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US 2023/0416844 A1Published application
Methods To Analyze Methylomes In Tumor And Plasma Cell-Free DNA
OwnerRegina Santella, Hui Zhou +2inventors
InventorsRegina Santella, Hui Zhou, Hui Chen Wu, Zhiguo Zhang
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Abstract
Disclosed herein are methods for detecting methylation in cell-free polynucleotides and methods for detecting the presence of cancer in a subject.
Claims19
A method for detecting methylation in cell-free polynucleotides, the method comprising:
- a. obtaining cell-free polynucleotides from a bodily sample from a subject, wherein the cell-free polynucleotides comprise single stranded DNA (ssDNA);
- b. ligating an ssDNA adaptor to the 3′ end of the ssDNA with an ssDNA ligase;
- c. converting the ssDNA from step c into double stranded DNA (dsDNA) with a DNA polymerase;
- d. ligating a dsDNA adaptor to the dsDNA from step c;
- e. denaturing the dsDNA from step d to obtain ssDNA;
- f immunoprecipitating the ssDNA from step e using one or more antibodies against 5 mC and/or 5 hmC;
- g. amplifying the immunoprecipitated DNA;
- and
- h. sequencing the amplified DNA to detect the presence of 5 mC and/or 5 hmC, thereby detecting methylation in the cell-free polynucleotides.
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A method for detecting the presence of cancer in a subject, the method comprising:
- a. obtaining cell-free polynucleotides from a bodily sample from the subject and from a control subject known to be cancer-free;
- b. ligating an ssDNA adaptor to the 3′ end of the ssDNA with an ssDNA ligase;
- c. converting the ssDNA from step c into double stranded DNA (dsDNA) with a DNA polymerase;
- d. ligating a dsDNA adaptor to the dsDNA from step c;
- e. denaturing the dsDNA from step d to obtain ssDNA;
- f immunoprecipitating the ssDNA from step e using one or more antibodies against 5 mC and/or 5 hmC;
- g. amplifying the immunoprecipitated DNA;
- h. sequencing the amplified DNA to detect the presence of 5 mC and/or 5 hmC, thereby detecting methylation in the cell-free polynucleotides of the subject and the cell-free polynucleotides of the control subject;
- and
- i. detecting cancer in the subject by comparing the presence of 5 mC and/or in the cell-free polynucleotides of the subject with that of the cell-free polynucleotides of the subject and control subject.
7 more elements
Dependencies are inferred from the claim text.