No government funds were used to make this invention.
The present invention encompasses a method of modifying a macromolecule without prior extraction from a sample by converting the macromolecule in the sample with a chemical, removing or converting chemical intermediates, if necessary; and purifying the resulting modified macromolecule.
One method used by vertebrates and higher plants to regulation gene expression is the methylation of cytosines found in CpG islands located in promoter regions of various genes. In order to study this method of gene regulation, techniques were developed to discriminate methylated cytosines from unmethylated cytosines. One method is to chemically treat DNA in such a way that the cytosines are converted to uracils while 5-methyl-cytosines are not significantly converted. Frommer et al. (1992). A systematic investigation on the critical parameters of the modification procedure has also been made. Grunau et al. (2001). The treated DNA may be used as template for methylation specific PCR (MSP). DNA methylation and methods related thereto are discussed for instance in US patent publication numbers 20020197639, 20030022215, 20030032026, 20030082600, 20030087258, 20030096289, 20030129620, 20030148290, 20030157510, 20030170684, 20030215842, 20030224040, 20030232351, 20040023279, 20040038245, 20040048275, 20040072197, 20040086944, 20040101843, 20040115663, 20040132048, 20040137474, 20040146866, 20040146868, 20040152080, 20040171118, 20040203048, 20040241704, 20040248090, 20040248120, 20040265814, 20050009059, 20050019762, 20050026183, 20050053937, 20050064428, 20050069879, 20050079527, 20050089870, 20050130172, 20050153296, 20050196792, 20050208491, 20050208538, 20050214812, 20050233340, 20050239101, 20050260630, 20050266458, 20050287553 and U.S. Pat. Nos. 5,786,146, 6,214,556, 6,251,594, 6,331,393 and 6,335,165.
DNA modification kits are commercially available, they convert purified genomic DNA with unmethylated cytosines into genomic lacking unmethylated cytosines but with additional uracils. The treatment is a two-step chemical process consisting a deamination reaction facilitated by bisulfite and a desulfonation step facilitated by sodium hydroxide. Typically the deamination reaction is performed as a liquid and is terminated by incubation on ice followed by adding column binding buffer. Following solid phase binding and washing the DNA is eluted and the desulfonation reaction is performed in a liquid. Adding ethanol terminates the reaction and the modified DNA is cleaned up by precipitation. However, both commercially available kits (Zymo and Chemicon) perform the desulfonation reaction while the DNA is bound on the column and washing the column terminates the reaction. The treated DNA is eluted from the column ready for MSP assay. The modification is tedious and has many steps that cause yield loss and increase operator error. All of the available modification procedures begin with purified genomic DNA, which is a tedious process that also has many steps that cause yield loss and increase operator error.
The present invention encompasses a method of modifying a macromolecule without prior extraction from a sample by converting the macromolecule in the sample with a chemical, removing or converting chemical intermediates, if necessary; and purifying the resulting modified macromolecule.
The present invention encompasses a method of modifying a macromolecule without prior extraction from a sample by converting the macromolecule in the sample with a chemical, removing or converting chemical intermediates, if necessary; and purifying the resulting modified macromolecule.
The macromolecules can be any known in the art including, without limitation, DNA, RNA, cellular metabolites, lipids, carbohydrates and proteins. The DNA can be any known in the art including, without limitation, viral, nucleic, mitochondrial, plastid, bacterial and synthetic. The RNA can be any known in the art including, without limitation, rtRNA, tRNA, miRNA, rRNA and mRNA. The cellular metabolite can be any known in the art including, without limitation, those produced by a metabolic cycle or enzymatic effects. The lipid can be any known in the art including, without limitation, liposomes, cell membrane lipids, intracellular membrane lipids and extracellular lipids. The carbohydrate can be any known in the art including, without limitation, protein-bound carbohydrates and nucleic acid-bound carbohydrates. The protein can be any known in the art including, without limitation, intracellular and extracellular.
