Identification of differentially methylated and mutated nucleic acids
a technology of methylation and mutation, applied in the field of methyl(or mutant) differential display (mdd) methods and nucleic acid probes, can solve the problems of no commercial assays for human samples, high repeatability, and high complexity of human genomes, so as to improve antigen binding characteristics, improve specificity, and improve affinity
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example 1
Materials and Methods
[0230] To test the MDD technique, a working model system was designed to isolate methyl-polymorphic DNA markers (see FIG. 1 for a schematic diagram of the MDD methods). There is evidence that in mammalian DNA, methylation patterns are both tissue-, and cell-type specific. Hence, DNA was isolated from two different types of cells from patients who have chronic lymphocytic leukemia (CLL). In CLL patients, the number of CD5+ B type lymphocytes is abnormally high. These CD5+ cells can be isolated by highly specific antibodies in a fluorescence activated cell sorter (FACs). For performing MDD, the DNA isolated from these CD5+ cells served as the tester, while DNA isolated from neutrophils of the same patient served as the driver. A methyl-polymorphic DNA probe, named CLL58, which exhibited methyl-differential displays was isolated from an individual CLL patient.
[0231] After the successful isolation of these methyl-polymorphic probes, MDD was again successfully used...
example 2
Different Methylation Patterns at Cpcpg Sites in Malignant B-Cells and Neutrophil Cells
[0252] DNA isolated from malignant B-cells and neutrophil cells of four CLL patients (#58, #111, #112, #128) was used to perform MDD as described above in the Materials and Methods provided in Example 1. The DNA from leukemic B-cells was used as the tester, and the DNA from neutrophil cells was used as the driver. Tester and driver DNAs were simultaneously digested with the restriction endonucleases, Msp I and Mse I. After ligating the first set of adapters to the digested tester and driver DNA fragments, tester and driver amplicons were generated by PCR amplification (see Materials and Methods). After two rounds of hybridization / subtraction and amplification, the difference products (DP) which were isolated from patient 111 appeared as individual DNA fragment bands on an agarose gel (FIG. 2). The DP fragments were then subcloned into the plasmid pUC118 (see Materials and Methods), and amplified ...
example 3
MDD-Isolated Probes from Human Breast Cancer Tumors Also Detect Ovarian and Colon Cancers
[0255] We have proven the existence of methylation of the external cytosine residue at CpCpG sites in the human genome, and have isolated CpCpG site related methyl-polymorphic markers in a working model system. MDD was then applied to successfully isolate CpCpG related methyl-polymorphic markers from human breast cancer biopsies.
Isolation of Probes From Breast Tumor DNA
[0256] MDD was used to test five pairs of DNA samples (tumor DNA and matched normal DNA) obtained from five breast cancer patients. To isolate methyl-polymorphic markers, tumor DNA served as the tester, and normal DNA served as the driver. MDD was performed in the same manner as described in Example 1. Individual DNA fragments were isolated by MDD from three breast cancer patients after two rounds of hybridization / subtraction and amplification (FIG. 5).
[0257] Three probes were further analyzed, BR50, BR104, and BR254, isolate...
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