Patent Application
20230399634
The present invention relates to an improved method and system for isolating cell-free DNA (cfDNA) present in a liquid body sample. More specifically, the present invention relates to a method and system for isolating cfDNA from blood plasma with size selection, to facilitate enrichment and recovery of small fragment cfDNA while at the same time minimizing recovery of high molecular weight, larger genomic DNA (gDNA) fragments.
Fragmented cfDNA molecules were first discovered in the human circulatory system in 1948 by Mandel and Metais. cfDNA also known as circulating free DNA or circulating cell-free DNA, are DNA fragments released into the bloodstream by the cells. Several mechanisms of the release of cfDNA molecules in blood have been proposed including necrosis, apoptosis, phagocytosis, active cellular secretion, exosome release, pyroptosis, mitotic catastrophe and autophagy, resulting in the presence of a cfDNA population with diverse physical properties in circulation. In healthy individuals, cfDNA fragments vary between 100-250 bp with the most prevailing size of 166 bp that corresponds a nucleosome complex of DNA molecule bound to the histone core. cfDNA can be used to describe various forms of fragmented DNA circulating freely in the bloodstream such as cell-free fetal DNA (cffDNA), circulating tumor DNA (ctDNA) or circulating cell-free mitochondrial DNA (ccf mtDNA).
Clinical significance of cfDNA was recognized when researchers observed differences between the characteristics of cfDNA from healthy and diseased individuals. Numerous studies have demonstrated that cancer patients generally have high levels of cfDNA in comparison to healthy subjects. Elevated levels of cfDNA in cancer patients is thought to be caused by excessive DNA release by apoptotic and necrotic cells and/or cfDNA accumulation due to chronic inflammation and excessive cell death. In healthy individuals, cfDNA levels are predominantly low, however they can get temporarily elevated after strenuous exercise. It has also been demonstrated that the size of cfDNA fragments that originate from tumor cells are shorter than cfDNA fragments that originate from non-malignant cells. Similarly, cfDNA of fetal origin contains a higher proportion of DNA smaller than 150 bp. Increased proportion of smaller fragments has also been reported in autoimmune disease and in donor derived fraction post transplantation. Thus, size-selection of smaller cfDNA fragments could be used to increase the amount of target cfDNA fragments (i.e. tumor derived cfDNA in cancer diagnostics or fetal cfDNA in noninvasive prenatal testing). cfDNA in cancer patients bear the unique genetic and epigenetic alterations that are characteristic of the tumor from which they originate. Genetic analysis and molecular profiling of cfDNA thus presents a promising clinical potential for cancer detection, prognosis, staging, monitoring and therapy selection. cfDNA as a biomarker for cancer management has been successfully demonstrated by two FDA-approved applications for cfDNA assays in routine clinical practice, namely the cobas EGFR Mutation Test v2 for lung cancer patients and Epi proColon, a colorectal cancer screening test based on the methylation status of the SEPT9 promotor. Fetal derived cfDNA present in maternal blood has also been successfully used to detect fetal abnormalities. cfDNA analysis has also shown potential for clinical use in organ transplant, autoimmune diseases and sepsis where cfDNA fraction is enriched in smaller DNA molecules.
In the blood of cancer patients, cfDNA originates from multiple sources including not just cancer cells but also cells from the tumor micro-environment and other non-cancer cells from various parts of the body. DNA from cancer cells is released most prominently by the mechanisms of apoptosis, necrosis, and active secretion. Apoptosis causes the systematic cleavage of chromosomal DNA into multiples of 160-180 bp stretches, resulting in the extracellular presence of mono-nucleosomes and poly-nucleosomes. The majority of cfDNA produced by apoptosis has a size of 167 bp (147 bp of DNA wrapped around a nucleosome plus a linker DNA of around 20 bp that links two nucleosome cores).
