Detection of methylated CpG rich sequences diagnostic for malignant cells

Abstract

The present invention provides methods for determining the methylation status of CpG-containing dinucleotides on a genome-wide scale using infrequent cleaving, methylation sensitive restriction endonucleases and two-dimensional gel electrophoretic display of the resulting DNA fragments. Such methods can be used to diagnose cancer, classify tumors and provide prognoses for cancer patients. The present invention also provides isolated polynucleotides and oligonucleotides comprising CpG dinucleotides that are differentially methylated in malignant cells as compared to normal, non-malignant cells. Such polynucleotides and oligonucleotides are useful for diagnosis of cancer. The present invention also provides methods for identifying new DNA clones within a library that contain specific CpG dinucleotides that are differentially methylated in cancer cells as compared to normal cells.

1 - 3 . (canceled)  
     
     
         4 . A method of preparing a polynucleotide or oligonucleotide for characterizing tissue obtained from a subject suspected of having cancer, comprising: synthesizing a polynucleotide or oligonucleotide which comprises a sequence which is identical to or substantially complementary to a target sequence on one of the strands of a diagnostic control fragment identified according to the method comprising: 
 a) digesting genomic DNA obtained from the malignant cells with an infrequently-cutting, methylation-sensitive, restriction enzyme to provide a set of malignant cell restriction fragments;    b) digesting genomic DNA obtained from non-malignant, control cells with an infrequently-cutting, methylation-sensitive, restriction enzyme to provide a set of control cell restriction fragments:    c) attaching a detectable label to the ends of the malignant cell restriction fragments and the control restriction fragments;    d) digesting the labeled malignant cell and control cell restriction fragments with a second restriction enzyme;    e) separating the labeled malignant cell restriction fragments and the labeled control cell restriction fragments, wherein the malignant cell restriction fragments and the control cell restriction fragments are separated by electrophoresis on two different gels;    f) digesting the restriction fragments in each of said gels with a third restriction enzyme;    g) electrophoresing the restriction fragments in each of said gels in a direction perpendicular to the first direction to provide a first pattern of detectable malignant cell restriction fragments and a second pattern of detectable control cell restriction fragments;    h) comparing the first pattern to the second pattern to identify diagnostic control cell restriction fragments in said second pattern which are absent or exhibit a decreased intensity in the first pattern, wherein said diagnostic control cell restriction fragments comprise a CpG island that is unmethylated in the DNA of the control cells and methylated in the DNA of the malignant cells; and    (i) determining the sequence of at least a portion of a diagnostic control cell restriction fragment, wherein said portion is located at or near an end of the fragment,    wherein the tumor or neoplasm is selected from the group: breast, colon, glioma, non-squamous cell head and neck, lung, and non-medulloblastoma primitive neuroectodermal tumors (PNET) and testicle, and    wherein said target sequence comprises at least two CpG dinucleotides, wherein said oligonucleotide is from 15 to 34 nucleotides in length, and wherein said polynucleotide is from 35 to 2000 nucleotides in length.    
     
     
         5 . The method of  claim 4  wherein said target sequence is located at or near the control restriction fragment end which was cleaved by the methylation-sensitive, restriction enzyme.  
     
     
         6 . The method of  claim 4  wherein the target sequence is located from about 100 nucleotides to about 500 nucleotides downstream of the control restriction fragment end that was cleaved by the methylation-sensitive, restriction enzyme.  
     
     
         7 . The method of  claim 4  wherein the control restriction fragment comprises a sequence selected from the group consisting of SEQ. ID. NO.:1, SEQ. ID. NO.:2, SEQ. ID. NO.:3, SEQ. ID. NO.:4, SEQ. ID. NO.:5, SEQ. ID. NO.:6, SEQ. ID. NO.:7, SEQ. ID. NO.:8, SEQ. ID. NO.:9, SEQ. ID. NO.:10, SEQ. ID. NO:11, SEQ. ID. NO.: 12, SEQ. ID. NO.:13, SEQ. ID. NO.: 14, SEQ. ID. NO.: 15, SEQ. ID. NO.: 16, SEQ. ID. NO.:17, SEQ. ID. NO.: 18, SEQ. ID. NO.: 19, SEQ. ID. NO.:20, SEQ. ID. NO.:21, SEQ. ID. NO.:22, SEQ. ID. NO.:23, SEQ. ID. NO.:24, SEQ. ID. NO.:25, SEQ. ID. NO.:26, SEQ. ID. NO.:27, SEQ. ID. NO.:28, SEQ. ID. NO.:29, SEQ. ID. NO.:30, SEQ. ID. NO.:31, SEQ. ID. NO.:32, SEQ. ID. NO.:33, SEQ. ID. NO.:34, SEQ. ID. NO.:35, SEQ. ID. NO.:36, SEQ. ID. NO.:37, SEQ. ID. NO.:38, SEQ. ID. NO.:39, SEQ. ID. NO.:40, SEQ. ID. NO.:41, SEQ. ID. NO.:42, SEQ. ID. NO.:43, SEQ. ID. NO.:44, SEQ. ID. NO.:45, SEQ. ID. NO.:46, SEQ. ID. NO.:47, SEQ. ID. NO.:48, SEQ. ID. NO.:49, SEQ. ID. NO.:50, SEQ. ID. NO.:51, SEQ. ID. NO.:52, SEQ. ID. NO.:53, SEQ. ID. NO.:54, SEQ. ID. NO.:55, SEQ. ID. NO.:56, SEQ. ID. NO.:57, SEQ. ID. NO.:58, SEQ. ID. NO.:59, SEQ. ID. NO.:60, SEQ. ID. NO.:61, SEQ. ID. NO.:62, SEQ. ID. NO.:63, SEQ. ID. NO.:64, SEQ. ID. NO.:65, SEQ. ID. NO.:66, SEQ. ID. NO.:67, SEQ. ID. NO.:68, SEQ. ID. NO.:69, SEQ. ID. NO.:70, SEQ. ID. NO.:71, SEQ. ID. NO.:72, SEQ. ID. NO.:73, SEQ. ID. NO.:74, SEQ. ID. NO.:75, SEQ. ID. NO.:76, SEQ. ID. NO.:77, SEQ. ID. NO.:78, SEQ. ID. NO.:79, SEQ. ID. NO.:80, SEQ. ID. NO.:81, SEQ. ID. NO.:82, SEQ ID NO: 83, SEQ ID NO. 84, SEQ ID. NO. 85, SEQ. ID. NO. 86, SEQ. ID. NO. 87, SEQ. ID. NO. 88, SEQ. ID. NO. 89, SEQ. ID. NO. 90, SEQ. ID. NO. 91, SEQ ID. NO. 92, and SEQ. ID. NO. 93.  
     