The modification is can be any known in the art including bisulfite and biotinylation of DNA or RNA, fluorination and methylation of RNA, heating, liposome formation, micelle formation, uni-layer formation and bilayer formation of lipids, oxidation, de-oxidation, amination and de-amination of carbohydrates and phosphorylation, dephosphorylation, methylation, biotinylation, amination, deamination, glycosylation and deglycosylation of proteins. 20070148670; Chuang et al. (2007); Emmerechts et al. (2007); Frommer et al. (1992); Grunau et al. (2001); Hurd et al. (2007); Jin et al. (2007); Oakeley (1999); Rathi et al. (2003); Rein et al. (1998); Sambrook et al. (2000); Wu et al. (2007).
The sample can be any known in the art including, without limitation, tissue, body fluid, a biopsy sample, and preserved tissue. The tissue can be any known in the art including, without limitation, whole organs, dissected organs, epithelium, neural, gastrointestinal, muscle, cardiac, mucosal and endothelium. The body fluid can be any known in the art including, without limitation, whole blood, plasma, urine, saliva, vitreous and serum. The biopsy sample can be any known in the art including, without limitation, fine needle aspirate, tissue section and skin sample. The preserved tissue can be any known in the art including, without limitation, fresh frozen, paraffin embedded and preserved in a preservation reagent. The preservation reagent can be any known in the art including, without limitation, formalin, RNAlater® and dimethylsulfoxide.
The purification can be any known in the art including, without limitation, particle-based, precipitation, centrifugation, electrophoretic and charge switch. The particle-based purification can be any known in the art including, without limitation, affinity, sizing and magnetic. The sizing particle can be any known in the art including, without limitation, silica-based and diatomaceous earth. The electrophoretic separation purification can be any known in the art including, without limitation, by size and/or charge. The electrophoretic separation purification can be any known in the art including, without limitation, by a gel formed of low molecular weight polymers and/or capillary.
The present invention provides a rapid and efficient method for obtaining bisulfite modified DNA. The method described herein effectively eliminates numerous steps of the previous methods thus reducing possible error while producing superior results. In addition considerable time savings of four to five hours are also realized.
The present invention provides a method of extracting and modifying DNA by obtaining a DNA sample; incubating the sample with an amount of a bisulfite and for a time and under conditions sufficient to convert at least ninety-five percent of the non-methylated cytosine residues in the DNA to uracil resides; binding the DNA in the sample to a column; washing the bound DNA to remove contaminants; incubating the column-bound DNA with a desulfonation reagent for a time and under conditions sufficient for desulfonation to occur; washing the bound DNA to remove the desulfonation reagent; and eluting the bisulfite modified DNA from the column.
The DNA can be at a concentration of from about 0.01 to about 30 μg and can be obtained by any method known in the art and can be purified DNA or DNA obtained directly from a cell lysate. For instance, the cell lysate can be formed from any suitable tissue by any method known in the art and directly treated with a bisulfite reagent. Cell lysis can be by for instance, proteinase and/or high salt concentration and/or detergent, sonication, freeze-thaw treatment or mechanical disruption. Any cell sample is suitable for use herein and can be obtained from tissue, body fluid, biopsy sample or preserved tissue.
The bisulfite reagent can be any known in the art, including, without limitation, sodium bisulfite or meta bisulfite. Other reagents are discussed for instance in US patent publications 20050089898, 20050095623 and 20050153308.
The incubation conditions of step b are about 1-16 hours at 50-95° C. with or without Thermocycling. Thermocycling can be for instance 3 hours at 70° C., 1 hour at 90° C. or cycling between 50° C. and 95° C.