Solid tumor biopsies are expensive and invasive, making them less than ideal for patients who are older or very young. On the other hand, cfDNA analysis as a disease biomarker can be done using non-invasive liquid biopsy which utilizes a liquid body sample from the patient like blood plasma, urine or serum. The amount of ctDNA in the whole pool of cfDNA may vary widely among the patients, cancer type, and cancer stage, from 0.01% to 90% in advanced metastasis. There is a prevailing consensus that ctDNA is fragmented to a higher extent than cfDNA derived from healthy cells and has shorter fragment size (less than 150 bp). Both low abundance and shorter fragment size of ctDNA presents a serious challenge to the isolation and further analysis of cfDNA. In addition, intra-tumoral genetic heterogeneity is yet another challenge in clinical oncology where identification of minor sub-clonal populations is essential for detection of emerging chemoresistance, minimal residual disease, and non-invasive monitoring of disease progression. The detection limit becomes negatively affected by the presence of contaminating high molecular weight gDNA that may be present in the plasma, originating from lysed blood cells. Therefore, it is important to select a cfDNA extraction method that not only delivers a high yield of cfDNA but also allows for efficient recovery of shorter cfDNA fragments and negatively selects against high molecular weight DNA. To detect some rare low-level resistance mutation, one is more likely to detect it when tumor originating cfDNA is enriched in the sample and blood cell-derived gDNA background is reduced to minimum. cfDNA is thus usually purified from the plasma or serum which is devoid of white blood cells (WBCs) to prevent gDNA contamination resulting from WBC lysis. gDNA contamination would dilute out the tumor cfDNA, preventing detection of rare variants. As increased fragmentation of cfDNA has been widely reported for fetal-derived cfDNA, donor-derived cfDNA following organ transplantation and in autoimmune disease, size selection-based cfDNA extraction provides a clear advantage outside cancer diagnostics.
The object of the present invention is to provide an improved and size-selective method for isolation of cfDNA from liquid body sample like blood plasma.
The distinctive advantage of the method is that it allows for efficient isolation of main cfDNA fraction together with smaller, highly degraded fragments over any high molecular weight gDNA that would be perceived as a contaminant. This size dependent DNA binding allows for the specific enrichment of extracted cfDNA in the fraction of interest, e.g. tumor derived cfDNA in cancer or fetal cfDNA during prenatal testing in pregnancies with suspected aneuploidy. This makes the method of the invention highly suitable for liquid biopsy-based diagnostics.
Another advantage of the method is that it requires a very small quantity of input plasma sample ranging from 0.5 ml-4 ml.
Another advantage of the method is that it is quick and cfDNA isolation procedure can be completed in less than 2 hours to yield high quality cfDNA suitable for downstream applications.
According to an aspect of the invention, a method for isolating cell-free DNA from liquid body sample comprises the following steps:
According to another aspect of the invention, a method for size-selective isolation of cell-free DNA from liquid body sample, comprises the following steps:
According to another aspect of the invention, guanidinium thiocyanate and Triton X-100 are used to form a binding buffer composition for size-selective binding of cell-free DNA present in blood plasma to silica-coated magnetic microbeads formulated in an aqueous suspension at 20-200 mg/ml, wherein said binding buffer is intended to be brought into contact with 2-propanol, blood plasma and magnetic microbeads to form a binding mixture comprising: around 1.5-2.5 M guanidinium thiocyanate; around 20-30% w/v of Triton X-100; around 15-25% v/v of 2-propanol; and around 25-40% v/v of blood plasma.
According to another aspect of the invention, a kit comprising silica coated microbeads capable of binding 50-400 bp DNA from a body sample in the presence of guanidinium thiocyanate, Triton X-100 and 2-propanol is described.
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:
To more clearly and concisely describe and point out the subject matter of the claimed invention, definitions are provided hereinbelow for specific terms used throughout the present specification and claims. Any exemplification of specific terms herein should be considered as a non-limiting example.
The terms “comprising” or “comprises” have their conventional meaning throughout this application and imply that the agent or composition must have the essential features or components listed, but that others may be present in addition. The term ‘comprising’ includes as a preferred subset “consisting essentially of” which means that the composition has the components listed without other features or components being present.