     
         8 . An isolated polynucleotide or oligonucleotide for characterizing cells that are obtained from a subject suspected of having a cancer which is associated with methylation of one or a plurality of CpG islands in the genomic DNA of malignant cells, wherein said polynucleotide or oligonucleotide comprises a sequence which is identical to or complementary to a target sequence on one of the strands of a diagnostic control fragment identified according to the method comprising: 
 a) digesting genomic DNA obtained from the malignant cells with an infrequently-cutting, methylation-sensitive, restriction enzyme to provide a set of malignant cell restriction fragments:    b) digesting genomic DNA obtained from non-malignant, control cells with an infrequently-cutting, methylation-sensitive, restriction enzyme to provide a set of control cell restriction fragments:    c) attaching a detectable label to the ends of the malignant cell restriction fragments and the control restriction fragments:    d) digesting the labeled malignant cell and control cell restriction fragments with a second restriction enzyme:    e) separating the labeled malignant cell restriction fragments and the labeled control cell restriction fragments, wherein the malignant cell restriction fragments and the control cell restriction fragments are separated by electrophoresis on two different gels:    f) digesting the restriction fragments in each of said gels with a third restriction enzyme:    g) electrophoresing the restriction fragments in each of said gels in a direction perpendicular to the first direction to provide a first pattern of detectable malignant cell restriction fragments and a second pattern of detectable control cell restriction fragments:    h) comparing the first pattern to the second pattern to identify diagnostic control cell restriction fragments in said second pattern which are absent or exhibit a decreased intensity in the first pattern, wherein said diagnostic control cell restriction fragments comprise a CpG island that is unmethylated in the DNA of the control cells and methylated in the DNA of the malignant cells; and    (i) determining the sequence of at least a portion of a diagnostic control cell restriction fragment, wherein said portion is located at or near an end of the fragment,    wherein the tumor or neoplasm is selected from the group: breast, colon, glioma, non-squamous cell head and neck, lung, and non-medulloblastoma primitive neuroectodermal tumors (PNET) and testicle, and    wherein said target sequence comprises at least two CpG dinucleotides, wherein said oligonucleotide is from 15 to 34 nucleotides in length; and wherein said polynucleotide is from 35 to 3000 nucleotides in length.    
     
     
         9 . The isolated polynucleotide or oligonucleotide of  claim 8  wherein said target sequence is located at or near the control restriction fragment end which was cleaved by the methylation sensitive restriction enzyme.  
     
     
         10 . The isolated polynucleotide or oligonucleotide of  claim 8  wherein the target sequence is located from about 100 nucleotides to about 500 nucleotides downstream of the control restriction fragment end that was cleaved by the methylation-sensitive, restriction enzyme.  
     
     
         11 . An isolated polynucleotide or oligonucleotide for characterizing cells which are obtained from a subject suspected of having a cancer which is associated with methylation of one or a plurality of CpG islands in the genomic DNA of malignant cells, wherein said polynucleotide or oligonucleotide comprises a sequence which is identical to or complementary to a modified target sequence on one of the strands of a diagnostic control fragment identified according to the method comprising: 
 a) digesting genomic DNA obtained from the malignant cells with an infrequently-cutting, methylation-sensitive, restriction enzyme to provide a set of malignant cell restriction fragments;    b) digesting genomic DNA obtained from non-malignant, control cells with an infrequently-cutting, methylation-sensitive, restriction enzyme to provide a set of control cell restriction fragments;    c) attaching a detectable label to the ends of the malignant cell restriction fragments and the control restriction fragments;    d) digesting the labeled malignant cell and control cell restriction fragments with a second restriction enzyme;    e) separating the labeled malignant cell restriction fragments and the labeled control cell restriction fragments, wherein the malignant cell restriction fragments and the control cell restriction fragments are separated by electrophoresis on two different gels;    f) digesting the restriction fragments in each of said gels with a third restriction enzyme;    g) electrophoresing the restriction fragments in each of said gels in a direction perpendicular to the first direction to provide a first pattern of detectable malignant cell restriction fragments and a second pattern of detectable control cell restriction fragments,    h) comparing the first pattern to the second pattern to identify diagnostic control cell restriction fragments in said second pattern which are absent or exhibit a decreased intensity in the first pattern, wherein said diagnostic control cell restriction fragments comprise a CpG island that is unmethylated in the DNA of the control cells and methylated in the DNA of the malignant cells; and    (i) determining the sequence of at least a portion of a diagnostic control cell restriction fragment, wherein said portion is located at or near an end of the fragment,    wherein the tumor or neoplasm is selected from the group: breast, colon, glioma, non-squamous cell head and neck, lung, and non-medulloblastoma primitive neuroectodermal tumors (PNET) and testicle, and    wherein said modified target sequence is derived from a target sequence that has been modified by treatment with sodium bisulfite, wherein said modified target sequence lacks cytosines and comprises at least two UpG dinucleotides, wherein said oligonucleotide is from 15 to 34 nucleotides in length; and wherein said polynucleotide is from 35 to 3000 nucleotides in length.    
     
