Patent Application
20210039087
The present invention relates to devices and methods for the improved storage and processing of nucleic acids, such as DNA or RNA, held on solid supports such as treated cellulous fibre materials.
Nucleic acids, such as deoxyribonucleic acids (DNA) or ribonucleic acids (RNA), have become of increasing interest as analytes for clinical or forensic uses. Powerful new molecular biology technologies enable one to detect for congenital diseases or infectious diseases. These same technologies can characterize DNA for use in settling factual issues in legal proceedings such as paternity suits and criminal prosecutions. Nucleic acid testing has been made possible due to powerful amplification methods. One can take small amounts of nucleic acids which, in and of themselves would be undetectable, and increase or amplify the amount to a degree where useful amounts are present for detection.
The most commonly employed amplification technique is known as a polymerase chain reaction, (PCR). Nucleic acid polymerases are used with template DNA from the sample in a cycled manner to create greater amounts of starting nucleic acid materials, which are easily detected, for example by electrophoresis techniques.
These known amplification techniques often provide a deliberate surplus of nucleic acids which are usually kept in cold conditions for preservation and for later possible use. Such is the scale of operations, particularly in the forensics field, that the amount of cold storage required and the energy needed to run that storage, has become a significant cost. Another option is to store surplus nucleic acids dried and at room temperature, stored on paper treated with preserving chemicals which do not significantly degrade the nucleic acids. Such papers are sold under the brand name of FTA, sold by Whatman Inc.
Such a treated paper is disclosed in U.S. Pat. No. 5,496,562 to Leigh A. Burgoyne, where an absorbent cellulose based matrix is treated with a combination of a weak base, a chelating agent, an anionic detergent, and, optionally, uric acid. The resulting product has an alkaline pH. DNA binds to this matrix and is protected against degradation.
One problem with the above mentioned paper storage is that the storage space needed is significant. For example, to prevent cross contamination between papers, the stored samples are often held in envelopes spaced from adjacent envelopes, which increases the volume of storage significantly. Another drawback is the need to manually handle stored samples or use complicated bespoke mechanisms to automate handling. Where automated handling is contemplated, the papers have a supporting card frame around them to keep them straight. The frames and/or envelopes and spaced storage means that the density of stored sample is low.
Another drawback with the above mentioned paper is that, for recovery of further amplifiable nucleic acids after storage, a portion or portions of the sample holding paper is/are removed, typically using a hollow punch, and then a number of wash, elution and amplification steps are needed. The punching step is a two step process—1) punch cleaning and 2) punching a portion usually of about 2 or 3 mm in diameter. Both steps are potential sources of cross contamination, although in practice the risk is insignificant, provided the cleaning is carried out correctly. Nevertheless, cleaning and punching take time, which slows down an automated process.
Once punched, the portion of paper can be processed according to known multi-step techniques to recover nucleic acids after said storage. However, handling of the relatively small punch paper portion(s) also requires manual intervention or bespoke handling equipment.
In place of the chemical treatment mentioned above chaotropic salts have been proposed to reduce the inhibitory burden of materials in the processing steps after punching and allow greater amounts of source DNA to be amplified, but this does not negate the practical problems of punching and sample handling after punching.
A process for isolating nucleic acids is shown in U.S. Pat. No. 5,234,809 to William R. Boom et alia, (Boom) (incorporated herein by reference). Recognizing that typical biological sources of nucleic acids can affect PCR reactions, Boom discloses using a combination of a biological source material, chaotropic salt, and a solid support, preferably finely divided glass. All three elements are combined in a liquid mixing device, with any nucleic acids present binding to the glass. After mixing, the solid support must be removed from the mixing device, washed, and the template nucleic acid eluted. Only then can it be exposed to amplification reactions.
