Field of the invention
This invention relates to Contamination free Cell free DNA (cfDNA) Isolation Protocol for Early Diagnosis of Rheumatoid Arthritis.
Background of the Invention
References which are cited in the present disclosure are not necessarily prior art and therefore their citation does not constitute an admission that such references are prior art in any jurisdiction. All publications, patents and patent applications herein are incorporated by reference to the same extent as if each individual or patent application was specifically and individually indicated to be incorporated by reference.
Several patents have been issued for cell free DNA but none of these are related to the present invention. For example, AU2020200418B2 provides methods and kits for preparing sequencing library to detect chromosomal abnormality using cell-free DNA (cfDNA) without the need of first isolating the cfDNA from a liquid fraction of a test sample. In some embodiments, the method involves reducing the binding between the cfDNA and nucleosomal proteins without unwinding the cfDNA from the nucleosomal proteins. In some embodiments, the reduction of binding may be achieved by treating with a detergent or heating. In some embodiments, the method further involves freezing and thawing the test sample before reducing the binding between the cfDNA and the nucleosomal proteins. In some embodiments, the test sample is a peripheral blood sample from a pregnant woman including cfDNA of both a mother and a fetus, wherein the methods may be used to detect fetal chromosomal abnormality such as copy number variation. In other embodiments, the test sample is a peripheral blood sample from a patient known or suspected to have cancer, wherein the methods can be used to detect chromosomal abnormalities in the cfDNA of the patient. Kits for detection of copy number variation of the fetus using the disclosed methods are also provided.
Another patent, US11091800B2 concerns a method of increasing detection of low-abundant fragments of cell-free DNA (ccfDNA) in a biological sample is disclosed and discussed. Such a method can include isolating an initial fraction of ccfDNA fragments from a biological sample, ligating a unique molecular identifier (UMI) to each of the ccfDNA fragments in the initial fraction, amplifying the plurality of ccfDNA fragments to generate a ccfDNA library, isolating a short fraction of ccfDNA fragments from the ccfDNA library, where the ccfDNA fragments in the short fraction are limited to a size of less than or equal to 160 base pairs (bp), amplifying the ccfDNA fragments in the short fraction, and sequencing the ccfDNA fragments in the short fraction to generate sequenced ccfDNA fragments.
Another patent, US10465183B2 provides novel methods and kits for isolating nucleic acids from biological samples, including cell-free DNA and/or cell-free DNA and nucleic acids including at least RNA from microvesicles, and for extracting nucleic acids from the microvesicles and/or from the biological samples.
Another patent, US10577655B2 include methods and compositions for producing standards for noninvasive prenatal genetic diagnostics and for the detection and monitoring of cancer. The compositions can include a plurality of different nucleosomal DNA fragments derived from either primary cells or cell lines and can include one or more synthetic oligonucleotides. The amount of the different nucleosomal DNA fragments can be varied so as to simulate naturally occurring cell free DNA samples obtained from the blood of the pregnant woman or naturally occurring cell free DNA samples obtained from the blood of cancer patients.
Another patent, WO2013123030A2 relates to methods and devices for stabilizing a biological sample for analysis, comprising the steps of obtaining in a sample collection device a biological sample from a subject, the biological sample including at (east one circulating cell-free first nucleic acid from the subject. The methods may include a step of contacting the biological sample while within the sample collection device with a protective agent composition that Includes a preservative agent, an optionai anticoagulant, and a quenching agent to form a mature that: includes the protective agent composition and the sample.
Another patent, US11525159B2 provides methods for determining the status of an allograft within a transplant recipient from genotypic data measured from a mixed sample of DNA comprising DNA from both the transplant recipient and from the donor. The mixed sample of DNA may be preferentially enriched at a plurality of polymorphic loci in a way that minimizes the allelic bias, for example using massively multiplexed targeted PCR.