     
         12 . The isolated polynucleotide or oligonucleotide of  claim 11  wherein the modified target sequence is derived from a target sequence located at or near the control restriction fragment end that was cleaved by the methylation sensitive restriction enzyme.  
     
     
         13 . The isolated polynucleotide or oligonucleotide of  claim 11  wherein the modifed target sequence is derived from a target sequence that is located from about 100 nucleotides to about 500 nucleotides downstream of the control restriction fragment end that was cleaved by the methylation-sensitive restriction enzyme.  
     
     
         14 . An isolated polynucleotide for characterizing cells which are obtained from a subject suspected of having a cancer selected from the group consisting of glioma, acute myeloid leukemia, primitive neuroectodermal tumors of childhood, breast cancer, colon cancer, head and neck cancer, testiclular cancer and lung cancer; wherein said polynucleotide is from 35 to 3000 nucleotides in length and comprises at least two CpG dinucleotides, and wherein said polynucleotide comprise a sequence which is identical to or complementary to a target sequence located within a sequence selected from the group consisting of SEQ. ID. NO.:1, SEQ. ID. NO.:2, SEQ. ID. NO.:3, SEQ. ID. NO.:4, SEQ. ID. NO.:5, SEQ. ID. NO.:6, SEQ. ID. NO.:7, SEQ. ID. NO.:8, SEQ. ID. NO.:9, SEQ. ID. NO.:10, SEQ. ID. NO:11, SEQ. ID. NO.: 12, SEQ. ID. NO.: 13, SEQ. ID. NO.: 14, SEQ. ID. NO.: 15, SEQ. ID. NO.: 16, SEQ. ID. NO.: 17, SEQ. ID. NO.:18, SEQ. ID. NO.: 19, SEQ. ID. NO.:20, SEQ. ID. NO.:21, SEQ. ID. NO.:22, SEQ. ID. NO.:23, SEQ. ID. NO.:24, SEQ. ID. NO.:25, SEQ. ID. NO.:26, SEQ. ID. NO.:27, SEQ. ID. NO.:28, SEQ. ID. NO.:29, SEQ. ID. NO.:30, SEQ. ID. NO.:31, SEQ. ID. NO.:32, SEQ. ID. NO.:33, SEQ. ID. NO.:34, SEQ. ID. NO.:35, SEQ. ID. NO.:36, SEQ. ID. NO.:37, SEQ. ID. NO.:38, SEQ. ID. NO.:39, SEQ. ID. NO.:40, SEQ. ID. NO.:41, SEQ. ID. NO.:42, SEQ. ID. NO.:43, SEQ. ID. NO.:44, SEQ. ID. NO.:45, SEQ. ID. NO.:46, SEQ. ID. NO.:47, SEQ. ID. NO.:48, SEQ. ID. NO.:49, SEQ. ID. NO.:50, SEQ. ID. NO.:51, SEQ. ID. NO.:52, SEQ. ID. NO.:53, SEQ. ID. NO.:54, SEQ. ID. NO.:55, SEQ. ID. NO.:56, SEQ. ID. NO.:57, SEQ. ID. NO.:58, SEQ. ID. NO.:59, SEQ. ID. NO.:60, SEQ. ID. NO.:61, SEQ. ID. NO.:62, SEQ. ID. NO.:63, SEQ. ID. NO.:64, SEQ. ID. NO.:65, SEQ. ID. NO.:66, SEQ. ID. NO.:67, SEQ. ID. NO.:68, SEQ. ID. NO.:69, SEQ. ID. NO.:70, SEQ. ID. NO.:71, SEQ. ID. NO.:72, SEQ. ID. NO.:73, SEQ. ID. NO.:74, SEQ. ID. NO.:75, SEQ. ID. NO.:76, SEQ. ID. NO.:77, SEQ. ID. NO.:78, SEQ. ID. NO.:79, SEQ. ID. NO.:80, SEQ. ID. NO.:81, SEQ. ID. NO.:82, SEQ ID NO: 83, SEQ ID NO. 84, SEQ ID. NO. 85, SEQ. ID. NO. 86, SEQ. ID. NO. 87, SEQ. ID. NO. 88, SEQ. ID. NO. 89, SEQ. ID. NO. 90, SEQ. ID. NO. 91, SEQ ID. NO. 92, and SEQ. ID. NO. 93.  
     
     
         15 . The isolated polynucleotide of  claim 14  wherein said subject is suspected of having glioma and said polynucleotide comprises a sequence which is identical to or complementary to a target sequence located within SEQ. ID. NO. 78, SEQ ID NO:71, SEQ ID NO:75, SEQ ID NO:18, SEQ ID NO:4, SEQ ID NO:22, SEQ ID NO:82, SEQ ID NO:27, SEQ ID NO:38, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:59, SEQ ID NO:36, SEQ ID NO:46, SEQ ID NO:2, SEQ ID NO:7, SEQ ID NO:20, SEQ ID NO:30, SEQ ID NO:45, SEQ ID NO:72, SEQ ID NO:70, SEQ ID NO:26, SEQ ID NO:54, or SEQ ID NO:69.  
     
     
         16 . The isolated polynucleotide of  claim 14  wherein said subject is suspected of having acute myeloid leukemia and said polynucleotide comprises a sequence which is identical to or complementary to a target sequence located within SEQ ID NO:10, SEQ ID NO:44, SEQ ID NO:48, SEQ ID NO:58, SEQ ID NO:73, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:14, SEQ ID NO:24, SEQ ID NO:33, SEQ ID NO:56, SEQ ID NO:68, SEQ ID NO:76, SEQ ID NO:17, SEQ ID NO:52, SEQ ID NO:57, SEQ ID NO:26, SEQ ID NO:38, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:59, SEQ ID NO:36, SEQ ID NO:46, SEQ ID NO:38, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:59, SEQ ID NO:36, SEQ ID NO:46, SEQ ID NO:2, SEQ ID NO:7, SEQ ID NO:20, SEQ ID NO:30, SEQ ID NO:45, SEQ ID NO:72, SEQ ID NO:16, SEQ ID NO:55, SEQ ID NO:61, SEQ ID NO:63, or SEQ ID NO:70.  
     