Paper solid under the brand name FTA Elute by Whatman Inc are treated with a chaotropic salt intended to preserve nucleic acids when dried on such supports, having been deposited thereon, usually as fluid samples, for subsequent genetic characterization, primarily by conventional amplification methods such as PCR. Those supports can be used in a known protocol to collect, store, or purify nucleic acids either from a biological source, for example a biological source having naturally occurring nucleic acid amplification inhibitors present, (including either a buccal swab, cerebrospinal fluid, feces, lymphatic fluid, a plasma sample, a saliva sample, a serum sample, urine, or a suspension of cells or viruses), or from a treated whole blood biological source that has naturally occurring nucleic acid amplification inhibitors present, as well as added blood stabilization components that also inhibit nucleic acid amplification. More importantly, these nucleic acids can be released after collection or storage in a manner that enables them to be amplified by PCR. In particular, the solid supports comprise an absorbent material that does not bind nucleic acids irreversibly, and is impregnated with the chaotropic salt. A biological source sample is contacted with the impregnated absorbent material. Any nucleic acids present in the biological source can be either eluted or resolubilized off the absorbent material.
U.S. Pat. No. 6,168,922 to Michael Harvey et alia (incorporated herein by reference), describes certain embodiments of said FTA Elute and wherein it is disclosed that an absorbent material such as cellulosics, porous glasses and woven/non-woven porous polymers, can be impregnated with a chaotropic salt, to provide a releasable support for amplifiable nucleic acids, even in the presence of naturally occurring amplification inhibitors. In more detail the disclosure describes techniques to collect, store, or purify nucleic acids either from a biological source other than untreated whole blood, the biological source having naturally occurring nucleic acid amplification inhibitors present other than hemoglobin, (including samples from either a buccal swab, cerebrospinal fluid, feces, lymphatic fluid, a plasma sample, a saliva sample, a serum sample, urine, or a suspension of cells or viruses) or from a treated whole blood source that has naturally occurring nucleic acid amplification inhibitors present, as well as added blood stabilization components that also inhibit nucleic acid amplification. It is proposed that the absorbent treated material disclosed can be used to detect pathogens such as bacteria or viruses that can be found in the circulatory system. More importantly, these nucleic acids can be released after collection or storage in a manner that enables them to be amplified by conventional techniques such as PCR either by elution or re-solubilisation off the absorbent material. The device described can collect nucleic acids not only from point sources such as humans or animals, but also can be used to collect widely disseminated sources such as fungal spores, viruses, or bacterial spores, or biological material, such as bodily fluids, present at crime scenes.
Embodiments of the present invention addresses the concerns mentioned above. The inventors have realized that an improved storage format is needed that allows easier handling, including storage of multiple samples, and convenient recovery of nucleic acids after storage. The inventors have also realized that the chemistry mentioned above employing chaotropic salts reduces the processing steps need to recover stored nucleic acids.
According to one aspect, the present invention provides a nucleic acids storage device comprising one or more sealable storage wells, the or each well containing one or more three dimensional solid supports capable of absorbing 5 μL or more of liquids containing any nucleic acids to be stored.
In an embodiment, said one or more solid supports is a single solid support having an absorbent volume of at least 7 millimeters cubed (mm3) and preferably about 7 to 180 mm3, and more preferably about 7 to 50 mm3.
In an embodiment, said one or more solid supports comprises plural solid supports, wherein each of the plural solids supports has an absorbent volume of at least 7 millimeters cubed (mm3) and preferably about 7 to 180 mm3, and more preferably about 7 to 50 mm3.
In an embodiment, the or each solid support has a thickness in each of three dimensions which three thicknesses are about equal, or where they are not equal, one dimension at least is at least 1 mm.
In an embodiment, said one or more solid supports comprises plural solid supports, wherein, in total the plural solids supports have an absorbent volume of at least 7 millimeters cubed (mm3) and preferably about 7 to 180 mm3, and more preferably about 7 to 50 mm3.
