Methods and nucleic acids for the analysis of gene expression associated with the development of prostate cell proliferative disorders
View Patent ↗The invention provides methods, nucleic acids and kits for detecting prostate cell proliferative disorders. The invention discloses genomic sequences the methylation patterns of which have utility for the improved detection of said disorder, thereby enabling the improved diagnosis and treatment of patients.
1. A method for detecting methylation of cytosine bases in at least 16 contiguous nucleic acids of SEQ ID NO: 2 from prostate cell genomic DNA from a subject comprising the steps of:
a) treating the prostate cell genomic DNA which is derived from a biological sample comprising prostate tumor DNA with bisulfite so that unmethylated cytosine bases are converted to uracil while methylated cytosine bases remain unconverted, wherein the prostate cell genomic DNA comprises a sequence of at least 16 contiguous nucleotides of SEQ ID NO: 2 and the 16 contiguous nucleotides comprise at least one CpG dinucleotide;
b) detecting unconverted cytosine bases in the at least 16 contiguous nucleic acids of SEQ ID NO: 2 by amplification of the treated DNA and subsequent hybridization or sequencing; and
c) detecting the presence of at least one unconverted cytosine comprised by a CpG dinucleotide in the at least 16 contiguous nucleic acids of SEQ ID NO: 2, wherein the amplification of the treated DNA comprises the use at least one primer oligonucleotide having a length of at least 9 nucleotides and comprising at least one CpG, CpA or TpG dinucleotide, wherein the primer is capable of hybridizing to a nucleic acid having a sequence selected from the group consisting of SEQ ID NOs: 7, 8, 15, and 16 or a complement thereof.
2. The method of claim 1 , wherein the biological sample is selected from the group consisting of histological slides, biopsies, paraffin embedded tissue, ejaculate, urine, blood plasma, blood serum and whole blood.
3. The method of claim 1 , wherein the amplification of the treated DNA further comprises the use of a blocker oligonucleotide having a length of at least 9 nucleotides and comprising at least one CpA or TpG dinucleotide, wherein the blocker oligonucleotide is capable of hybridizing to a nucleic acid having a sequence selected from the group consisting of SEQ ID NOs: 7, 8, 15 and 16 or a complement thereof.
4. The method of claim 1 , wherein the amplification of the treated DNA further comprises the use of a probe oligonucleotide having a length of at least 9 nucleotides and comprising at least one CpG dinucleotide, wherein the probe oligonucleotide is capable of hybridizing to a nucleic acid having a sequence selected from the group consisting of SEQ ID Nos:7, 8, 15 and 16 or a complement thereof.