Early diagnosis and treatment of rheumatoid arthritis (RA) can change the course of the condition, prevent joint erosions from forming, and even put the condition into remission. Early RA diagnosis is essential to the beginning of treatment since it stops the disease from advancing to more severe forms, which demand more intensive therapy and are also extremely expensive. Over 40% of RA patients have another ailment, according to a study based on research. As joint swelling is usually an issue in obese people, it can be difficult to tell whether a patient has RA or other diseases such osteoarthritis, psoriatic arthritis, viral arthritis, Lyme disease, fibromyalgia, or lupus. Although RA patients often have normal levels of inflammatory markers like ESR or C-Reactive Protein (CRP),
RA in Early stages of rheumatoid arthritis. "Cell-free DNA" refers to all bloodstream DNA that is not encapsulated (or "cfDNA"). Cell-free DNA (cfDNA) was discovered in blood plasma for the first time by Mandel and Metais in 1948. During necrosis or apoptosis, nucleic acid fragments known as cfDNA are released into the bloodstream. These fragments are taken out by macrophages. Extracellular DNA can originate from both healthy and diseased cells and can be discovered in bodily fluids. Variable amounts of cfDNA are present. They can be quite low during critical periods, such as early recurrence or the formation of resistance. In plasma, human nuclear DNA has a wider size distribution than cfDNA. While taking treatment, the plasma cfDNA may remain constant for the first week before completely disappearing in two to three weeks. We standardized the cell free DNA Isolation Protocol for Diagnosis of RA at earlier stages.
Till now there is no Gene based protocol, for detection of Rheumatoid Arthritis in India and not in even Uttarakhand. The standard Protocol of cell free DNA Isolation algorithm is not available specifically for RA diagnostics. Still the biochemical tests, inflammatory markers, anti ccp and radiograph diagnosis is used to detect this disorder and mostly in later stages. Cell free DNA technique can help in early diagnosis of RA and other autoimmune disorders.
The primary object of the present invention is contamination free cell free DNA (cfDNA) isolation protocol for early diagnosis of Rheumatoid Arthritis.
Another object of the present invention is cell free DNA technique can help in early diagnosis of RA and other autoimmune disorders.
These and other objects and advantages of the present invention will become readily apparent from the following detailed description.
Summary of Invention
This summary is not a comprehensive overview of the disclosure and does not reflect the main/essential features of the establishment or specify the scope of the establishment. Its sole purpose is to present some of the concepts presented here in a simpler way as a precursor to more detailed explanations presented later.
The primary object of the present invention is contamination free cell free DNA (cfDNA) isolation protocol for early diagnosis of Rheumatoid Arthritis.
In some embodiments of the present invention, the Rheumatoid Arthritis sample collection of 34 patients of Uttarakhand Region from various clinics and hospitals.
In some embodiments of the present invention, for the cell free DNA isolation protocol various reagents and chemicals were purchased from Option India pvt ltd. The plasma was separated at 5000 rpm initially from Whole Blood Sample.
In some embodiments of the present invention, as the freely floating cell free DNA can be easily damaged by excessive concentration of lysis buffer moderate amount of lysis was used.
These and other aspects of the embodiments herein will be better appreciated and understood when considered in concurrence with the following explanation and the accompanying drawings. It should be understood, however, that the following descriptions, while indicating preferred embodiments and numerous specific details thereof, are given by way of illustration and not of limitation. Many changes and modifications may be made within the scope of the embodiments herein without departing from the spirit thereof, and the embodiments herein include all such modifications.
Brief Description of the Drawings
Fig. 1 & 2: Gel Picture of Cell Free DNA Concentration visualized under UV Transilluminator
Detailed Description of the Invention
These embodiments are described in sufficient detail to enable those skilled in the art to practice the embodiments and all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
The primary object of the present invention is contamination free cell free DNA (cfDNA) isolation protocol for early diagnosis of Rheumatoid Arthritis.
In some embodiments of the present invention, the Rheumatoid Arthritis sample collection of 34 patients of Uttarakhand Region belonging to various different areas, mainly Daguli, Tikochi in Distt. Uttarkashi, Tyuni Jhajhra, Premnagar, Balawala, Chakrata, Selaqui, Suddhowala, Ataal, District Pauri from various clinics and hospitals. For the cell free DNA isolation protocol various reagents and chemicals were purchased from Option India pvt ltd. The plasma was separated at 5000 rpm initially from Whole Blood Sample. As the freely floating cell free DNA can be easily damaged by excessive concentration of lysis buffer moderate amount of lysis was used.