In an embodiment the solid support is coated or sorbed with a chaotropic agent, such as one or more of n-Butanol; Ethanol; Guanidinium chloride; Guanidinium/Guanidine (i so)thiocyanate; Guanidine hydrochloride; Lithium perchlorate; Lithium acetate; Magnesium chloride; Phenol 2-propanol; Sodium (iso)thiocyanate; Sodium iodide; Sodium dodecyl sulfate; Sodium perchlorate; Potassium iodide; Thiourea; and/or Urea, or a salt or salts thereof. Other chaotropic agents could be used.
In an embodiment, the storage volume and solid support have dimensions or a complementary shape which allow the placing of the, or at least one of the solid supports into the bottom of the well, such that the solid support is in contact with the lowermost part of the bottom of the well.
In an embodiment, the or each solid support is a spherical or cylindrical shape or a polyhedral shape.
In an embodiment, the one or more storage well comprises plural storage wells formed together in an array of spatially separated wells, for example a 24, 48 or 96 well array, for example each well having a closed bottom and a top opening formed in a common supporting plate.
According to a second aspect, the present invention provides a method for storing nucleic acids, the method comprising, in any suitable order, the steps of:
a) providing a storage device including plural storage wells each containing at least one absorbent solid support;
b) adding liquids, including any nucleic acids to be stored, to one or more of the storage wells and thereby to be absorbed by a respective solid support in the storage well;
c) allowing said liquids to dry substantially, optionally at a temperature above room temperature, for example up to 80 degrees Celsius and optionally in the presence of a desiccant;
d) following step c), optionally sealing each storage well; and
e) storing the device at room temperature.
In an embodiment, the above method has further step of recovering stored nucleic acids, including the steps of:
a) optionally opening the sealed storage well or where a plurality of storage wells are provided, one or more of the storage wells;
b) optionally moving at least a portion of the contents of the, or one of the storage wells into a processing well for elution or direct amplification
In an embodiment, the above method has the further step of elution of nucleic acid for amplification, including the steps of:
a) optionally adding additional wash liquids to the storage or processing well and then discarding said wash liquids but keeping the solid support;
b) adding additional liquids to the storage or processing well;
c) heating and agitating the solid support along with the additional liquids in the storage or processing well and collecting the resultant liquids for analysis.
Additional nucleic acid recovery processing steps could be employed as disclosed in co-pending patent application CN2017/085296 filed at the Chinese State Intellectual Property Office under the rules of the PCT on 22nd May 2017 in the name of General Electric Company and incorporated herein by reference.
In embodiments, storage of solid supports in separated in individual wells helps to prevent cross contamination of e.g., forensic samples. This is advantageous over the current procedures whereby papers or cards need to be stored individually in pouches to prevent cross contamination, then processed by removing a small disc or punch from each card using a punching device prior to processing. This process is cumbersome, time consuming and poses a greater risk of cross-contamination. This multiplexed format is suitable for storage of forensic crime scene purified DNA samples at room temperature.
The invention extends to any features described herein. Where features are mentioned in combination herein, a claim which includes just one or a subset of said combined features is expressly considered to fall within the ambit of the invention disclosed herein.
More advantages and benefits of the present invention will become readily apparent to the person skilled in the art in view of the detailed description below.
The invention will now be described in more detail with reference to the appended drawings, wherein:
Another alternative is to use a pouch enclosing the well (or plural wells), instead of a seal/lid 16. At this stage the storage well 10′ can be stored indefinitely at room temperature without the risk is significant degradation of any nucleic acids on the ball 20′.
For simplicity, the ball 20″ is shown in the storage well 10″ in
Solid supports can be transferred manually, or by automatic means, for example using a stake to pierce the ball and move it, or without contact for example by using a nozzle emitting a gentle flow of clean air which when in close proximity to said ball 20′/120′ accelerates sufficiently to reduce pressure below atmospheric pressure and therefore allow the ball to be held in the close proximity but not touch the nozzle. Electrostatic attraction is another alternative means for lifting a solid support. Where wells 110 are removeable from the remaining array 100, there will be no need to handle the solid supports, but rather the individual well can be handled instead.
