

[0027] In step 100 in this example, one or more multi-vessel well plates, and/or associated vessels, are labeled with a bar code that is associated in a computing device with a unique patient or subject identifier. By labeling each of the multi-vessel well plates, the multi-vessel well plates can be tracked using the computing device as the multi-vessel well plates are processed. An exemplary two-dimensional scanner 200 of a liquid handling system is shown in FIG. 2. Optionally, four multi-vessel well plates are used for each iteration of the steps described and illustrated with reference to FIG. 1. In this example, the well plates are 96-sample multi-vessel well plates, although other numbers of well plates and other sizes of well plates can also be used.

[0028] In step 102 in this example, samples including human cells are introduced into the individual vessels in each of the plurality of labeled multi-vessel well plates. In this example, the samples including the human cells include body fluids, body wastes, body excretions, or blood, although other sample types can also be used.

[0029] In step 104 in this example, at least a subset of the human cells in the plurality of samples is lysed without the use of heat. In one example, the lysing includes introducing one or more chemical reagents to the plurality of samples. Exemplary chemical reagents can include a chaotropic salt solution, a protease enzyme, such as Proteinase K, or a combination thereof. Other chemical reagents and other protease enzymes can also be used.

[0030] In an unheated extraction step, a detergent solution is applied to the sample to effect cell lysis at room temperature. In some cases, the detergent concentration may be increased from that used in a heated method. Detergent and proteinase concentration may both be increased to complete unheated extraction. Generally, SDS (Sodium dodecyl sulfate) is used as an extraction detergent. A more aggressive detergent may be substituted into a lysis buffer or additional extraction reagents may be added, including deoxycholate, cholate, sarkosyl, triton X-100, DDM (n-Dodecyl -D-maltoside), digitonin, tween 20, tween 80, CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate), and/or urea.

[0031] In some cases, the sample may be mixed with a first portion of detergent solution, agitated, mixed with a second portion of detergent solution and agitated again, such that repetition of detergent and agitation steps may replace a heating step. Repetitive aspiration by pipette can facilitate lysis instead of, or in addition to repetitive detergent additions. Subsequent to or at the same time as the detergent addition, proteinase K may be added to the sample solution to break down protein contaminants in solution. Typically, a heating step facilitates proteinase K activity through the denaturation of proteins in solution. In the high-throughput, automated system and method described here, however, the reagents may be applied at room temperature, or unheated, to conserve resources.

[0032] The removal of heating steps increases throughput by decreasing overall extraction time from at least 2.5 hours per incubation to less than 2 hours per incubation. The complete unheated extraction facilitates DNA extraction from a plurality of samples in a plurality of vessels in less than 2 hours. The samples may be in a 96-sample well container, with a plurality of 96-well containers per extraction run on the automated liquid handling system. In an 8-hour shift, at least one additional extraction run may be completed using the unheated method versus the standard heated method with a potential for 384 additional samples extracted in a sample handling unit handling 4 96-well plates. In a 24-hour period, 2-3 additional extraction runs may be completed, with a potential of >1000 additional samples extracted by each sample handling unit handling 4 96-well plates. The unheated high-throughput DNA extraction system and method may therefore facilitate extraction of 1000, 2000, 3000, 4000 or more samples on parallel liquid handling systems per 24-hour period.

[0033] In step 106 in this example, DNA in the at least a subset of lysed human cells is isolated with the use of a plurality of paramagnetic beads. The paramagnetic beads can be Mag-Bind beads available from Omega Bio-Tek Inc. of Norcross, Ga., although other paramagnetic beads can also be used. The paramagnetic beads can be introduced to the multi-vessel well plates and attracted to four magnetic devices on each carrier of the liquid handling system. Exemplary magnetic devices 300(1)-(4) of a liquid handling system are shown in FIG. 3. Accordingly, the liquid handling system can include a magnetic device 300(1)-(4) for each of the four multi-vessel well plates used in this example. Each of the magnetic devices 300(1)-X(4) includes 24 magnetic vertical prongs and, accordingly, each prong fits between four wells on the 96-sample multi-vessel well plates.

[0034] The paramagnetic beads of the vertical prongs in this example are static, although in other examples other orientations and/or other mobile magnets or paramagnetic particles could also be used. The paramagnetic beads in this example allow for rapid isolation of high quality genomic DNA from 1-200 L of whole blood samples utilizing reversible binding properties. The isolated DNA can be used without modifications in downstream applications such as Polymerase Chain Reaction (PCR), for example.

[0035] Optionally, unbound substances such as proteins, polysaccharides, and cellular debris, for example, are removed by a high salt wash and/or an ethanol wash, for example, although other methods for washing unbound substances can also be used. The isolated DNA can then be eluted from the paramagnetic beads in a low ionic strength buffer, for example, although other elution methods can also be used.