In some embodiment of this aspect of the present invention, for preparation of lysis buffer 45% of Guanidine hydrochloride, 15% of Polyoxyethylene sorbitan fatty acid ester, 3% hydrochloride salts of amino alcohol and balanced water concentration was used. Few reagents below 1% were also used. Physical properties and solubility in water was analysed. 88% of Protease with stable Ph of 10-12.5 was used for removal of contaminating proteins.
In some embodiments of the present invention, for washing buffer 46 Mm of Monosodium Phosphate (NaH2 PO4) with Ph 8.0 ,290mM NaCl, 19Mm Imidazole, 0.04% Triton*-100, 1Mm EDTA and 1 mm DTT. For Binding buffer for 5.0 mm of Tris-HCL (PH 7.9 ,0.5 mm of EDTA and 1m NaCl) and for Final Elution Buffer 10 mm of Tris EDTA, Tris SDS 1mm and 1% dH2O.
A process of contamination free cell free DNA (cfDNA) isolation protocol for early diagnosis of Rheumatoid Arthritis consists of steps: -
placing 18 µL of Proteinase K into a 1.5 mL microcentrifuge (MCT) tube;
adding 150 µL of plasma into the MCT separated from the whole blood sample, and 150 µL of Lysis Buffer was added and vortexed for 10 secs.
transferring the whole sample to Silica Column and 300 µL of Binding Buffer was dispensed in Silica Binding Column and centrifuged at 10.000 rpm for 1 minute;
adding 200 µL of 95–100% ethanol to the lysate vortex for 5–10 seconds and centrifuge at 10,000 rpm for 1 minute;
transferring the column to a fresh collection tube, and 600 µL of Wash Buffer was added and centrifuged at 10,000 rpm for 3 minutes;
the flow through was discarded and the column was transferred to a fresh collection tube;
adding again 600 µL of Wash Buffer to the column and centrifuged at 10,000 rpm for 3 minutes;
centrifuging the column again for dry wash to remove any leftover lysate, and the column was transferred to the new MCT;
adding 65 µL of Elution Buffer to the column and centrifuged for 1 minute at 10,000 rpm to elute out the Cell free DNA;
collecting the eluted Cell free DNA in MCT and stored the Isolated DNA Template at -20oC; and
staining the cfDNA with an intercalation Ethidium Bromide dye to visualize under UV Transilluminator.
The process as claimed in claim 1, wherein the yield of cfDNA bands were obtained was at 151 bp and 190 bp visualized under UV Transilluminator.
The process as claimed in claim 1, wherein for the preparation of lysis buffer 45% of Guanidine hydrochloride, 15% of Polyoxyethylene sorbitan fatty acid ester, 3% hydrochloride salts of amino alcohol and balanced water concentration was used.
The process as claimed in claim 1, wherein 88% of Protease with stable Ph of 10-12.5 was used for removal of contaminating proteins
EXAMPLE 1
EXPERIMENTAL SECTION
The Rheumatoid Arthritis sample collection of 34 patients of Uttarakhand Region belonging to various different areas, mainly Daguli, Tikochi in Distt. Uttarkashi, Tyuni Jhajhra, Premnagar, Balawala, Chakrata, Selaqui, Suddhowala, Ataal, District Pauri from various clinics and hospitals. For the cell free DNA isolation protocol various reagents and chemicals were purchased from Option India pvt ltd. The plasma was separated at 5000 rpm initially from Whole Blood Sample. As the freely floating cell free DNA can be easily damaged by excessive concentration of lysis buffer. Moderate amount of lysis was used thus for preparation of lysis buffer 45% of Guanidine hydrochloride, 15% of Polyoxyethylene sorbitan fatty acid ester, 3% hydrochloride salts of amino alcohol and balanced water concentration was used. Few reagents below 1% were also used. Physical properties and solubility in water was analysed. 88% of Protease with stable Ph of 10-12.5 was used for removal of contaminating proteins. For washing buffer 46 Mm of Monosodium Phosphate (NaH2PO4) with Ph 8.0, 290mM NaCl, 19Mm Imidazole, 0.04% Triton*-100, 1Mm EDTA and 1 mm DTT. For Binding buffer for 5.0 mm of Tris-HCL (PH 7.9, 0.5 mm of EDTA and 1m NaCl) and for Final Elution Buffer 10 mm of Tris EDTA, Tris SDS 1mm and 1% dH2O. Further all concentrations were standardized upto 50 ml final concentration.