     
         17 . The isolated polynucleotide of  claim 14  wherein said subject is suspected of having a primitive neuroectodermal tumor of childhood and said polynucleotide comprises a sequence which is identical to or complementary to a target sequence located within SEQ ID NO:39, SEQ ID NO:42, SEQ ID NO:50, SEQ ID NO:9, SEQ ID NO:14, SEQ ID NO:24, SEQ ID NO:37, SEQ ID NO:4, SEQ ID NO:36, SEQ ID NO:46, SEQ ID NO:72, SEQ ID NO:26, SEQ ID NO:15, SEQ ID NO:19, or SEQ ID NO:61.  
     
     
         18 . The isolated polynucleotide of  claim 14  wherein said subject is suspected of having breast cancer and said polynucleotide comprises a sequence identical or complementary to a target sequence which is located within SEQ ID NO:21, SEQ ID NO:28, SEQ ID NO:41, SEQ ID NO:80, SEQ ID NO:37, SEQ ID NO:63, SEQ ID NO:71, SEQ ID NO:75, SEQ ID NO:18, SEQ ID NO:4, SEQ ID NO:22, SEQ ID NO:82, SEQ ID NO:12, SEQ ID NO:23, SEQ ID NO:31, SEQ ID NO:34, SEQ ID NO:43, SEQ ID NO:60, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:77.  
     
     
         19 . The isolated polynucleotide of  claim 14  wherein said subject is suspected of having colon cancer and said polynucleotide comprises a sequence which is identical to or complementary to a target sequence located within SEQ ID NO:11, SEQ ID NO:40, SEQ ID NO:74, SEQ ID NO:81, SEQ ID NO:53, SEQ ID NO:62, SEQ ID NO:76, SEQ ID NO:17, SEQ ID NO:52, SEQ ID NO:57, SEQ ID NO:37, SEQ ID NO:75, SEQ ID NO:18, SEQ ID NO:4, SEQ ID NO:27, SEQ ID NO:38, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:59, SEQ ID NO:36, or SEQ ID NO:46.  
     
     
         20 . The isolated polynucleotide of  claim 14  wherein said subject is suspected of head and neck cancer and said polynucleotide comprises a sequence which is identical to or complementary to a target sequence located within in SEQ ID NO:1, SEQ ID NO. 79, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:62, SEQ ID NO:76, SEQ ID NO:8, or SEQ ID NO:13.  
     
     
         21 . The isolated polynucleotide of  claim 14  wherein said subject is suspected of testiclular cancer and said polynucleotide comprises a sequence which is identical to or complementary to a target sequence located within in SEQ ID NO. 29, SEQ ID NO:33, SEQ ID NO:56, SEQ ID NO:68, SEQ ID NO:51, SEQ ID NO:57, SEQ ID NO:70, SEQ ID NO:54, or SEQ ID NO:69  
     
     
         22 . The isolated polynucleotide of  claim 14  wherein said subject is suspected of having a lung cancer and said polynucleotide comprises a sequence which is identical to or complementary to a target sequence located within SEQ ID NO: 83, SEQ ID NO. 84, SEQ ID. NO. 85, SEQ. ID. NO. 86, SEQ. ID NO. 87, SEQ. ID. NO. 88, SEQ. ID. NO. 89, SEQ. ID. NO. 90, SEQ. ID NO. 91, SEQ ID. NO. 92, and SEQ. ID. NO. 93.  
     
     
         23 . An isolated CpG diagnostic oligonucleotide for characterizing cells which are obtained from a subject suspected of having a cancer selected from the group consisting of glioma, acute myeloid leukemia, primitive neuroectodermal tumors of childhood, breast cancer, colon cancer, head and neck cancer, testiclular cancer and lung cancer; wherein said olignoucleotide is from 15 to 34 nucleotides in length and comprises at least two CpG dinucleotides, and wherein said oligonucleotide comprise a sequence which is identical to a target sequence located within a region extending from nucleotide 1 through nucleotide 99 of a sequence selected from the group consisting of SEQ. ID. NO.:1, SEQ. ID. NO.:2, SEQ. ID. NO.:3, SEQ. ID. NO.:4, SEQ. ID. NO.:5, SEQ. ID. NO.:6, SEQ. ID. NO.:7, SEQ. ID. NO.:8, SEQ. ID. NO. 9, SEQ. ID. NO.:10, SEQ. ID. NO:11, SEQ. ID. NO.: 12, SEQ. ID. NO.:13, SEQ. ID. NO.: 14, SEQ. ID. NO.: 15, SEQ. ID. NO.: 16, SEQ. ID. NO.:17, SEQ. ID. NO.: 18, SEQ. ID. NO.: 19, SEQ. ID. NO.:20, SEQ. ID. NO.:21, SEQ. ID. NO.:22, SEQ. ID. NO.:23, SEQ. ID. NO.:24, SEQ. ID. NO.:25, SEQ. ID. NO.:26, SEQ. ID. NO.:27, SEQ. ID. NO.:28, SEQ. ID. NO.:29, SEQ. ID. NO.:30, SEQ. ID. NO.:31, SEQ. ID. NO.:32, SEQ. ID. NO.:33, SEQ. ID. NO.:34, SEQ. ID. NO.:35, SEQ. ID. NO.:36, SEQ. ID. NO.:37, SEQ. ID. NO.:38, SEQ. ID. NO.:39, SEQ. ID. NO.:40, SEQ. ID. NO.:41, SEQ. ID. NO.:42, SEQ. ID. NO.:43, SEQ. ID. NO.:44, SEQ. ID. NO.:45, SEQ. ID. NO.:46, SEQ. ID. NO.:47, SEQ. ID. NO.:48, SEQ. ID. NO.:49, SEQ. ID. NO.:50, SEQ. ID. NO.:51, SEQ. ID. NO.:52, SEQ. ID. NO.:53, SEQ. ID. NO.:54, SEQ. ID. NO.:55, SEQ. ID. NO.:56, SEQ. ID. NO.:57, SEQ. ID. NO.:58, SEQ. ID. NO.:59, SEQ. ID. NO.:60, SEQ. ID. NO.:61, SEQ. ID. NO.:62, SEQ. ID. NO.:63, SEQ. ID. NO.:64, SEQ. ID. NO.:65, SEQ. ID. NO.:66, SEQ. ID. NO.:67, SEQ. ID. NO.:68, SEQ. ID. NO.:69, SEQ. ID. NO.:70, SEQ. ID. NO.:71, SEQ. ID. NO.:72, SEQ. ID. NO.:73, SEQ. ID. NO.:74, SEQ. ID. NO.:75, SEQ. ID. NO.:76, SEQ. ID. NO.:77, SEQ. ID. NO.:78, SEQ. ID. NO.:79, SEQ. ID. NO.:80, SEQ. ID. NO.:81, SEQ. ID. NO.:82; SEQ ID NO: 83, SEQ ID NO. 84, SEQ ID. NO. 85, SEQ. ID. NO. 86, SEQ. ID. NO. 87, SEQ. ID. NO. 88, SEQ. ID. NO. 89, SEQ. ID. NO. 90, SEQ. ID. NO. 91, SEQ ID. NO. 92, and SEQ. ID. NO. 93; or a sequence which is the reverse complement of a target sequence located in a region extending from about nucleotide 100 through nucleotide 500 in said SEQ ID NO.  
     