This written description uses examples to disclose the invention, including the best mode, and also to enable any person skilled in the art to practice the invention, including making and using any devices or systems and performing any incorporated methods. The patentable scope of the invention is defined by the claims, and may include other examples that occur to those skilled in the art. Such other examples are intended to be within the scope of the claims if they have structural elements that do not differ from the literal language of the claims, or if they include equivalent structural elements with insubstantial differences from the literal languages of the claims.
cfDNA: cell-free DNA
cffDNA: cell-free fetal DNA
ccf mtDNA: circulating cell-free mitochondrial DNA
PBS: phosphate-buffered saline
GuSCN: guanidinium thiocyanate
bp: base pair
EDTA: Ethylenediaminetetraacetic acid
gDNA: Genomic DNA
ddPCR: Digital Droplet PCR
All tubes and pipette tips used were of DNase-free grade. Good laboratory practices were followed to avoid sample contamination.
The cfDNA isolation method of the invention allows for rapid extraction and purification of cfDNA from small quantities of liquid body sample such as blood plasma ranging from 0.5 ml-4 ml and provides high-resolution cfDNA size selection. The method has been specifically designed to select for short-fragment cfDNA (50 bp-400 bp) over longer high-molecular weight contaminating gDNA. The isolation procedure of the invention can be completed in less than 2 hours to yield high quality cfDNA suitable for downstream applications such as PCR, digital droplet PCR (ddPCR), genotyping and next generation sequencing (NGS).
The predominant type of cfDNA found in plasma is derived from the nuclear genome and has a fragment size that corresponds to a single nucleosome. These macromolecular complexes need to be dissociated in order to release cfDNA and promote binding of cfDNA to a DNA binding solid phase. In an embodiment of the invention, the solid phase was preferably silica coated magnetic microbeads where the silica bead surface is directly involved in DNA binding via surface silane (Si—OH) groups. Some of the cfDNA is also believed to be encapsulated in lipid vesicles and needs to be released prior to the binding step. The release of cfDNA from these diverse macromolecule complexes and lipid vesicles is achieved by using a combination of chaotropic agents and detergents. Chaotropic agents disrupt the nucleosomal unit to release cfDNA and detergents help to solubilize and denature proteins to release non-covalently bound cfDNA. Where the blood is collected in Streck cfDNA blood collection tubes, proteinase K treatment is additionally required to reverse the effects of Streck cfDNA stabilization chemistry by removing the crosslinks which would otherwise prevent efficient recovery of cfDNA during the isolation process. As a person skilled in the art would appreciate, Proteinase K treatment might not be required when using other blood collection tubes. Denatured contaminants are then removed by subsequent washing of the silica beads with wash buffers followed by air-drying of the silica beads. The purified cfDNA is then eluted from the silica beads using an elution buffer. In a preferred embodiment of the invention, SeraSil-Mag 700 beads by GE Healthcare Life Sciences were used for binding the released cfDNA, GuSCN was used as the chaotropic agent and 20% SDS (sodium dodecyl sulfate) was used as the detergent. As would be appreciated by the skilled person, any other DNA binding solid phase could be used instead of silica beads, for example,
the solid phase could be beads, particles, sheets and membranes having inherent DNA binding or added DNA binding capability.
As the levels of cfDNA encountered in blood plasma are very low, a significant volume reduction is needed during the isolation process to generate sufficient concentration of cfDNA for analysis. Efficient binding of the cfDNA from blood plasma, gentle washing and minimal elution are key to providing purified cfDNA that is suitable for downstream applications. Due to typically low levels of cfDNA in the final extract, UV-absorbance based analysis is not usually recommended.
Instead, cfDNA concentration is evaluated using qPCR or fluorescence-based methods such as Qubit™ (Invitrogen™). Qubit™ dsDNA HS Assay Kit, that is compatible with any fluorometer or fluorescence plate reader, allows for accurate estimation of total DNA concentrations down to 10 pg/μL. To assess the quality and yield of cfDNA in addition to the presence of gDNA, assessment can be performed using the Agilent 2100 Bioanalyzer system, with the Agilent High Sensitivity DNA Analysis Kit.
Protocol for Purification of cfDNA from 1.0-4.0 mL Blood Plasma
Whole blood sample collected in Streck cfDNA blood collection tubes was processed to separate the plasma as described above. The following steps of the cfDNA isolation method were then performed to obtain purified cfDNA from the plasma sample. Each step was performed using three different input plasma volumes of 1 ml, 2 ml and 4 ml of the plasma sample.
