Methods and nucleic acids for analyses for cellular proliferative disorders
View Patent ↗Provided herein are methods, nucleic acids and kits for detecting a cell proliferative disorder. Also provided herein are genomic sequences of RASSF′-2, the methylation patterns of which have utility for the improved detection of cell proliferative disorders, thereby enabling the improved diagnosis and treatment of patients.
1. A method for detecting CpG methylation in genomic DNA comprising a RASSF2 gene, comprising:
a) isolating genomic DNA comprising a RASSF2 gene from a biological sample obtained from a subject, wherein the biological sample is selected from the group consisting of urine, blood plasma, blood serum, whole blood, blood, cells isolated from the blood and combinations thereof;
b) treating the genomic DNA comprising the RASSF2 gene, or a fragment thereof comprising the RASSF2 gene, with bisulfite such that non-methylated cytosines are converted to uracil;
c) contacting the bisulfite-treated genomic DNA comprising the RASSF2 gene, or the bisulfite-treated fragment thereof comprising the RASSF2 gene, with an amplification enzyme and:
at least two methylation-specific primers comprising a contiguous sequence of at least 9 nucleotides, wherein each of the at least two methylation-specific primers is identical across its entire length or is fully complementary across its entire length to a sequence selected from the group consisting of SEQ ID NOs: 6, 7, 16, 17, or
at least two primers comprising a contiguous sequence of at least 9 nucleotides wherein each of the at least two primers is identical across its entire length or is fully complementary across its entire length to a sequence selected from the group consisting of SEQ ID NOs: 6, 7, 16, 17, and at least one methylation-specific blocking nucleic acid molecule or peptide nucleic acid molecule comprising in each case a contiguous sequence at least 9 nucleotides in length that is identical across its entire length or is fully complementary across its entire length to a sequence selected from the group consisting of SEQ ID NO: 6, 7, 16, 17, wherein the nucleic acid molecule or peptide nucleic acid molecule suppresses amplification of a nucleic acid to which it is hybridized,
wherein the bisulfite-treated genomic DNA or the bisulfite-treated fragment thereof is either amplified to produce at least one amplificate comprising a sequence selected from the group consisting of SEQ ID NOs: 6, 7, 16, 17 or a fragment thereof, or is not amplified; and
d) determining a methylation state or level of the RASSF2 gene or a portion thereof, based on a presence or absence of the at least one amplificate, or on a property of the at least one amplificate, wherein increased methylation in the RASSF2 gene or portion thereof, compared to that of a normal control, indicates CpG methylation in the genomic DNA comprising the RASSF2 gene, thereby detecting CpG methylation in genomic DNA comprising a RASSF2 gene.
2. The method of claim 1 , wherein contacting the bisulfite-treated genomic DNA comprising the RASSF2 gene, or the bisulfite-treated fragment thereof comprising the RASSF2 gene, comprises use of at least one method selected from the group consisting of use of a heat-resistant DNA polymerase as the amplification enzyme, use of a polymerase lacking 5′-3′ exonuclease activity, use of a polymerase chain reaction (PCR), and generation of an amplificate nucleic acid molecule carrying a detectable label.
3. The method of claim 1 , wherein determining the methylation state or level of the RASSF2 gene or portion thereof comprises hybridization of at least one nucleic acid molecule or peptide nucleic acid molecule in each case comprising a contiguous sequence at least 9 nucleotides in length that is identical across its entire length or is fully complementary across its entire length to a sequence selected from the group consisting of SEQ ID NO: 6, 7, 16, 17.
4. The method of claim 3 , wherein at least one of the nucleic acid molecules or peptide nucleic acid molecules is bound to a solid phase.
5. The method of claim 3 , further comprising extending at least one of the nucleic acid molecules by at least one nucleotide base.
6. The method of claim 1 , wherein determining the methylation state or level of the RASSF2 gene or portion thereof comprises sequencing of the amplificate.
7. The method of claim 1 , wherein the at least two primers comprise a first primer having the sequence of SEQ ID NO: 26 and a second primer having the sequence of SEQ ID NO: 27.
8. The method of claim 1 , wherein contacting the bisulfite-treated genomic DNA comprising the RASSF2 gene, or the bisulfite-treated fragment thereof comprising the RASSF2 gene, comprises use of a blocking nucleic acid having the sequence of SEQ ID NO: 85 or a probe oligonucleotide having the sequence of SEQ ID NO: 86 or SEQ ID NO: 87.
9. The method of claim 1 , wherein the subject has an increased risk of colorectal cancer.
10. The method of claim 1 , wherein the subject has an increased risk of prostate cancer.
11. The method of claim 1 , wherein the subject has colorectal cancer.
12. The method of claim 1 , wherein the subject has prostate cancer.