The column can be any known in the art, preferably, it is silica-based or diatomaceous earth. The desulfonation can be by any method known in the art and is preferably performed with sodium hydroxide and an alcohol. Preferably, the alcohol is isopropanol or ethanol. More preferably, when the column is silica-based, the alcohol is ethanol and when the column is diatomaceous earth, the alcohol is isopropanol. The desulfonation preferably occurs from about 0-30, preferably about 5-15 and more preferably about 15 minutes at about 0° C. to about 50° C. Preferably, the temperature is about room temperature.
The modified macromolecule can be eluted by any method known in the art, including, without limitation with water or a suitable buffer.
The following examples are provided to illustrate but not limit the claimed invention. All references cited herein are hereby incorporated herein by reference.
Microfuge tube containing cell culture pellet in PBS
The efficiency of the procedure is assayed using quantitative PCR and β-Actin using GSTP1 as markers. The β-Actin promoter is not methylated and the marker is designed to serve as a control for the modification procedure. The Ct value produced by this marker is reflective of the number of genome equivalents added to the assay. Whereas the GSTP1 promoter is methylated epigenetically and may be reflective of a cancerous state. Therefore the GHSTP1 marker Ct value is more variable and usually greater than the β-Actin Ct value. The Prostate FFPE blocks were obtained from Asterand. The “Zymo” treatment refers commercially available DNA modification kit sold as EZ DNA Methylation Kit from Zymo Research. A 2 Step procedure refers to two separate procedures that use a DNA purification kit (Qiagen QiaAmp mini DBNA purification kit) and a DNA modification kit such as the Zymo kit. The Zymo 1 step procedure is identical to the ID procedure until the 3M NaOH step where the Zymo DNA modification procedure is followed replacing the 3M NaOH with M-Dilution buffer.
The results shown in Table 1 using cell culture pellets indicate that the 1 Step method produces lower Ct values and thus superior results when compared to the Qiagen/Zymo 2 Step procedure. Statistically, a paired T test indicates the methods, using the β-Actin marker, are significantly different from each other with a P value of 0.00006 while the GSTP1 marker had a P value of 0.0001. The results shown in Table 2 indicate that ID 1 step produces lower Ct values when compared to the Zymo 1 Step method with the β-Actin marker produces a P value of 0.02 and the GSTP1 marker results in a P value of 0.003
However, the results shown in Table 1 using prostate FFPE blocks indicate the opposite results from the cell cultures with the Zymo 2 Step producing lower Ct values then the 1 step method. The β-Actin marker results indicate that the methods are significantly different with a P value of 0.021 while the GSTP1 results suggest that they maybe different with a P value of 0.054. The different results suggested that the extraction buffer for cell culture might not be sufficient to lysis tissue blocks. Table 4 results compares the ID 1 Step and the Zymo 1 Step methods using a more aggressive extraction buffer switching the Tween to SDS while using prostate tissue blocks. The ID 1 Step method once again shows superior results suggesting that the failure to produce superior results in Table 3 was due to the extraction buffer. The β-Actin marker results indicates that the methods are significantly different with a P value of 0.0008 while the GSTP1 results suggest that they maybe different with a P value of 0.056. Since the QPCR assay method only has 40 cycles, once an assay fails to produce Ct's then it is not significant to compare with assays that produce Ct's. If the two samples that had assays that failed then the P values would be 0.0025, which indicates the methods using the GSTP1 marker are significantly different.
The purpose of this experiment was to compare the DEM kit versus the Zymo EZ Modification kit on purified DNA obtained from LnCAP cells and urine. Ten normalized random samples were divided between the two kits
5 Samples per Kit (DEM and Zymo)
PCR performed in duplicate using Fast Start Taq (GSTP1, β-Actin, and APC)
ANOVA analysis indicates a P value of 0.663 and a Paired T-Test indicates that there is no statistical difference between DEM and Zymo for GSTP1.
ANOVA analysis indicates a P value of 0.008 and a Paired T-Test indicates that there is a statistical difference between DEM and Zymo for β-Actin.
