Methods and systems for detecting methylation changes in DNA samples
Methods and systems for highly sensitive detection of methylation changes in DNA samples are provided, particularly in DNA samples obtained from biological fluids such as plasma and urine.
1. A method for sensitive detection of methylation changes in a DNA sample, the method comprising:
(a) providing a DNA sample, wherein the DNA sample contains less than 5% single-stranded DNA (ssDNA); and
(b) subjecting the DNA sample to methylation ratio analysis by digesting the DNA sample with at least one methylation-sensitive restriction endonuclease or at least one methylation-dependent restriction endonuclease, co-amplifying from the digested DNA at least one restriction locus differentially methylated between normal and disease DNA and at least one control locus in a single reaction mixture, and comparing a ratio between signal intensities of the amplification products of each of said at least one restriction locus and the control locus to at least one reference ratio,
thereby detecting methylation changes in the DNA sample at a detection sensitivity of at least 1:100.
2. The method of claim 1 , wherein the DNA is cell-free DNA extracted from a biological fluid sample selected from plasma, serum and urine.
3. The method of claim 1 , wherein the DNA sample contains less than 1% ssDNA.
4. The method of claim 1 , wherein the DNA sample contains less than 0.1% ssDNA.
5. The method of claim 1 , wherein the DNA sample contains less than 0.01% ssDNA or is free of ssDNA.
6. The method of claim 1 , wherein methylation changes are detected in the sample at a detection sensitivity of at least 1:500.
7. The method of claim 1 , wherein methylation changes are detected in the sample at a detection sensitivity of at least 1:1,000.
8. The method of claim 1 , wherein detecting methylation changes comprises determining whether the DNA sample is a normal or disease DNA sample.
9. The method of claim 8 , comprising determining whether the DNA sample is a normal DNA sample or a cancer DNA sample.
10. The method of claim 1 , wherein the methylation ratio analysis in step (b) is performed using real-time PCR.
11. The method of claim 1 , wherein the methylation ratio analysis in step (b) is performed using Next Generation Sequencing (NGS).
12. The method of claim 1 , comprising amplifying in step (b) a plurality of restriction loci differentially methylated between normal and disease DNA and a single control locus.
13. A system for detecting methylation changes in a DNA sample, the system comprising:
(a) a DNA sample, wherein the DNA sample contains less than 5% single-stranded DNA (ssDNA); and
(b) components for carrying out a methylation ratio analysis, comprising:
(i) at least one methylation-sensitive restriction endonuclease or at least one methylation-dependent restriction endonuclease for digesting the DNA sample; (ii) a plurality of primer pairs for co-amplification of a plurality of genomic loci in a single reaction mixture from the DNA sample following digestion, wherein the plurality of genomic loci comprises at least one restriction locus differentially methylated between normal and disease DNA and at least one control locus; and (iii) computer software stored on non-transitory computer readable medium, the computer software directs a computer processor to determine methylation changes in the DNA sample based on a comparison of a ratio of signal intensities of the restriction locus and the control locus following amplification to a reference ratio,
with the methylation changes determined at a detection sensitivity of at least 1:100.
14. The system of claim 13 , wherein the DNA is cell-free DNA extracted from a biological fluid sample selected from plasma, serum and urine.
15. The system of claim 13 , wherein the plurality of genomic loci comprises a plurality of restriction loci differentially methylated between normal and diseased DNA and a single control locus.
16. The system of claim 13 , wherein the methylation changes are determined by performing the following steps: calculating signal intensities for amplification products of the restriction locus and the control locus; calculating a ratio between the signal intensities of the amplification products; and comparing the calculated ratio to one or more reference ratios obtained from DNA samples of known sources.
17. The system of claim 13 , wherein determining methylation changes in the DNA sample comprises providing an indication whether the DNA sample is a normal or diseased DNA sample.
18. The system of claim 13 , wherein determining methylation changes in the DNA sample comprises providing an indication whether the DNA sample is a normal DNA sample or a cancer DNA sample.