     
         24 . The isolated oligonucleotide of  claim 23  wherein said subject is suspected of having glioma and said oligonucleotide comprises a sequence which is identical to a target sequence located within a region extending from nucleotide 1 through nucleotide 99 in SEQ ID 78, SEQ ID NO:71, SEQ ID NO:75, SEQ ID NO:18, SEQ ID NO:4, SEQ ID NO:22, SEQ ID NO:82, SEQ ID NO:27, SEQ ID NO:38, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:59, SEQ ID NO:36, SEQ ID NO:46, SEQ ID NO:2, SEQ ID NO:7, SEQ ID NO:20, SEQ ID NO:30, SEQ ID NO:45, SEQ ID NO:72, SEQ ID NO:70, SEQ ID NO:26, SEQ ID NO:54, or SEQ ID NO:69, or said oligonucleotide comprises a sequence which is the reverse complement of a target sequence located in a region extending from about nucleotide 100 through nucleotide 500 in said SEQ ID NO.  
     
     
         25 . The isolated oligonucleotide of  claim 23  wherein the subject is suspected of having acute myeloid leukemia; and said oligonucleotide comprises a sequence which is identical to a target sequence located within a region extending from nucleotide 1 through nucleotide 99 SEQ ID NO:10, SEQ ID NO:44, SEQ ID NO:48, SEQ ID NO:58, SEQ ID NO:73, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:14, SEQ ID NO:24, SEQ ID NO:33, SEQ ID NO:56, SEQ ID NO:68, SEQ ID NO:76, SEQ ID NO:17, SEQ ID NO:52, SEQ ID NO:57, SEQ ID NO:26, SEQ ID NO:38, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:59, SEQ ID NO:36, SEQ ID NO:46, SEQ ID NO:38, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:59, SEQ ID NO:36, SEQ ID NO:46, SEQ ID NO:2, SEQ ID NO:7, SEQ ID NO:20, SEQ ID NO:30, SEQ ID NO:45, SEQ ID NO:72, SEQ ID NO:16, SEQ ID NO:55, SEQ ID NO:61, SEQ ID NO:63, or SEQ ID NO:70, or said oligonucleotide comprises a sequence which is the reverse complement of a target sequence located in a region extending from about nucleotide 100 through nucleotide 500 in said SEQ ID NO.  
     
     
         26 . The isolated oligonucleotide of  claim 23  wherein said subject is suspected of having primitive neuroectodermal tumors of childhood; and said oligonucleotide comprises a sequence which is identical to a target sequence located within a region extending from nucleotide 1 through nucleotide 99 of SEQ ID NO:39, SEQ ID NO:42, SEQ ID NO:50, SEQ ID NO:9, SEQ ID NO:14, SEQ ID NO:24, SEQ ID NO:37, SEQ ID NO:4, SEQ ID NO:36, SEQ ID NO:46, SEQ ID NO:72, SEQ ID NO:26, SEQ ID NO:15, SEQ ID NO:19, or SEQ ID NO:61, or said nucleotide comprises a sequence which is the reverse complement of a target sequence located in a region extending from about nucleotide 100 through nucleotide 500 in said SEQ ID NO.  
     
     
         27 . The isolated oligonucleotide of  claim 23  wherein said subject is suspected of having breast cancer; and said oligonucleotide comprises a sequence which is identical to a target sequence located within a region extending from nucleotide 1 through nucleotide 99 of SEQ ID NO:21, SEQ ID NO:28, SEQ ID NO:41, SEQ ID NO:80, SEQ ID NO:37, SEQ ID NO:63, SEQ ID NO:71, SEQ ID NO:75, SEQ ID NO:18, SEQ ID NO:4, SEQ ID NO:22, SEQ ID NO:82, SEQ ID NO:12, SEQ ID NO:23, SEQ ID NO:31, SEQ ID NO:34, SEQ ID NO:43, SEQ ID NO:60, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:77, or said oligonucleotide comprises a sequence which is the reverse complement of a target sequence located in a region extending from about nucleotide 100 through nucleotide 500 in said SEQ ID NO.  
     