The spherical solid supports 20/120 if used singly should have a diameter of about 3.5 mm (
The material the solid supports is preferably fibrous and liquid porous in nature. Many materials are suitable for use. The main characteristics needed for the solid support material are that it is or can be made hydrophilic, and does not substantially bind nucleic acids irreversibly through either hydrophobic, ionic, covalent, or electrostatic means. The matrix must not by itself inhibit or bind amplification reactants, release substances that effect amplification reactants or otherwise affect PCR and other amplification reactions. Suitable materials include cellulosics, woven porous polymers, or non-woven porous polymers, including polyesters and polypropylenes. Cellulose fiber materials can be used, for example cellulose acetate fibers made from bleached cotton or wood pulp esterified with acetic acid. Other polymers could be used or glass fibers could be used. Some degree of absorption is preferred, but for larger wells, the solid supports can be made bigger and so just their surfaces could be made absorptive. Thus solid supports with hollow or non-absorptive cores could be used. For example a plastics polymer core could be used having a fibrous outer layer spun around it, or the polymer could be mechanically or chemically treated such that its outer surfaces have a porous or semi porous quality.
iFTAe micro cards, GE Healthcare catalogue no WB120412/WB120411
Ambion nulcease-free water (Lot—1408160)
Invitrogen Ultrapure 0.5 M EDTA, pH 8.0, catalogue no 15575-038 #1852916
Gibco 1 M TRIS, pH 8.0, catalogue no 15568-025 #1849607
Purified gDNA @50 ng/ul
1 g sachets of Desiccant, GE Healthcare WB100003
Multibarrier pouches, GE HealthcareHC WB100037
Technipaq foil pouches (for AA storage).
Preparation of TE buffer: (10 mM Tris, 1 mM EDTA, pH 8.0): 1 ml 1 M Tris; 200 ul 0.5 M EDTA; 98.8 ml nuclease free water=100 ml final volume.
Preparation of TE-4 buffer: (10 mM Tris, 0.1 mM EDTA, pH 8.0): 1 ml 1 M Tris; 20 ul 0.5 M EDTA; 98.98 ml nuclease free water=100 ml final volume
Preparation of DNA dilutions: 20 ng/ul, add 1.2 ml 50 ng/ul stock solution to 1.8 ml TE buffer; 2 ng/ul, add 250 ul solution 1 to 2.25 ml TE buffer.
For the 2 ng/ul gDNA solution, 25 ul was spotted onto multiple iFTAe cards from GE Healthcare. These cards were allowed to dry in the biosafety cabinet at room temperature for 2-3 hours. Once completely dried, the cards were stored in zip lock bags with 1 g desiccant (in desiccator cabinet) at room temperature until further use.
FTA elute cards used: #9795867
2 ng/ul gDNA solution
4×3 mm diameter disk were cut from uncontaminated FTA elute cards and each was supported on a sterile needle to form a continuous stack of punches forming a cylindrical shape. Six stacks were formed, 15 ul gDNA solution @2 ng/ul was spotted onto each stack of 4 discs (note: volume was optimised previously using sterile water):
Stack 1)—Three of the stacks were each spotted with 15 ul gDNA and placed inside a 1.5 ml Eppendorf tube to dry; and
Stack 2)—three of the stacks were spotted with 15 ul gDNA and placed upside down in Eppendorf rack to air dry.
In addition, using the control experiment, a further FTAe micro-card was spotted with 25 ul gDNA (@2 ng/ul) & dried. Then 3 mm diameter disks were cut and formed into further stacks on sterile needles, making:
Stack 3)—Three control stacks which were formed from cards which were already charged with gDNA resulting from the control experiment mentioned above. Samples were left to dry in the biosafety cabinet overnight. The following day, all samples were stored in a desiccator cabinet until required for testing.