This step is performed to release cfDNA from macromolecular complexes and to reverse the Streck DNA stabilization chemistry. Proteinase K (20 mg/mL) solution and plasma sample were added into a 15 mL Streck cfDNA blood collection tube and mixed by brief vortexing. 20% Sodium Dodecyl Sulfate (SDS) was then added into the tube. Either the proteinase K or the plasma may be added to the tube first. However, 20% SDS should not be allowed to contact the proteinase K solution directly to prevent enzyme inactivation. The tube was pulse vortexed 2-3 times and the contents mixed thoroughly by vortexing for 15 seconds. The tube was then incubated at about 55-65° C. for around 20-30 minutes. Table 1 below shows different plasma input volumes that were used and the corresponding quantities of proteinase K and 20% SDS.
Step 2: Binding Mixture Preparation
Magnetic microbeads (Sera-Sil Mag 700 by GE Healthcare Life Sciences) were fully resuspended by vortexing before dispensing. A binding mixture was prepared by combining the plasma of step 1, a binding buffer, an aqueous suspension of the magnetic beads and 2-propanol. In this example, a composite reagent was first prepared by combining the binding buffer, aqueous suspension of the magnetic beads and 2-propanol. This pre-mixed composite reagent was then added to the plasma containing tube of step 1 and mixed thoroughly by pulse vortexing to form the binding mixture. A person skilled in the art would appreciate that the binding mixture could also be prepared by adding the binding buffer, magnetic beads and 2-propanol one by one into the plasma containing tube followed by thorough pulse vortexing rather than using the pre-mixed composite reagent. In an embodiment of the invention, the microbeads and the binding buffer are added to the plasma sample before adding 2-propanol.
The relative amount of each component in the binding mixture is critical for the maximum cfDNA recovery and minimum binding of gDNA. Table 2 below shows the quantities of the pre-mixed composite reagent used corresponding to the three different input plasma volumes to form the binding mixture. Table 3 shows quantities of individual components of the composite reagent as shown in Table 2.
The binding buffer is typically composed of a detergent and a chaotropic agent. In this example, Triton X-100 was used as the detergent and GuSCN was used as the chaotropic agent. Triton X-100 is a non-ionic surfactant, for example, a surfactant having hydrophilic polyethylene oxide chain and an aromatic hydrocarbon lipophilic or hydrophobic group (C14H22O(C2H4O)n where n=9-10). As would be appreciated by a person skilled in the art, other detergents and chaotropic agents could be used with a similar effect. Some examples of alternate detergents are Triton X-114, Nonidet P-40 and Igepal CA-630. An example of an alternate chaotropic agent is sodium perchlorate.
The tube containing the binding mixture was then incubated in a thermomixer (25° C., 1400 rpm) for 10 minutes after which it was briefly spun and placed on a magnetic rack for at least 5 minutes. Once the beads containing bound cfDNA were collected against the magnet to form a bead pellet, the clear supernatant comprising of denatured proteins/lipids was carefully aspirated to waste.
The tube was removed from the magnetic rack and 400 μL of Wash Buffer 1 was added to the tube directly on the bead pellet. Wash Buffer 1 was composed of 50% of ethanol and 50% of a solution containing GuSCN at around 2.0M and a non-ionic surfactant such as Triton X-100 at about 22% w/v. The beads were fully resuspended by pulse vortexing and brief spinning. The bead suspension was pipetted up and down and the content of the tube was transferred into a 1.5 mL microtube. Due to the liquid viscosity, the content of the tip was expelled slowly to ensure complete transfer of the bead suspension. A second aliquot of Wash Buffer 1 (400 μL) was added to the tube. The tube was vortexed, briefly spun and the content transferred to the same 1.5 mL microtube. The microtube was then placed on a magnetic rack for 1 minute to allow the beads to collect against the magnet before discarding the supernatant.
