Methods for detecting DNA originating from different individuals
In a first aspect, the present invention features methods for differentiating DNA species originating from different individuals in a biological sample. These methods may be used to differentiate or detect fetal DNA in a maternal sample or to differentiate DNA of an organ donor from DNA of an organ recipient. In preferred embodiments, the DNA species are differentiated by observing epigenetic differences in the DNA species such as differences in DNA methylation. In a second aspect, the present invention features methods of detecting genetic abnormalities in a fetus by detecting fetal DNA in a biological sample obtained from a mother. In a third aspect, the present invention features methods for differentiating DNA species originating from an organ donor from those of an organ recipient. In a fourth aspect, the present invention features kits for differentiating DNA species originating from different individuals in a biological sample.
1. A system for detecting a methylation difference between DNA species originating from cells of a pregnant female and an unborn fetus comprising:
(i) a cell free biological sample comprising DNA species originating from cells of a pregnant female and an unborn fetus; and
(ii) a reagent for detecting a methylation difference between the DNA species, wherein the reagent modifies the DNA species differentially depending on the DNA methylation status of the DNA species.
2. The system according to claim 1 , wherein the methylation difference is associated with a fetal or maternal disorder.
3. The system according to claim 2 , wherein the fetal or maternal disorder is preeclampsia, a chromosomal aneuploidy, trisomy 21 (Down syndrome), Prader Willi Syndrome or Angelman Syndrome.
4. The system according to claim 1 , wherein the biological sample is obtained from a second trimester pregnancy.
5. The system according to claim 1 , wherein the biological sample is obtained from a third trimester pregnancy.
6. The system according to claim 1 , wherein the biological sample is obtained from a pregnant female heterozygous for a biallelic polymorphism.
7. The system according to claim 1 , wherein the biological sample is plasma or serum.
8. The system according to claim 1 , wherein the DNA species of the fetus comprises an allele that the fetus has inherited from the pregnant female.
9. The system according to claim 1 , wherein the DNA species of the fetus comprises a paternally-inherited allele.
10. The system according to claim 1 , wherein the reagent comprises sodium bisulfite.
11. The system according to claim 1 , further comprising: (iii) a reagent for extracting the DNA species from the biological sample.
12. The system according to claim 1 , further comprising: (iii) a nucleic acid mimicking one of the DNA species.
13. The system according to claim 1 , further comprising: (iii) PCR primers for amplifying the DNA species to produce an amplicon.
14. The system according to claim 13 , wherein the PCR primers are for performing a methylation-specific polymerase chain reaction.
15. The system according to claim 13 , wherein the PCR primers are for distinguishing a sequence difference between methylated and unmethylated DNA following bisulfite conversion of the DNA species.
16. The system according to claim 13 , wherein at least one of the PCR primers is designed for a methylated DNA sequence.
17. The system according to claim 13 , wherein at least one of the PCR primers is designed for an unmethylated DNA sequence.
18. The system according to claim 13 , wherein the amplicon is specific for the unborn fetus.
19. The system according to claim 13 , further comprising: (iv) a reagent for purifying the amplicon.
20. The system according to claim 11 , further comprising: iv) a reagent for sequencing the amplicon.
21. The system according to claim 13 , further comprising: (iv) a reagent for detecting the amplicon.
22. The system according to claim 21 , wherein the reagent for detecting the amplicon is a gel.
23. The system according to claim 13 , further comprising: (iv) a reagent for performing primer extension.
24. The system according to claim 13 , further comprising: (iv) a reagent for performing real-time PCR.
25. The system according to claim 13 , further comprising: (iv) a marker.
26. The system according to claim 13 , further comprising: (iv) EDTA.
27. A system for differentiating human DNA species comprising:
(i) a cell free biological sample comprising human DNA species originating from cells of a pregnant female and an unborn fetus;
(ii) PCR primers for amplifying the human DNA species; and
(iii) a reagent for detecting a methylation difference between the human DNA species, wherein the reagent modifies the DNA species differentially depending on the DNA methylation status of the DNA species.
28. The system according to claim 27 , wherein the reagent comprises sodium bisulfite.
29. The system according to claim 27 , wherein the biological sample is plasma or serum.
30. A system for amplification of a fetal DNA species comprising:
(i) a cell free biological sample comprising DNA species originating from cells of a pregnant female and an unborn fetus;
(ii) a reagent for isolating the DNA species from the biological sample;
(iii) a reagent for amplifying the fetal DNA species; and
(iv) a regent for detecting a methylation difference between the DNA species originating from cells of the pregnant female and the unborn fetus,
wherein the reagent in (iv) modifies the DNA species differentially depending on the methylation status of the DNA species.
31. The system according to claim 30 , wherein the reagent comprises sodium bisulfite.
32. The system according to claim 30 , wherein the biological sample is plasma or serum.