Detecting colorectal neoplasm
Provided herein is technology relating to detecting neoplasia and particularly, but not exclusively, to methods, compositions, and related uses for detecting premalignant and malignant neoplasms such as colorectal cancer.
1. A method comprising:
extracting genomic DNA from a biological sample of a human individual suspected of having or having colorectal cancer,
treating the extracted genomic DNA with bisulfite,
amplifying the bisulfite-treated genomic DNA using primers specific for two to eleven different markers, wherein one of the two to eleven different markers is selected from ankyrin repeat domain 33B (ANKRD33B), leucine-rich repeat-containing protein 4 (LRRC4), protein phosphatase 2 regulatory subunit (PPP2R5C), and zinc finger protein 568 (ZNF568), wherein if LRRC4 is one of the two to eleven different markers than at least one of ANKRD33B, PPP2R5C, and ZNF568 must be one of the two to eleven different markers,
wherein amplifying the bisulfite-treated genomic DNA using primers specific for a CpG site for ANKRD33B is a set of primers that specifically binds at least a portion of a genetic region comprising chromosome 5:10565042-10565191 or chromosome 5:10564655-10564807,
wherein amplifying the bisulfite-treated genomic DNA using primers specific for a CpG site for LRRC4 is a set of primers that specifically binds at least a portion of a genetic region comprising 7:127671918-127672318,
wherein amplifying the bisulfite-treated genomic DNA using primers specific for a CpG site for ZNF568 is a set of primers that specifically binds at least a portion of a genetic region comprising 19:37407197-37407365,
wherein amplifying the bisulfite-treated genomic DNA using primers specific for a CpG site for PPP2R5C is a set of primers that specifically binds at least a portion of a genetic region comprising 14:102248062-102248216,
measuring the methylation level of the CpG site for the two to eleven different markers by methylation-specific PCR, quantitative methylation-specific PCR, methylation-sensitive DNA restriction enzyme analysis, or bisulfite genomic sequencing PCR.
2. The method of claim 1 , wherein the biological sample is a stool sample, a tissue sample, a colorectal cyst sample, a colorectal tumor sample, a blood sample, or a urine sample.
3. The method of claim 1 , wherein the sample comprises colorectal tissue.
4. The method of claim 1 , wherein the two to eleven different markers is 2-8 different markers.
5. The method of claim 1 , wherein the two to eleven different markers is 2-6 different markers.
6. The method of claim 1 , wherein the two to eleven different markers is 2-4 different markers.
7. A method comprising:
extracting genomic DNA from a biological sample of a human individual suspected of having or having colorectal cancer,
treating the extracted genomic DNA with bisulfite,
amplifying the bisulfite-treated genomic DNA using primers specific for two to eleven different markers, wherein one of the two to eleven different markers is selected from ankyrin repeat domain 33B (ANKRD33B), protein phosphatase 2 regulatory subunit (PPP2R5C), and zinc finger protein 568 (ZNF568),
wherein amplifying the bisulfite-treated genomic DNA using primers specific for a CpG site for ANKRD33B is a set of primers that specifically binds at least a portion of a genetic region comprising chromosome 5:10565042-10565191 or chromosome 5:10564655-10564807,
wherein amplifying the bisulfite-treated genomic DNA using primers specific for a CpG site for ZNF568 is a set of primers that specifically binds at least a portion of a genetic region comprising 19:37407197-37407365,
wherein amplifying the bisulfite-treated genomic DNA using primers specific for a CpG site for PPP2R5C is a set of primers that specifically binds at least a portion of a genetic region comprising 14:102248062-102248216,
measuring the methylation level of the CpG site for the two to eleven different markers by methylation-specific PCR, quantitative methylation-specific PCR, methylation-sensitive DNA restriction enzyme analysis, or bisulfite genomic sequencing PCR.
8. The method of claim 7 , wherein the biological sample is a stool sample, a tissue sample, a colorectal cyst sample, a colorectal tumor sample, a blood sample, or a urine sample.
9. The method of claim 7 , wherein the sample comprises colorectal tissue.
10. The method of claim 7 , wherein the two to eleven different markers is 1-8 different markers.
11. The method of claim 7 , wherein the two to eleven different markers is 1-6 different markers.
12. The method of claim 7 , wherein the two to eleven different markers is 1-4 different markers.
13. The method of claim 1 , wherein the primers capable of binding at least a portion of the bisulfite-treated genomic DNA from chromosome 14:102248062-102248216 region of the PPP2R5C gene are SEQ ID NOS: 18 and 19.