Methods for detecting fetal DNA in a plasma or serum sample from a pregnant woman
This application describes the discovery that, in a pregnant woman, certain genes (such as RASSF1A, APC, CASP8, RARB, SCGB3A1, DAB2IP, PTPN6, THY1, TMEFF2, and PYCARD) originated from a fetus are highly methylated, whereas the same genes of maternal origin are unmethylated. This discovery allows the easy detection of one or more of these methylated fetal genes in a biological sample from a pregnant woman, serving as a universal indicator of the presence of fetal DNA in the sample. These fetal methylation markers are particularly useful as positive controls for a non-invasive analytical process during which the quality and quantity of fetal DNA are monitored. These newly identified fetal markers can also be measured directly for diagnosis of certain pregnancy-related conditions.
1. A method for detecting the presence of fetal DNA in a sample from a pregnant woman, comprising the sequential steps of:
(a) treating a first plasma or serum sample from the woman with an agent that differentially modifies methylated and unmethylated DNA;
(b) detecting the presence of methylated DNA sequence of RASSF1A in the first sample;
(c) determining that the sample contains fetal DNA based on the presence of methylated RASSF1A DNA sequence in step (b); and
(d) when and only when methylated RASSFIA DNA sequence is present in the first sample in step (b), determining the amount of a fetal DNA sequence other than RASSFIA in the same sample or in a second, identical plasma or serum sample from the woman.
2. The method of claim 1 , wherein the first sample is a plasma sample.
3. The method of claim 1 , wherein the first sample is a serum sample.
4. The method of claim 1 , wherein the agent digests unmethylated DNA but not methylated DNA.
5. The method of claim 4 , wherein the agent is a methylation sensitive enzyme.
6. The method of claim 5 , wherein the methylation sensitive enzyme is a methylation sensitive restriction enzyme.
7. The method of claim 6 , wherein the methylation sensitive restriction enzyme is Hpa II or BstU I.
8. The method of claim 1 , wherein the agent comprises bisulfite.
9. The method of claim 1 , wherein step (b) comprises an amplification process.
10. The method of claim 9 , wherein the amplification process is a polymerase chain reaction (PCR).
11. The method of claim 10 , wherein the PCR is real-time PCR.
12. The method of claim 1 , wherein step (b) further comprises determining the amount of the methylated RASSF1A DNA sequence.
13. The method of claim 1 , wherein the first sample and the second sample are the same sample.
14. The method of claim 1 , wherein the second sample in step (d) is not treated with any agent that differentially modifies methylated and unmethylated DNA.
15. The method of claim 1 , wherein the second sample in step (d) is treated with the agent before the amount of the fetal DNA sequence other than RASSF1A is determined.
16. The method of claim 1 , wherein the second sample in step (d) is treated with a second, different agent that differentially modifies methylated and unmethylated DNA before the amount of the fetal DNA sequence other than RASSF1A is determined.
17. The method of claim 1 , wherein the fetal DNA sequence other than RASSF1A is a gene of a RhD blood type, of an ABO blood type, of a RhC blood type, of a RhE blood type, of a HLA type, on the Y chromosome, or containing a pre-determined mutation.
18. The method of claim 17 , wherein the second sample in step (d) is not treated with any agent that differentially modifies methylated and unmethylated DNA.
19. The method of claim 17 , wherein step (d) comprises an amplification process.
20. The method of claim 19 , wherein the amplification process is a polymerase chain reaction (PCR).
21. The method of claim 20 , wherein the PCR is real-time PCR.
22. The method of claim 1 , wherein the fetal DNA sequence other than RASSF1A is distinguished from its maternal counterpart by a difference in sequence or methylation.