The DNA Isolation was performed Biosafety Level II B2, Cooling Centrifuge of BR Biochem made for centrifugation was used for the whole process. After the reagent preparation the extraction algorithm was developed as:
1. 18 µL of Proteinase K was placed into a 1.5 mL microcentrifuge (MCT) tube.
2. 150 µL of plasma was added into the MCT separated from the whole blood sample.
3. 150 µL of Lysis Buffer was added and vortexed for 10 secs.
4. The whole sample was transferred to Silica Column and 300 µL of Binding Buffer was dispensed in Silica Binding Column and centrifuged at 10.000 rpm for 1 minute.
The flow-through liquid in collection tube was discarded.
5. 200 µL of 95–100% ethanol was added to the lysate vortex for 5–10 seconds and Centrifuge at 10,000 rpm for 1 minute.
6. The collection tube and flow-through was discarded and the column was transferred to a fresh collection tube.
7. 600 µL of Wash Buffer was added and centrifuged at 10,000 rpm for 3 minutes.
8. The flow through was discarded and the column was transferred to a fresh collection tube.
9. 600 µL of Wash Buffer was again added to the column and centrifuged at 10,000 rpm for 3 minutes.
10. The flow through was discarded and the column was centrifuged again for dry wash to remove any leftover lysate.
11. The column was transferred to the new MCT.
12. 65 µL of Elution Buffer was added to the column and centrifuged for 1 minute at 10,000 rpm to elute out the cfDNA.
13. The eluted cfDNA was collected in MCT and the column was discarded.
The Isolated DNA Template was stored at -20oC. The Concentration and yield were determined by Agarose Gel Electrophoresis and intensity of template sample cell free DNA was compared to Quantitation standard DNA. The standard Quantitation DNA was of 100 bp DNA difference The Cell free DNA were stained with an intercalation Ethidium Bromide dye to visualize under UV Transilluminator. The yield of cfDNA bands were obtained was at 151 bp and 190 bp (Figure 1 and Figure 2) respectively when visualized under UV Transilluminator, the protocol was capable of isolating good yields of cfDNA and thus can be used for analyzing the gene-based diagnosis in RA Patients.

We Claims:

1. A process of contamination free cell free DNA (cfDNA) isolation protocol for early diagnosis of Rheumatoid Arthritis consists of steps: -
a) placing 18 µL of Proteinase K into a 1.5 mL microcentrifuge (MCT) tube;
b) adding 150 µL of plasma into the MCT separated from the whole blood sample, and 150 µL of Lysis Buffer was added and vortexed for 10 secs.
c) transferring the whole sample to Silica Column and 300 µL of Binding Buffer was dispensed in Silica Binding Column and centrifuged at 10.000 rpm for 1 minute;
d) adding 200 µL of 95–100% ethanol to the lysate vortex for 5–10 seconds and centrifuge at 10,000 rpm for 1 minute;
e) transferring the column to a fresh collection tube, and 600 µL of Wash Buffer was added and centrifuged at 10,000 rpm for 3 minutes;
f) the flow through was discarded and the column was transferred to a fresh collection tube;
g) adding again 600 µL of Wash Buffer to the column and centrifuged at 10,000 rpm for 3 minutes;
h) centrifuging the column again for dry wash to remove any leftover lysate, and the column was transferred to the new MCT;
i) adding 65 µL of Elution Buffer to the column and centrifuged for 1 minute at 10,000 rpm to elute out the Cell free DNA;
j) collecting the eluted Cell free DNA in MCT and stored the Isolated DNA Template at -20oC; and
k) staining the cfDNA with an intercalation Ethidium Bromide dye to visualize under UV Transilluminator.
2. The process as claimed in claim 1, wherein the yield of cfDNA bands were obtained was at 151 bp and 190 bp visualized under UV Transilluminator.
3. The process as claimed in claim 1, wherein for the preparation of lysis buffer 45% of Guanidine hydrochloride, 15% of Polyoxyethylene sorbitan fatty acid ester, 3% hydrochloride salts of amino alcohol and balanced water concentration was used.
4. The process as claimed in claim 1, wherein 88% of Protease with stable Ph of 10-12.5 was used for removal of contaminating proteins.