     
         28 . The isolated oligonucleotide of  claim 23  wherein said subject is suspected of having colon cancer; and said oligonucleotide comprises a sequence which is identical to a target sequence located within a region extending from nucleotide 1 through nucleotide 99 of SEQ ID NO:11, SEQ ID NO:40, SEQ ID NO:74, SEQ ID NO:81, SEQ ID NO:53, SEQ ID NO:62, SEQ ID NO:76, SEQ ID NO:17, SEQ ID NO:52, SEQ ID NO:57, SEQ ID NO:37, SEQ ID NO:75, SEQ ID NO:18, SEQ ID NO:4, SEQ ID NO:27, SEQ ID NO:38, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:59, SEQ ID NO:36, SEQ ID NO:46 or said oligonucleotide comprises a sequence which is the reverse complement of a target sequence located in a region extending from about nucleotide 100 through nucleotide 500 in said SEQ ID NO.  
     
     
         29 . The isolated oligonucleotide of  claim 23  wherein said subject is suspected of having head and neck cancer; and said oligonucleotide comprises a sequence which is identical to a target sequence located within a region extending from nucleotide 1 through nucleotide 99 in SEQ ID NO:1, SEQ ID NO. 79, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:62, SEQ ID NO:76, SEQ ID NO:8, or SEQ ID NO:13., or said nucleotide a sequence which is the reverse complement of a target sequence located in a region extending from about nucleotide 100 through nucleotide 500 in said SEQ ID NO.  
     
     
         30 . The isolated oligonucleotide of  claim 23  wherein said subject is suspected of having testiclular cancer; and said oligonucleotide comprises a sequence which is identical to a target sequence located within a region extending from nucleotide 1 through nucleotide 99 in SEQ ID NO. 29, SEQ ID NO:33, SEQ ID NO:56, SEQ ID NO:68, SEQ ID NO:51, SEQ ID NO:57, SEQ ID NO:70, SEQ ID NO:54, or SEQ ID NO:69, or said oligonucleotide comprises a sequence which is the reverse complement of a target sequence located in a region extending from about nucleotide 100 through nucleotide 500 in said SEQ ID NO.  
     
     
         31 . The isolated oligonucleotide of  claim 23  wherein said subject is suspected of having lung cancer; and said oligonucleotide comprises a sequence which is identical to a target sequence located within a region extending from nucleotide 1 through nucleotide 99 of SEQ ID NO SEQ ID NO: 83, SEQ ID NO. 84, SEQ ID. NO. 85, SEQ. ID. NO. 86, SEQ. ID. NO. 87, SEQ. ID. NO. 88, SEQ. ID. NO. 89, SEQ. ID. NO. 90, SEQ. ID. NO. 91, SEQ ID. NO. 92, and SEQ. ID. NO. 93, or said oligonucleotide comprises a sequence which is the reverse complement of a target sequence located in a region extending from about nucleotide 100 through nucleotide 500 in said SEQ ID NO.  
     
     
         32 . A method for determining whether cells obtained from a subject suspected of having a cancer are malignant or non-malignant, comprising: 
 a) digesting DNA which has been isolated from the cells with a methylation-sensitive restriction enzyme to provide a set of restriction fragments;    b) hybridizing said restriction fragments with a CpG diagnostic polynucleotide comprising a sequence which is identical to or complementary to a target sequence on one of the strands of a diagnostic control fragment identified according to the method comprising:    a) digesting genomic DNA obtained from the malignant cells with an infrequently-cutting, methylation-sensitive, restriction enzyme to provide a set of malignant cell restriction fragments;    b) digesting genomic DNA obtained from non-malignant, control cells with an infrequently-cutting, methylation-sensitive, restriction enzyme to provide a set of control cell restriction fragments;    c) attaching a detectable label to the ends of the malignant cell restriction fragments and the control restriction fragments;    d) digesting the labeled malignant cell and control cell restriction fragments with a second restriction enzyme;    e) separating the labeled malignant cell restriction fragments and the labeled control cell restriction fragments, wherein the malignant cell restriction fragments and the control cell restriction fragments are separated by electrophoresis on two different gels;    f) digesting the restriction fragments in each of said gels with a third restriction enzyme;    g) electrophoresing the restriction fragments in each of said gels in a direction perpendicular to the first direction to provide a first pattern of detectable malignant cell restriction fragments and a second pattern of detectable control cell restriction fragments;    h) comparing the first pattern to the second pattern to identify diagnostic control cell restriction fragments in said second pattern which are absent or exhibit a decreased intensity in the first pattern, wherein said diagnostic control cell restriction fragments comprise a CpG island that is unmethylated in the DNA of the control cells and methylated in the DNA of the malignant cells; and    (i) determining the sequence of at least a portion of a diagnostic control cell restriction fragment, wherein said portion is located at or near an end of the fragment,    wherein the tumor or neoplasm is selected from the group: breast, colon, glioma, non-squamous cell head and neck, lung, and non-medulloblastoma primitive neuroectodermal tumors (PNET) and testicle, and    wherein said method employed said methylation-sensitive restriction enzyme, wherein said target sequence comprises at least two CpG dinucleotides, wherein said polynucleotide is from 35 to 3000 nucleotides in length, and wherein said reaction is conducted under stringent hybridization conditions; and    c) assaying the reaction products of step b to determine the size or the sequence of the restriction fragment to which the CpG diagnostic polynucleotide has hybridized.    
     