The results indicate that Zymo and the DEM kit are equivalent for GSTP1
The DEM kit was optimized for tissue as opposed to purified DNA further optimization within ProMU could lead to lower CT values.
The DEM kit demonstrates a higher β-Actin value
The binding of crude lysate containing DNA or purified genomic DNA is uniquely bound to the silica-gel based column utilizing the high concentration of salt that is present from the bisulfite conversion.
Ethanol is added to the sample prior to binding only to dissolve the conversion reagent.
Large scale DNA modification may be necessary to make panels for quality control testing of methylation specific PCR methods and kits.
1. Denature 20 μg of Prostate Cell Culture Cell line 22Rv1 genomic DNA (ATCC) in a total volume of 225 μl TE, plus 27.5 μl of a 3.0 M NaOH solution. Incubate 10 minutes 37° C.
2. Add 2× volume Conversion reagent (Zymo); incubate 3 hr 70° C. followed by 10 min on ice.
Bind the DNA to a solid phase support by adding 2 ml of binding buffer containing the support matrix (Promega) and adding it to the syringe column vacuum apparatus. Using the vacuum, filter the matrix.
Using the vacuum, wash with the matrix with 1 ml 80% IPA
Add 2 ml OD desulfonation buffer (0.3 M NaOH in 80% IPA) and incubate at room temperature for 10 minutes. Using the vacuum, remove the buffer.
Using the vacuum, wash with 1 ml 80% IPA.
3. Remove the column from the syringe and place it in a 1.5 ml microfuge tube Elute 5×200 μl followed by addition of 1 ml EB Table 5 depicts the results obtained.
In this cell line the GSTP1 promoter is known to be unmethylated therefore the lack of Ct values for this marker is expected.
A Modified Protocol for Fast and Efficient Bisulfite Modification of Genomic DNA
The EZ DNA Methylation Kit is provided by Zymo Research (Orange, Calif.) to perform bisulfite modification of DNA. As per manufacturer's recommendation the DNA sample to be modified is incubated with the bisulfite conversion reagent at 50° C. for 12-16 hrs. These conditions have been modified to generate comparable quality bisulfite converted DNA in much less time. Several temperatures for different times were tested and demonstrated that incubation of DNA sample with bisulfite conversion reagent at 70° C. for 1-3 hr provides efficient bisulfite modification comparable to modification conditions recommended in the kit. The data below show methylation specific PCR analysis with DNA samples incubated with bisulfite reagent at different temperatures for different times.
Extraction of genomic DNA and its bisulfite modification prior to being used in a MSP reaction comprise very significant upstream procedures that are part of this in vitro diagnostic assay. These procedures can be time consuming involving many tedious steps and could also increase chances of sample contamination. By combining the use of a lysis buffer, 10 mM Tris pH 8.0, 150 mM NaCl, 2 mM EDTA, 0.5% SDS including proteinase K and a bisulfite modification kit, a quick and simple sample processing protocol to recover and modify minimal amounts of DNA available from these sample types has been developed.
The following steps were performed:
During the 15 min incubation, prepare CT Conversion Reagent (as per manufacturer's instructions).
The protocol described above excludes the use of a DNA purification kit prior to bisulfite modification, thereby, reducing sample processing times, preventing DNA losses during purification, reducing cost, and reducing chances of contamination.
Table 6 shows that using the tissue lysate directly for bisulfite modification of DNA gives comparable and even lower Cts than purified DNA using Qiagen DNA isolation kit. Thus further purification of DNA is not required prior to DNA modification using ZymoResearch EZ DNA methylation kit. Also, the data show that combining TNES/PK digestion and EZ DNA methylation kit yields more DNA sample.
Table 7 shows that direct lysate from FFPE biopsy tissue can be used successfully for downstream DNA modification with comparable and even better results as compared to using Qiagen DNA isolation kit, thus avoiding unnecessary DNA purification steps and losses.