Elution of DNA from Control Cards and Stacks
Stacks 1, 2 and 3 were then processed as follows
a) Place each stack into the bottom of a 1.5 mL microcentrifuge tube
b) Pipette 500 μL of TE-4 buffer into the microcentrifuge tube containing the 3 mm punches.
c) Close the tube and vortex the microcentrifuge tube for 5 sec.
d) Pipette off excess TE-4 buffer and discard.
e) Repeat steps 3-5 (for a total of three washes with TE-4 buffer).
f) Pipette 150 ul of TE-4 buffer into the microcentrifuge tube containing the sample.
g) Place the microcentrifuge tube on a heated mixer/shaker at 95° C. for 30 min at 1,000 rpm.
h) After incubation, briefly centrifuge the microcentrifuge tube to remove any excess liquid from the cap.
i) Place a clean spin basket into a new microcentrifuge tube.
j) Transfer the punches and eluate to the spin basket and spin at maximum speed (13 k rpm in Heraeus biofuge) for 2 min.
j) Remove the spin basket, discard the punches, and proceed with quantification and/or amplification.
l) Extracts were stored at +4° C., then quantified using a Quantifiler Human DNA Quantification Kit as per manufacturer's instructions.
Lines 1, 2 and 3 above represent stacks 1, 2 and 3 respectively. It should be noted that for stacks 1 and 2, the quantity DNA added to the stacks was 30 ng, whereas for stack 3 the amount was 18 ng. Therefore the percentage yield (last column) reflects this starting amount of DNA. The results demonstrate that acceptable yields of DNA can be had from a three dimensional volume of solid support, in this case a stack of paper solid supports, even if the stack is left in a well to dry.
The skilled person will appreciate that the present invention can incorporate any combination of the preferred features described above. All publications or unpublished patent applications mentioned herein are hereby incorporated by reference thereto. Other embodiments of the present invention are not presented here which are obvious to those of ordinary skill in the art, now or during the term of any patent issuing from this patent specification, and thus, are within the spirit and scope of the present invention. The invention is not to be seen as limited by the embodiments described above, but can be varied within the scope of the appended claims as is readily apparent to the person skilled in the art, for example, indefinite storage can be maintained in dry conditions, for example, by storing the sample-containing solid support in a sealed container optionally along with desiccant material, for example incorporated into the sealing film 16/116.
Testing was undertaken of SBS standard 96 well polypropylene plates filled with a stack of 7 or 8×6 mm diameter FTA Elute discs. The plate was covered with foil containing a 5 mm diameter hole above each well.
All pipettes—calibration due end September 2018
1. Preparation of TE-1 buffer (10 mM Tris, 1 mM EDTA, pH 8.0), comprising:
98.8 ml nuclease free water
100 ml final volume
2. Preparation of TE-4 buffer (10 mM Tris, 0.1 mM EDTA, pH 8.0), comprising:
98.98 ml nuclease free water
100 ml final volume
3. All FTAe microcards were spotted, punched and processed as below:—
NOTE: If the punches remain at the bottom of the microcentrifuge tube during vortexing, they will not be washed adequately.
5. Pipette off excess TE-4 buffer and discard.
Note: Remove ALL excess buffer between wash steps.
6. Repeat steps 3-5 (for a total of three washes with TE-4 buffer).
7. Pipette 400 ul of TE-4 buffer into the microcentrifuge tube containing the sample punches.
8. Place the microcentrifuge tube on a heated mixer/shaker at 95° C. for 30 min at 1,000 rpm.
9. After incubation, briefly centrifuge the microcentrifuge tube to remove any excess liquid from the cap.
10. Place a clean spin basket into a new microcentrifuge tube. Transfer the punches and eluate to the spin basket and spin at maximum speed for 2 min.
11. Remove the spin basket, discard the punches, and proceed with quantification and/or amplification.
NOTE: If the sample is too dilute to meet the DNA input needed for PCR amplification, the sample can be concentrated.
Concentrate any DNA extracts <0.033 ng/ul (equivalent to 0.5 ng/15 ul) using Millipore Microcon DNA Fast Flow devices.