[0036] In step 108, the isolated DNA is analyzed to identify one or more single nucleotide polymorphisms (SNPs). The SNPs can include APOE 112, APOE 158, MTHFR C677T, FII, FVL, CYP2C19*2, CYP2C19*3, CYP2C19*17, CYP2C9*2, CYP2C9*3, and/or VKORC1, for example, although other SNPs can also be identified from the isolated DNA.

[0037] In step 110, the one or more SNPs identified in step 108 are analyzed to assess disease state, effectiveness of disease treatment, and/or risk of developing a disease, for example. Exemplary diseases can include cardiovascular disease, diabetes, or fatty liver disease, for example, although the SNPs can also be used to asses other diseases.

EXAMPLE 1

[0038] In one exemplary implementation of steps 102-106 of FIG. 1, the supplies, equipment, and reagents included in Table 1 are used, although other supplies, equipment, and/or reagents from other vendors could also be used.

TABLE-US-00001 TABLE 1 Supplies CO-RE Tips 12 480 Standard Volume (300 L) with Filter CO-RE Tips 8 480 Standard Volume (1000 L) with Filter Reagent container (50 mL) Waste bags Cap Holder Racks 2 10 RNAse/DNAse/Pyrogen-free Matrix 0.5 mL 2D Screw tubes PP, V Bottom with Cap-Latch Rack Plate, 96 Deep Well, 1.2 mL Axygen Reservoir 96 Row, Pyramid Bottom, Single Well, Sterile Thermo Clear Seal 3730 BD Sterile Culture Tubes 12 75 Adhesive Covers (similar alternative is suitable) Equipment list MicroLab Star/StarLet Liquid Handling System (Hamilton Robotics, Inc.) ALPS-3000 Heat Sealer (Thermo Fisher Scientific Inc.) Compact 106 Air Compressor InfinityXL Platform Rocker (Next Advance, Inc.) Nexar (Douglas Scientific) Capper/Decapper unit (Hamilton Robotics, Inc.) Reagents Mag-Bind Blood DNA HDQ Kit and Proteinase K (Omega Bio-Tek Inc.) Ethanol (Anhydrous Alcohol) C2H5OH (IBI Scientific) Isopropyl Alcohol (Isopropanol) C3H7OH (IBI Scientific) Molecular grade (nuclease free) glass distilled reagent water (Teknova)

[0039] In this example, in step 102, 96-sample multi-vessel well plates with blood are loaded into a source carrier of a liquid handling system. Exemplary source carriers 400(1)-400(4) of a liquid handling system are shown in FIG. 4. Next, the well plates are transported to a shaking device, shaken, and transported back to the source carrier. Exemplary shaker devices 500(1)-500(4) of a liquid handling system are shown in FIG. 5.

[0040] In step 104 in this example, a lysis buffer containing a chaotropic salt, such as guanidinium hydrocholoride, is added to a reagent reservoir of the liquid handling system, which then aspirates the reagent and dispenses into the well plates. The well plates are then transported by the liquid handling system to shakers, shaken, and transported back to the source carrier.

[0041] In step 106 in this example, a Mag-Bind HDQ mix (prepared as a mastermix with HDQ Beads, Isopropanol and HDQ binding buffer) is added to a reagent reservoir of the liquid handling system. The liquid handling system then aspirates the HDQ Mix and dispenses into the well plates. Next, the well plates are transported to shakers, shaken, and transported to magnets for magnetic separation, and then the liquid handling system then aspirates waste from the well plates.

[0042] In this example, aqueous Guanidine Hydrochloride solution (VHB) buffer is then added to the reagent reservoir of the liquid handling system which then aspirates and dispenses into the well plates. Subsequently, the well plates are transported to shakers, shaken, and transported to the magnets for magnetic separation, and then the liquid handling system aspirates waste from the well plates. Optionally, more VHB buffer can be added and the aspirating, dispensing, transporting to the shaker, shaking, and transporting to the magnets, and aspirating waste steps can be repeated one or more times.

[0043] Subsequent to utilizing the VHB buffer, in this example an SPM wash buffer is added to the reagents reservoir of the liquid handling system which then aspirates, and dispenses into the well plates. Subsequently, the well plates are transported to shakers, shaken, and transported to the magnets for magnetic separation, and then the liquid handling system then aspirates waste from the well plates.

[0044] Finally, in this example, an elution buffer can be added to the reagent reservoir of the liquid handling system which then aspirates and dispenses into the well plates. Subsequently, the well plates are transported to shakers, shaken, and transported to the magnets for magnetic separation, and then the liquid handling system aspirates waste from the well plates. Accordingly, any number of buffers can be used in the isolation of the DNA. Additionally, the shakers are not heated in this example. With this technology, at least 4000 samples can advantageously be analyzed in a 24 hour period.