DNA methylation assay is developed to be used on patient samples such as archived formalin-fixed, paraffin-embedded tissues, freshly collected urine and blood samples that comprise an invaluable resource for translational studies of cancer and a variety of other diseases. Sample processing is a key upstream part of this diagnostic assay. Conventionally, DNA is purified from these sample types by using standard phenol-chloroform extraction or column based procedures and then subjected to bisulfite modification procedures. Several commercial kits exist for purification of DNA from paraffin embedded archived tissues, and body fluids. However, loses during such extensive purification procedures can significantly reduce DNA yields when very small amount of starting tissue or body fluid sample type is available. Low DNA yields can severely impact the downstream assay performance and can also be time consuming. To avoid DNA loses some studies have used digested tissue lysate directly for bisulfite modification of genomic DNA using standard in solution bisulfite modification protocols. The protocol developed in this invention (referred to as TNES protocol) quickly and efficiently extracts and bisulfite modifies genomic DNA by using the deproteinized lysed tissue extract or lysed cells from urine sediment and directly using this with a commercially available DNA methylation kit such as ZymoResearch EZ for downstream bisulfite modification without any further purification steps. In addition, the present invention improves multiplex PCR assay performance by minimizing loss during additional purification steps.
A. Protocol for DNA Extraction from Urine Samples (TNES Protocol)
The EZ DNA Methylation Kit is provided by Zymo Research (Orange, Calif.) to perform bisulfite modification of DNA. As per manufacturer's recommendation the DNA sample to be modified is incubated with the bisulfite conversion reagent at 50° C. for 12-16 hrs. These conditions have now been modified to generate comparable quality bisulfite converted DNA in much less time. Several temperatures and different times were tested and it was demonstrated that incubation of DNA sample with bisulfite conversion reagent at 70° C. for 1-3 hr provides efficient bisulfite modification comparable to modification conditions recommended in the kit
The protocol is as follows for processing urine samples:
M-Wash Buffer (Prepare before starting using the kit)
Conversion Reagent (after briefly spinning) to each sample (or add 400 μl CT reagent for a scaled up protocol) and vortex lightly (the sample may turn cloudy). Spin the sample briefly. Incubate the sample at 70° C. for 3 hr with the heating block (shaking at 1100 rpm) covered with aluminum foil. (The CT Conversion Reagent is light sensitive, so try to minimize reaction's exposure to light).
Column and place column into a 2 ml collection tube and centrifuge at maximum speed for 15-30 seconds. Discard the flow-through. For a higher sample volume, this step is repeated by adding supernatant from each aliquoted tube one at a time on the column and centrifuging until all of the sample is loaded onto the column.
The protocol described above excludes the use of a DNA purification kit prior to bisulfite modification, thereby, reducing sample processing times, preventing DNA losses during purification, reducing cost, and reducing chances of contamination).
Table 8 shows end results from MSP assay on Cepheid Smart Cycler with DNA samples processed by TNES protocol from 50 ml of urine. Better β-actin and GSTP1 Cts (up to 3 Cts lower) are observed using above described TNES/PK digestion protocol over use of purified DNA using commercially available Qiagen DNA isolation kit (QiAmp Viral RNA kit). Thus further purification of DNA is not required prior to DNA modification using this method. Also, the data show that combining TNES/PK digestion and EZ DNA methylation kit yields more DNA sample.
Results with TNES extraction protocol are even more compelling on serial dilutions of LNCaP prostate cells (range of 10,000 to 100 spiked per 50 ml pooled urine from healthy donors). Combined protocol for DNA extraction followed directly by bisulfite modification allows to improve sensitivity of the prostate methylation assay by 10 fold (Table 9) as compared with two commercial kits combined. TNES protocol allows detection of 100 cells per 50 ml urine, the level which is undetectable by Qiagen protocol using both Ct value and copy number analysis.
Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, the descriptions and examples should not be construed as limiting the scope of the invention.