The microtube was removed from the magnetic rack and 700 μL of Wash Buffer 1 was added to the microtube. The microtube was incubated in the thermomixer at 25° C./1400 rpm for 1 minute, vortexed and then briefly spun. The microtube was then placed on the magnetic rack for 1 minute before discarding the supernatant. The microtube was removed from the magnetic rack and 700 μL of Wash Buffer 2 was added to the microtube. Wash Buffer 2 was composed of 80% of ethanol and 20% of a solution containing tris-HCl at around 10 mM, EDTA at around 1.0 mM and a polysorbate-type non-ionic surfactant such as TWEEN-20 at around 0.5% w/v. As a skilled person would appreciate, alternate non-ionic surfactants could also be used with a similar effect. Some of the examples are Tween-80 or Tween-60. Alternatively, the surfactant could be omitted altogether. The microtube was incubated in a thermomixer at 25° C./1400 rpm for 1 minute, vortexed and briefly spun. The microtube was then placed on the magnetic rack for 1 minute before discarding the supernatant. Another round of washing was done using the Wash Buffer 2.
The microtube was briefly spun to collect any residual Wash Buffer 2 at the bottom of the microtube. The microtube was placed on the magnetic rack for 1 minute to allow the beads to collect against the magnet. The clear residual supernatant was carefully removed from the very bottom of the microtube using a small pipette tip. The bead pellet was then allowed to air-dry for minutes while on the magnetic rack.
The microtube was removed from the magnetic rack. Elution buffer was added to the microtube and mixed well by vortexing to ensure the bead pellet was fully resuspended. The elution buffer contained tris-HCl at around 10 mM and EDTA at around 0.5 mM and the pH adjusted to 8.0. The microtube was incubated in the thermomixer at 25° C./1400 rpm for 3 minutes and briefly spun to bring the bead suspension to the bottom of the tube. The tube was placed on the magnetic rack for 1 minute to allow for the beads to collect against the magnet. Once the beads were collected against the magnet, the supernatant containing the isolated cfDNA was carefully transferred into a fresh microtube. Table 4 below shows the amount of elution buffer used corresponding to the three different volumes of input plasma.
Protocol for Purification of cfDNA from 500 μL (0.5 ml) Plasma
Whole blood sample was processed to separate the plasma as described previously. The following steps of the cfDNA isolation method were then performed to obtain purified cfDNA from 0.5 ml of plasma sample.
10 μL of Proteinase K (20 mg/mL) and 0.5 ml of plasma were added into a 2 ml microcentrifuge tube and mixed by brief vortexing. 25 μL of 20% SDS was then added to the tube. Either the Proteinase K or the plasma may be added to the tube first. However, 20% SDS should not be allowed to contact the Proteinase K solution directly to prevent enzyme inactivation. The tube was pulse vortexed 2-3 times and contents mixed thoroughly by vortexing for 15 seconds. The tube was then incubated at about 55-65° C. for around 20-30 minutes.
The magnetic microbeads (Sera-Sil Mag 700 by GE Healthcare Life Sciences) were fully resuspended by vortexing before dispensing. A composite reagent was prepared by combining the below three components and mixing thoroughly by pulse vortexing.
1. 0.725 mL Binding buffer×(No. of samples to be processed+10%)
2. 0.35 mL 2-Propanol×(No. of samples to be processed+10%)
3. 3.75 μL Magnetic Bead suspension (No. of samples+10%)
1.05 ml of freshly prepared composite reagent was added into the plasma containing tube of step 1 and contents mixed thoroughly by pulse vortexing to prepare the binding mixture. As mentioned in Example 1 above, the binding buffer, magnetic bead suspension and 2-propanol could be added one by one to the plasma containing tube of step 1 instead of using the pre-mixed composite reagent.
The tube was then incubated in the thermomixer at 25° C./1400 rpm for 10 minutes. The tube was then briefly spun and placed on a magnetic rack for at least 5 minutes. Once the beads containing bound cfDNA were collected against the magnet, the clear supernatant was carefully aspirated to waste.
The tube was removed from the magnetic rack and 700 μL of wash buffer was added into the tube. Multiple washing rounds were performed using Wash buffers 1 and 2 as shown in Table 5 below.
Wash Buffers 1 and 2 used were as described in Example 1 above. The tube was incubated in a thermomixer at 25° C./1400 rpm for 1 minute followed by vortexing and brief spinning. The tube was then placed on the magnetic rack for 1 minute before discarding the supernatant.