Measuring DNA Yield from Samples Using Quantifiler Human DNA Quantification Kit Using an ABI™ 7900HT Fast Real-Time PCR System.
2 ul DNA extracts were added to 23 ul of the following reaction mix:—
Qfiler PCR reaction mix: 1250 ul
Total volume: 2300 ul
Add 23 ul per well
95° C. for 10 mins, then:
95° C. for 15 secs
60° C. for 60 secs
For 40 cycles
Standard curve prepared as per Manufacturer's instructions.
Note: samples of diluted gDNA (i.e., solutions that were spotted onto cards) were included in the qPCR.
Following qPCR analysis, all samples spiked with gDNA at 100 pg/ul were concentrated as described below:—
Concentrate any DNA extracts where concentration was <0.033 ng/ul using Millipore Microcon DNA Fast Flow devices.
Note: 0.033 ng/ul is equivalent to 0.5 ng/15 ul. The Powerplex Fusion kit allows 15 ul sample addition, minimum quantity of DNA is 0.5 ng.
NOTE: For Microcon® DNA Fast Flow PCR Grade devices, use aseptic technique when opening packages and throughout the procedure. Carefully reseal pouches to protect unused samples from contamination.
1. Insert Microcon® device into tube.
2. Pipette solution into device (0.5 mL maximum volume), taking care not to touch the membrane with the pipette tip. Seal with attached cap.
3. Place assembly in a compatible centrifuge (described in the Equipment Required section) and counterbalance with a similar device.
NOTE: When placing the assembled device into the centrifuge rotor, align the cap strap toward the center of the rotor.
4. Spin at 500×g for DNA Fast Flow devices=2,300 rpm in Hereaus Biofuge for 20 mins.
5. Remove assembly from centrifuge. Separate tube from filter device.
6. Place a new tube over the top of the device. Invert the assembly and centrifuge for 3 minutes at 1,000×g (or pulse briefly) to transfer concentrate to tube=3,200 rpm in Hereaus Biofuge.
7. Remove from centrifuge. Separate tube from filter device. Close sealing cap to store sample for later use.
For volumes of DNA extract eluted from Microcon devices & calculations to provide 0.5 ng per PCR reaction—refer to attachment 1
Calculate volume of DNA extract to provide 0.5 ng DNA. Make final volume of sample up to 15 ul using sterile water. Add 15 ul sample to appropriate wells of a 96 well plate:
Note:
For details of actual qty of gDNA added per STR reaction, refer to attached Excel spreadsheet containing qPCR data.
Total volume: 1000 ul
Add 10 ul per well
(plus 15 ul sample=25 ul per well)
96° C. for 1 minute, then:
94° C. for 10 secs
72° C. for 30 secs
For 30 cycles, then:
60° C. for 10 minutes
4° C. soak
Total volume: 10 ml
Add 10 ul per well
Note: control DNA was added to appropriate wells at 1 ng/ul, 100 pg/ul and 20 pg/ul.
Summary of qPCR Data:—
DNA yield from all 96-well prototypes samples were equivalent to, or better than (p>0.05) the microcard control (Mann Whitney non parametric t-test AND unpaired t-test with Welch's correction).
Note: in the tables above, % recovery was calculated using two methods:
Graphs of DNA yield for different initial concentrations of DNA are illustrated in
Note: however ALL liquid control ratios are below 2.0
DNA yield from all 96-well prototypes samples were equivalent to, or better than (p>0.05) the microcard control (Mann Whitney non parametric t-test AND unpaired t-test with Welch's correction).
It was found that DNA yield and quality from 96-well plate prototypes obtained bt Example 2 were comparable (if not better) than FTA Elute microcards used conventionally.
| Number | Date | Country | Kind |
|---|---|---|---|
| 1716135.7 | Oct 2017 | GB | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2018/076932 | 10/3/2018 | WO | 00 |
