DNA methylation analysis by digital bisulfite genomic sequencing and digital methylight
Provided are novel sensitive methylation assays referred to herein as Digital MethyLight, comprising stochastically distributing and compartmentalizing bisulfite-treated genomic DNA over multiple PCR reaction wells for detection of individually methylated DNA molecules in a large background of unmethylated DNA. Digital Bisulfite Genomic DNA Sequencing methods are also provided for high-resolution DNA methylation information without subcloning. Background signal and PCR contaminants are diluted, while the ratio of primer to methylated template DNA is kept high. Preferably, biological fluid (e.g., urine, blood-based (e.g., plasma and/or serum)) samples are analyzed for cancer diagnosis, prognosis and surveillance. Multiplexed PCR formats may be implemented to enhance when using small DNA amounts. Compositions and methods for diagnosis and/or prognosis of breast cancer, comprising the use of FOXE1, CLDN5 and/or RUNX3 gene markers are also provided (SEQ ID NOS: 17, 16 and 18, respectively for respective CpG island sequences), and in preferred embodiments plasma or serum samples are used.
1. A method for analyzing a small number of methylated DNA molecules in a large background of unmethylated DNA, comprising:
obtaining a sample having genomic DNA comprising a small number of methylated genomic DNA molecules in a large background of unmethylated genomic DNA;
contacting the genomic DNA molecules with a reagent or series of reagents suitable to distinguish between methylated and nonmethylated CpG dinucleotides to provide for treated genomic DNA molecules;
compartmentalizing the treated DNA molecules over multiple PCR compartments or wells by stochastically distributing individual treated DNA molecules such that, on average, there is less than one treated DNA molecule per reaction compartment or well, and wherein at least one well or compartment has no more than a single treated DNA molecule;
amplifying, in a first amplification reaction, portions or fragments of said compartmentalized single treated DNA molecule by means of a polymerase chain reaction (PCR) and one or more sets of primer oligonucleotides to provide for at least one amplificate of the compartmentalized single, treated DNA molecule; and
detecting, in real-time during the first amplification, of the at least one amplificate, wherein the methylation state of at least one CpG dinucleotide sequence thereof is determined, and wherein analyzing a small number of methylated genomic DNA molecules in a large background of unmethylated genomic DNA is provided for.
2. The method of claim 1 , wherein the reagent or series of reagents comprises at least one reagent selected from the group consisting of bisulfite, hydrogen sulfite and disulfite.
3. The method of claim 1 , comprising multiplexing with a plurality of primer sets to provide for amplification of a respective plurality of different DNA amplificate fragments.
4. The method of claim 3 , wherein at least three, at least five, or at least ten different fragments, each having a length of about 100 to about 2000 base pairs, are amplified.
5. The method of claim 1 , wherein the sample comprising genomic DNA is a plasma or serum sample.
6. The method of claim 5 , further comprising determining the number of methylated molecules of a particular locus relative to a specific volume of plasma or serum.
7. The method of claim 5 , wherein the plasma sample is at least one selected from the group consisting of a breast cancer patient, pancreatic patient, and a colorectal cancer patient.
8. The method of claim 7 , wherein the breast cancer patient is a Stage II or Stage IV breast cancer patient.
9. The method of claim 1 , wherein the ratio of primer to methylated template DNA is kept high.
10. The method of claim 1 , wherein the PCR reactions are compartmentalized into a plurality of individual microfluidic reaction chambers to provide for microfluidic analysis.
11. The method of claim 1 , wherein, prior to compartmentalizing, determining the Ct value to determine the amount of treated DNA to load into each PCR compartments or well.
12. The method of claim 11 , wherein the Ct values are calibrated, using a suitable control reaction, to the C-LESS signal using genomic DNA as a standard.
13. The method of claim 1 , wherein about 20 to about 30 DNA molecules are stochastically distributed and compartmentalized over the multiple PCR compartments or wells.
14. The method of claim 1 , comprising determining the methylation status of one or more CpG dinucleotides within at least one of FOXE1 (SEQ ID NO:17), CLDN5 (SEQ ID NO:16), and RUNX3 (SEQ ID NO:18).
15. The method of claim 1 , comprising:
obtaining a sample having genomic DNA comprising a small number of methylated genomic DNA molecules in a large background of unmethylated genomic DNA;
contacting the genomic DNA molecules with a reagent or series of reagents suitable to distinguish between methylated and nonmethylated CpG dinucleotides to provide for treated genomic DNA molecules;
compartmentalizing the treated DNA molecules over multiple PCR compartments or wells by stochastically distributing individual treated DNA molecules such that, on average, there is less than one treated DNA molecule per reaction compartment or well, and wherein at least one well or compartment has no more than a single treated DNA molecule;
amplifying, in a first amplification reaction, portions or fragments of said compartmentalized single treated DNA molecule by means of a polymerase chain reaction (PCR) and one or more sets of primer oligonucleotides to provide for at least one amplificate of the compartmentalized single, treated DNA molecule; and
sequencing the at least one amplificate, wherein at least one individual genomic DNA molecule CpG methylation pattern of a methylated genomic DNA molecule in a large background of unmethylated genomic DNA is determined.
16. The method of claim 15 , wherein about 20 to about 30 individual molecules are assayed and sequenced to determine methylation patterns at a candidate gene locus.
17. The method of claim 15 , further comprising the use of melting curve analysis to identify the melting curve of the PCR product in at least one of the compartments or wells.