     
         33 . The method of  claim 32  wherein said subject is suspected of having a cancer selected from the group consisting of glioma, acute myeloid leukemia, primitive neuroectodermal tumors of childhood, breast cancer, colon cancer, head and neck cancer, and testiclular cancer; 
 wherein said DNA sample is digested with NotI; and    wherein said polynucleotide comprises a sequence which is identical to or complementary to a target sequence located in SEQ. ID. NO.: 1, SEQ. ID. NO.:2, SEQ. ID. NO.:3, SEQ. ID. NO.:4, SEQ. ID. NO.:5, SEQ. ID. NO.:6, SEQ. ID. NO.:7, SEQ. ID. NO.:8, SEQ. ID. NO.:9, SEQ. ID. NO.:10, SEQ. ID. NO:11, SEQ. ID. NO.: 12, SEQ. ID. NO.:13, SEQ. ID. NO.:14, SEQ. ID. NO.:15, SEQ. ID. NO.: 16, SEQ. ID. NO.: 17, SEQ. ID. NO.:18, SEQ. ID. NO.: 19, SEQ. ID. NO.:20, SEQ. ID. NO.:21, SEQ. ID. NO.:22, SEQ. ID. NO.:23, SEQ. ID. NO.:24, SEQ. ID. NO.:25, SEQ. ID. NO.:26, SEQ. ID. NO.:27, SEQ. ID. NO.:28, SEQ. ID. NO.:29, SEQ. ID. NO.:30, SEQ. ID. NO.:31, SEQ. ID. NO.:32, SEQ. ID. NO.:33, SEQ. ID. NO.:34, SEQ. ID. NO.:35, SEQ. ID. NO.:36, SEQ. ID. NO.:37, SEQ. ID. NO.:38, SEQ. ID. NO.:39, SEQ. ID. NO.:40, SEQ. ID. NO.:41, SEQ. ID. NO.:42, SEQ. ID. NO.:43, SEQ. ID. NO.:44, SEQ. ID. NO.:45, SEQ. ID. NO.:46, SEQ. ID. NO.:47, SEQ. ID. NO.:48, SEQ. ID. NO.:49, SEQ. ID. NO.:50, SEQ. ID. NO.:51, SEQ. ID. NO.:52, SEQ. ID. NO.:53, SEQ. ID. NO.:54, SEQ. ID. NO.:55, SEQ. ID. NO.:56, SEQ. ID. NO.:57, SEQ. ID. NO.:58, SEQ. ID. NO.:59, SEQ. ID. NO.:60, SEQ. ID. NO.:61, SEQ. ID. NO.:62, SEQ. ID. NO.:63, SEQ. ID. NO.:64, SEQ. ID. NO.:65, SEQ. ID. NO.:66, SEQ. ID. NO.:67, SEQ. ID. NO.:68, SEQ. ID. NO.:69, SEQ. ID. NO.:70, SEQ. ID. NO.:71, SEQ. ID. NO.:72, SEQ. ID. NO.:73, SEQ. ID. NO.:74, SEQ. ID. NO.:75, SEQ. ID. NO.:76, SEQ. ID. NO.:77, SEQ. ID. NO.:78, SEQ. ID. NO.:79, SEQ. ID. NO.:80, SEQ. ID. NO.:81, and SEQ. ID. NO.:82.    
     
     
         34 . The method of  claim 32  wherein the subject is suspected of having lung cancer; 
 wherein the DNA is digested with AscI;    and wherein said polynucleotide comprises a sequence which is identical to or complementary to a target sequence located in, SEQ ID NO: 83, SEQ ID NO. 84, SEQ ID. NO. 85, SEQ. ID. NO. 86, SEQ. ID. NO. 87, SEQ. ID. NO. 88, SEQ. ID. NO. 89, SEQ. ID. NO. 90, SEQ. ID. NO. 91, SEQ ID. NO. 92, and SEQ. ID. NO. 93.    
     
     
         35 . A method of determining whether cells contained within a tissue sample obtained from a subject suspected of having cancer are malignant, comprising: 
 a) treating DNA isolated from the tissue sample with a compound which converts non-methylated cytosines to a different nucleotide base;    b) reacting a portion of the treated DNA with a CpG diagnostic oligonucleotide which is complementary to a target sequence which comprises CpG islands that are preferentially methylated in malignant cells of subjects known to have said cancer;    c) reacting a portion of the treated DNA with a modified CpG diagnostic oligonucleotide which is complementary to a modified target sequence in which the cytosines in said target sequence are replaced with the different nucleotide base; and    d) assaying the reaction products of step b and step c to determine whether the treated DNA has hybridized with the CpG diagnostic oligonucleotide or the modified CpG diagnostic oligonucleotide; wherein hybridization of the treated DNA with the CpG diagnostic oligonucleotide as opposed to the modified CpG diagnostic oligonucleotide indicates that the DNA has been obtained from malignant cells.    
     
     
         36 . The method of  claim 35  wherein the chemical compound is sodium bisulfite and the non-methylated cytosines are converted to uracil.  
     
     
         37 . The method of  claim 35  wherein the assay is a polymerase chain reaction, 
 wherein a portion of the treated DNA is reacted with a first primer set which comprises two diagnostic CpG olignonucleotides; and    wherein a portion of the treated DNA is reacted with a second primer set which comprises two modified diagnostic CpG oligonucleotides.    
     