[0045] In order to analyze the efficacy of this example, genomic DNA from whole blood samples was isolated using the methods described and illustrated in this example and a reference method, using the same liquid handling system, analyzed for the APOE 112, APOE 158, MTHFR C677T, FII, FVL,CYP2C19*2, *3 and *17, and Warfarin (CYP2C9*2, *3 and VKORC1) SNPs, and the concordance was compared. The reference method included automated transfers of sample materials wherein the materials are heated in either a water bath or on a heating block after addition of the lysis buffer.

[0046] At least 95% of the samples extracted using the method of this example (referred to herein as HDQ method) resulted in a genotype call for each one of the above-identified SNPs. There was no negative concordance in genotype results for all of the SNP assays between the two instruments. Samples that were marked as non-concordance/unable to assay had undetermined status for one of their results. The result of the comparison is illustrated in the following Tables 2-10.

TABLE-US-00002 TABLE 2 APO-E 112 Summary HDQ-Bahamas HDQ-Haiti Samples 384 384 Not Analyzed 1 2 n 383 382 Positive Concordance 381 381 Non Concordance 2 1 Negative Concordance 0 0 % Positive 99.48% 99.74% Concordance

TABLE-US-00003 TABLE 3 APO-E 158 Summary HDQ-Bahamas HDQ-Haiti Samples 384 384 Not Analyzed 1 2 n 383 382 Positive Concordance 383 382 Non Concordance 0 0 Negative Concordance 0 0 % Positive 100.00% 100.00% Concordance

TABLE-US-00004 TABLE 4 CYP2C19*2 Summary HDQ-Bahamas HDQ-Haiti Samples 380 380 Not Analyzed 3 4 n 377 376 Positive Concordance 376 376 Non Concordance 1 0 Negative Concordance 0 0 % Positive 99.73% 100.00% Concordance

TABLE-US-00005 TABLE 5 CYP2C19*3 Summary HDQ-Bahamas HDQ-Haiti Samples 380 380 Not Analyzed 1 2 n 379 378 Positive Concordance 379 378 Non Concordance 0 0 Negative Concordance 0 0 % Positive 100.00% 100.00% Concordance

TABLE-US-00006 TABLE 6 CYP2C19*17 Summary HDQ-Bahamas HDQ-Haiti Samples 380 380 Not Analyzed 2 3 n 378 377 Positive Concordance 376 376 Non Concordance 2 1 Negative Concordance 0 0 % Positive 99.47% 99.73% Concordance

TABLE-US-00007 TABLE 7 FVL Summary HDQ-Bahamas HDQ-Haiti Samples 380 380 Not Analyzed 1 2 n 379 378 Positive Concordance 377 378 Non Concordance 2 0 Negative Concordance 0 0 % Positive 99.47% 100.00% Concordance

TABLE-US-00008 TABLE 8 Factor II Summary HDQ-Bahamas HDQ-Haiti Samples 380 380 Not Analyzed 1 2 n 379 378 Positive Concordance 379 376 Non Concordance 0 2 Negative Concordance 0 0 % Positive 100.00% 99.47% Concordance

TABLE-US-00009 TABLE 9 MTHFR Summary HDQ-Bahamas HDQ-Haiti Samples 382 382 Not Analyzed 2 3 n 380 379 Positive Concordance 379 379 Non Concordance 1 0 Negative Concordance 0 0 % Positive 99.74% 100.00% Concordance

TABLE-US-00010 TABLE 10 Warfarin Summary HDQ-Bahamas HDQ-Haiti Samples (all 3 SNPs) 279 279 Not Analyzed 0 0 n 279 279 Positive Concordance 279 279 Non Concordance 0 0 Negative Concordance 0 0 % Positive 100.00% 100.00% Concordance

[0047] Accordingly, by this technology, DNA can be rapidly extracted from a human sample and prepared for use in a subsequent high-throughput genotyping assay is provided. With this technology, cells are lysed without requiring heat thereby reducing the time and required energy for performing the lysing. Additionally, all of the steps required to isolate the DNA can be performed on the same liquid handling system using a bar code tracking system thereby avoiding the need for manual pipetting and manual matching of sample numbers. Accordingly, extraction time can be significantly reduced and throughput can be increased, thereby allowing more samples to be analyzing over the same period of time.

[0048] Having thus described the basic concept of the invention, it will be rather apparent to those skilled in the art that the foregoing detailed disclosure is intended to be presented by way of example only, and is not limiting. Various alterations, improvements, and modifications will occur and are intended to those skilled in the art, though not expressly stated herein. These alterations, improvements, and modifications are intended to be suggested hereby, and are within the spirit and scope of the invention. Additionally, the recited order of processing elements or sequences, or the use of numbers, letters, or other designations therefore, is not intended to limit the claimed processes to any order except as may be specified in the claims. Accordingly, the invention is limited only by the following claims and equivalents thereto.