Methods and compositions for detecting gastrointestinal and other cancers
View Patent ↗This application describes methods and compositions for detecting and treating vimentin-associated neoplasia. Differential methylation of the vimentin nucleotide sequences has been observed in vimentin-associated neoplasia such as neoplasia of the upper or lower gastrointestinal tract, pancreas, and/or bladder. Nucleic acid sequences for regions of vimentin that are found to be differentially methylated are described. The present disclosure further describes bisulfite-converted vimentin template DNA sequences. Oligonucleotide primer sequences for use in assays (e.g., methylation-sensitive PCR assays or HpaII assays) designed to detect the methylation status of the vimentin gene are also described.
1. An isolated polynucleotide comprising a bisulfite-converted nucleic acid; wherein the polynucleotide is 20-3000 nucleotides in length; wherein the polynucleotide comprises a region having:
a) a nucleotide sequence comprising the bisulfite-converted methylated nucleotide sequence of SEQ ID NO: 41, SEQ ID NO: 42, or SEQ ID NO: 44, a complement thereof, or a fragment thereof; wherein said nucleotide sequence, said complement or said fragment is at least 20 nucleotides in length; or
b) a nucleotide sequence that is at least 95% identical to the bisulfite-converted methylated nucleotide sequence of SEQ ID NO: 41, SEQ ID NO: 42, or SEQ ID NO: 44, a complement thereof, or a fragment thereof; wherein said nucleotide sequence, said complement or said fragment is at least 20 nucleotides in length;
and wherein said nucleic acid, prior to bisulfite conversion, comprises at least one methylated cytosine and at least one unmethylated cytosine.
2. The isolated polynucleotide of claim 1 , wherein said polynucleotide consists of a nucleotide sequence that is at least 95% identical to the bisulfite-converted methylated nucleotide sequence of SEQ ID NO: 41, SEQ ID NO: 42, or SEQ ID NO: 44, or a complement thereof or a fragment thereof.
3. The isolated polynucleotide of claim 2 , wherein said polynucleotide consists of a nucleotide sequence that is at least 95% identical to the bisulfite-converted methylated nucleotide sequence of SEQ ID NO: 41, or a fragment thereof or a complement thereof.
4. The isolated polynucleotide of claim 2 , wherein said polynucleotide consists of a nucleotide sequence that is at least 95% identical to the bisulfite-converted methylated nucleotide sequence of SEQ ID NO: 42, or a fragment thereof or a complement thereof.
5. The isolated polynucleotide of claim 2 , wherein said polynucleotide consists of a nucleotide sequence that is at least 95% identical to the bisulfite-converted methylated nucleotide sequence of SEQ ID NO: 44, or a fragment thereof or a complement thereof.
6. The isolated polynucleotide of claim 1 , wherein said complement thereof consists of the bisulfite-converted methylated nucleotide sequence of SEQ ID NO: 48, or a fragment thereof.
7. The isolated polynucleotide of claim 1 , wherein the polynucleotide is generated by amplifying at least a portion of the bisulfite-converted methylated nucleic acid derived from the vimentin gene locus of SEQ ID NO: 51, and wherein said polynucleotide may be amplified using polynucleotide primers having the nucleotide sequences of SEQ ID NOs: 62 and 63.
8. The isolated polynucleotide of claim 1 , wherein the polynucleotide is generated by amplifying at least a portion of the bisulfite-converted methylated nucleic acid derived from the vimentin gene locus of SEQ ID NO: 51, and wherein said polynucleotide may be amplified using polynucleotide primers having the nucleotide sequences of SEQ ID NOs: 72 and 71.
9. The isolated polynucleotide of claim 1 , wherein the polynucleotide is generated by amplifying at least a portion of the bisulfite-converted methylated nucleic acid derived from the vimentin gene locus of SEQ ID NO: 51, and wherein said polynucleotide may be amplified using polynucleotide primers having the nucleotide sequences of SEQ ID NOs: 68 and 71.
10. The isolated polynucleotide of claim 1 , wherein the polynucleotide is between 50 to 500 nucleotides in length.
11. The isolated polynucleotide of claim 1 , wherein the polynucleotide is between 80 to 150 nucleotides in length.
12. The isolated polynucleotide of claim 1 , wherein the polynucleotide is a bisulfite-converted fragment of a nucleic acid obtained from a biological sample taken from a patient suspected of having a colon neoplasia.