     
         38 . The method of  claim 35  wherein the subject is suspected of having a cancer selected from the group consisting of glioma, acute myeloid leukemia, primitive neuroectodermal tumors of childhood, breast cancer, colon cancer, head and neck cancer, testiclular cancer and lung cancer; and 
 wherein the CpG diagnostic oligonucleotide comprises a sequence which is identical to a target sequence located between nucleotide 1 and 100 in a sequece selected from the group consisting said polynucleotide comprises a sequence which is identical to or complementary to a target sequence located in SEQ. ID. NO.: 1, SEQ. ID. NO.:2, SEQ. ID. NO.:3, SEQ. ID. NO.:4, SEQ. ID. NO.:5, SEQ. ID. NO.:6, SEQ. ID. NO.:7, SEQ. ID. NO.:8, SEQ. ID. NO.:9, SEQ. ID. NO.:10, SEQ. ID. NO:11, SEQ. ID. NO.: 12, SEQ. ID. NO.:13, SEQ. ID. NO.:14, SEQ. ID. NO.:15, SEQ. ID. NO.: 16, SEQ. ID. NO.:17, SEQ. ID. NO.:18, SEQ. ID. NO.: 19, SEQ. ID. NO.:20, SEQ. ID. NO.:21, SEQ. ID. NO.:22, SEQ. ID. NO.:23, SEQ. ID. NO.:24, SEQ. ID. NO.:25, SEQ. ID. NO.:26, SEQ. ID. NO.:27, SEQ. ID. NO.:28, SEQ. ID. NO.:29, SEQ. ID. NO.:30, SEQ. ID. NO.:31, SEQ. ID. NO.:32, SEQ. ID. NO.:33, SEQ. ID. NO.:34, SEQ. ID. NO.:35, SEQ. ID. NO.:36, SEQ. ID. NO.:37, SEQ. ID. NO.:38, SEQ. ID. NO.:39, SEQ. ID. NO.:40, SEQ. ID. NO.:41, SEQ. ID. NO.:42, SEQ. ID. NO.:43, SEQ. ID. NO.:44, SEQ. ID. NO.:45, SEQ. ID. NO.:46, SEQ. ID. NO.:47, SEQ. ID. NO.:48, SEQ. ID. NO.:49, SEQ. ID. NO.:50, SEQ. ID. NO.:51, SEQ. ID. NO.:52, SEQ. ID. NO.:53, SEQ. ID. NO.:54, SEQ. ID. NO.:55, SEQ. ID. NO.:56, SEQ. ID. NO.:57, SEQ. ID. NO.:58, SEQ. ID. NO.:59, SEQ. ID. NO.:60, SEQ. ID. NO.:61, SEQ. ID. NO.:62, SEQ. ID. NO.:63, SEQ. ID. NO.:64, SEQ. ID. NO.:65, SEQ. ID. NO.:66, SEQ. ID. NO.:67, SEQ. ID. NO.:68, SEQ. ID. NO.:69, SEQ. ID. NO.:70, SEQ. ID. NO.:71, SEQ. ID. NO.:72, SEQ. ID. NO.:73, SEQ. ID. NO.:74, SEQ. ID. NO.:75, SEQ. ID. NO.:76, SEQ. ID. NO.:77, SEQ. ID. NO.:78, SEQ. ID. NO.:79, SEQ. ID. NO.:80, SEQ. ID. NO.:81, SEQ. ID. NO.:82, SEQ ID NO: 83, SEQ ID NO. 84, SEQ ID. NO. 85, SEQ. ID. NO. 86, SEQ. ID. NO. 87, SEQ. ID. NO. 88, SEQ. ID. NO. 89, SEQ. ID. NO. 90, SEQ. ID. NO. 91, SEQ ID. NO. 92, and SEQ. ID. NO. 93; or a sequence which is the reverse complement of a target sequence located in a region extending from about nucleotide 100 through nucleotide 500 in said SEQ ID NO.    
     
     
         39 . An isolated polynucleotide for characterizing cells which are obtained from a subject suspected of having a cancer selected from the group consisting of glioma, acute myeloid leukemia, primitive neuroectodermal tumors of childhood, breast cancer, colon cancer, head and neck cancer, testiclular cancer and lung cancer; wherein said polynucleotide is from 35 to 1000 nucleotides in length and comprises at least two CpG dinucleotides, and wherein said polynucleotide comprise a sequence which is identical to or complementary to a target sequence which originates between nucleotide 1 and nucleotide 15 of SEQ. ID. NO.:1, SEQ. ID. NO.:2, SEQ. ID. NO.:3, SEQ. ID. NO.:5, SEQ. ID. NO.:6, SEQ. ID. NO.:7, SEQ. ID. NO.:8, SEQ. ID. NO.:9, SEQ. ID. NO.:10, SEQ. ID. NO:11, SEQ. ID. NO.: 12, SEQ. ID. NO.:13, SEQ. ID. NO.:14, SEQ. ID. NO.:17, SEQ. ID. NO.:18, SEQ. ID. NO.: 19, SEQ. ID. NO.:20, SEQ. ID. NO.:21, SEQ. ID. NO.:22, SEQ. ID. NO.:23, SEQ. ID. NO.:24, SEQ. ID. NO.:25, SEQ. ID. NO.:27, SEQ. ID. NO.:28, SEQ. ID. NO.:29, SEQ. ID. NO.:30, SEQ. ID. NO.:31, SEQ. ID. NO.:32, SEQ. ID. NO.:33, SEQ. ID. NO.:34, SEQ. ID. NO.:35, SEQ. ID. NO.:36, SEQ. ID. NO.:37, SEQ. ID. NO.:38, SEQ. ID. NO.:40, SEQ. ID. NO.:41, SEQ. ID. NO.:42, SEQ. ID. NO.:43, SEQ. ID. NO.:44, SEQ. ID. NO.:45, SEQ. ID. NO.:47, SEQ. ID. NO.:48, SEQ. ID. NO.:49, SEQ. ID. NO.:50, SEQ. ID. NO.:51, SEQ. ID. NO.:52, SEQ. ID. NO.:53, SEQ. ID. NO.:54, SEQ. ID. NO.:55, SEQ. ID. NO.:56, SEQ. ID. NO.:58, SEQ. ID. NO.:60, SEQ. ID. NO.:62, SEQ. ID. NO.:63, SEQ. ID. NO.:64, SEQ. ID. NO.:65, SEQ. ID. NO.:66, SEQ. ID. NO.:67, SEQ. ID. NO.:69, SEQ. ID. NO.:70, SEQ. ID. NO.:72, SEQ. ID. NO.:73, SEQ. ID. NO.:75, SEQ. ID. NO.:76, SEQ. ID. NO.:77, SEQ. ID. NO.:78, SEQ. ID. NO.:79, SEQ. ID. NO.:80, SEQ. ID. NO.:81, SEQ. ID. NO.:82, SEQ ID NO: 83, SEQ. ID. NO. 86, SEQ. ID. NO. 87, SEQ. ID. NO. 88, SEQ. ID. NO. 89, SEQ. ID. NO. 90, SEQ. ID. NO. 91, SEQ ID. NO. 92, and SEQ. ID. NO. 93.