The tube was briefly spun to bring any residual Wash buffer 2 droplets to the bottom of the tube. The tube was then placed on a magnetic rack for 1 minute to allow for the beads to collect against the magnet. Clear residual supernatant was carefully removed from the very bottom of the microtube using a small pipette tip and the bead pellet was allowed to air-dry for 5 minutes while on the magnetic rack.
The tube was removed from the magnetic rack. 15 μL of elution buffer was added into the tube and the contents of the tube mixed well by vortexing to ensure the bead pellet was fully resuspended. The elution buffer used was the same as described in Example 1 above. The tube was incubated in the thermomixer at 25° C./1400 rpm for 3 minutes and then briefly spun to bring bead suspension to the bottom of the tube. The tube was placed on the magnetic rack for 1 minute to allow for the beads to collect against the magnet. Once the beads were collected against the magnet, the supernatant containing the isolated cfDNA was carefully transferred into a fresh microcentrifuge tube.
The method of the invention has been designed to maximize the recovery of small cfDNA fragments, for example as reported to be present in plasma of patients with advanced stage cancer, and to represent a fraction enriched in DNA of tumour origin. At the same time, the method design considerably reduces any co-purification of higher molecular weight gDNA that may be present, originating from lysed blood cells. This synergistic effect where small fragment recovery is elevated while large fragment recovery is depressed is demonstrated in Examples 3 and 4 described below and illustrated in
2 ml of plasma was obtained from the blood collected from two healthy human subjects in Streck cfDNA blood collection tubes. Both plasma samples were spiked with 50 bp DNA Ladder at a concentration of 10 ng/mL of plasma and each plasma sample was processed according to the method of the invention to extract the DNA. 1 μl of the isolated DNA from each sample was then run on High Sensitivity DNA chip in Bioanalyzer 2100. The results are as illustrated in
Plasma was obtained from the blood collected from two healthy human subjects in Streck cfDNA blood collection tubes. Both plasma samples were spiked with 50 bp DNA Ladder at a concentration of 10 ng/mL of plasma and each plasma sample was processed according to the method of the invention to extract cfDNA. Percent recovery of spiked-in 50 bp DNA Ladder for selected fragments of 50 bp, 100 bp and 2.5 kbp, based on 8 independent experiments was measured.
Synergistic Effect: Elevated Recovery of Small cfDNA Fragments Along with Depressed Recovery of Larger High Molecular Weight DNA
The inventors of the current invention surprisingly found that by manipulating the relative proportion of Triton X-100, 2-propanol and GuSCN in the binding mixture, it is possible to obtain the desired DNA fragment recovery profile from plasma samples. It was found that increasing the proportion of both Triton X-100 and 2-propanol in the binding mixture, recovery of cfDNA having short fragment size improved and recovery of contaminating gDNA decreased. It was also found that increasing the amount of guanidinium ions above a certain level increases binding of higher molecular weight fragments. As described previously, the binding mixture is a combination of binding buffer, 2-propanol, aqueous suspension of magnetic beads and blood plasma.
In this experiment, the proportion of 2-propanol in the binding mixture was varied to determine the effect it had on cfDNA recovery profile. The various combinations tested are summarized in Table 6 below. Proportion of Triton X-100 was fixed at −8.8% in the binding mixture. GuSCN in the binding mixture was fixed at 2M for Prep. Nos. 10A-10D. For Prep. Nos. 10E-10H, GuSCN in the binding mixture was fixed at 2.4M.
The extracted cfDNA was run on Bioanalyzer 2100 to see the recovery profile.
In this experiment, the effect of increasing 2-propanol from 22% to 25.2% in the binding mixture was tested while keeping GuSCN fixed at 2M and Triton X-100 fixed at 8.8% in the binding mixture.
This is summarized in Table 7 as provided below.
The extracted cfDNA was run on Bioanalyzer 2100 to see the recovery profile.
In this experiment, the effect of 2-propanol at 17.5%, 19%, 20.6% and 22.2% in the binding mixture was tested while keeping GuSCN fixed at 2M and Triton X-100 fixed at 11.1% in the binding mixture. This is summarized in Table 8 below.
The extracted cfDNA was run on Bioanalyzer 2100 to see the recovery profile.
In this experiment, the proportion of Triton X-100 in the binding mixture was varied to determine the effect it had on cfDNA recovery profile. The various combinations tested are summarized in Table 9 below. Proportions of Triton X-100 in the binding mixture were tested at 8.8% and 11.1% while keeping 2-propanol fixed at 22% and GuSCN fixed at 2M in the binding mixture.
cfDNA extracts were run on Bioanalyzer 2100 to see the effect of proportion of Triton X-100 in the binding mixture on cfDNA recovery profile.
In this experiment, proportions of Triton X-100 in the binding mixture were tested at 8.8% and 4.5% while keeping 2-propanol fixed at ˜25.2% and GuSCN fixed at 2M in the binding mixture. The various combinations tested are summarized in Table 10 below. Extracts of cfDNA obtained were run on Bioanalyzer 2100 to see the effect on cfDNA recovery profile.
It was noted by the inventors that plasma is required in the binding mixture to achieve the desired cfDNA recovery profile. This is explained in Example 10 below.
In this experiment, GuSCN was fixed at 2M, Triton X-100 was fixed at 11.1% and 2-propanol was fixed at 22.2% in the binding mixture as shown in Table 11 below. Size selection of cfDNA was tested in absence of plasma. This was done by substituting plasma once with NaCl and once with PBS. Each of plasma, NaCl and PBS containing samples were spiked with 50 bp DNA Ladder to monitor size selection and DNA recovery.
Extracted DNA was run on Bioanalyzer to see the effect of plasma on size selection.
Scalability of the cfDNA Isolation Method
Plasma obtained from blood collected in Streck cfDNA blood collection tubes was spiked with 50 bp DNA Ladder at a concentration of 10 ng/ml of plasma. The spiked plasma was then processed according to the method of the invention. Four different plasma input volumes were used for this experiment (0.5 ml, 1 ml, 2 ml and 4 ml) to demonstrate the scalability of the isolation method. The elution volumes were scaled to the input plasma volume for comparable DNA concentrations in the extracts as shown below in Table 12.
1 μl of each extract was run on High Sensitivity DNA chip on the Bioanalyzer 2100.
Expected Results from Plasma Collected in Standard EDTA Blood Collection Tubes
The method of the invention works best for extraction of cfDNA from blood plasma collected in Streck cfDNA blood collection tubes. However, it is possible to efficiently extract cfDNA from blood plasma collected in standard EDTA tubes as well as mentioned previously. However, in these instances, the recovery of the smaller fragments might fall below levels expected for Streck cfDNA blood collection tubes.
2 ml of plasma collected in standard EDTA tubes was spiked with 50 bp DNA Ladder (10 ng/mL of plasma) and processed using the cfDNA isolation method of the invention. 1 μl of the extract was run on High Sensitivity DNA chip alongside 50 bp DNA Ladder input.
The method of the invention allows for a highly efficient extraction of cfDNA and minimal carry-over of gDNA. This unique feature gives a distinctive advantage in liquid biopsy-based applications allowing for detection of mutations present at a very low level which otherwise might be missed if standard isolation methods are used. This is described in Example 13 below.
1 ml of plasma from 3 cancer patients, collected in standard EDTA blood collection tubes was obtained from commercial sources and cfDNA was isolated using the method of the invention as well as a standard commercial kit with no size selection. 1 μl of the isolated cfDNA was run on High Sensitivity DNA chip and the results were as illustrated in
The invention is not to be seen as limited by the embodiments and examples 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 instance, the blood collection tubes could be standard EDTA tubes or Heparin tubes. A person skilled in the art could vary the wash buffer compositions to get essentially the same results. For example, a wash buffer could just be 70-80% aqueous ethanol. Similarly, an elution buffer could be water or any standard dilute tris-HCl or tris-EDTA buffer. It is also to be understood that the skilled person can use any suitable solid phase other than silica coated microbeads, for example, glass microbeads and glass-fibre membranes which are also DNA binding. Several alternative examples of detergents and chaotropic agents are known in the art and a skilled person could use them without departing from the scope of the claims.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2001034.4 | Jan 2020 | GB | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2021/051046 | 1/19/2021 | WO |
