Methods and compositions for detecting genetic material
The present disclosure provides methods and compositions for detecting polynucleotides in a sample and for quantifying polynucleotide load in a sample. The polynucleotides can be associated with a disease, disorder, or condition. In some applications, methylated DNA is quantified, e.g., in order to determine the load of polynucleotides in a sample. The present disclosure also provides methods and compositions for determining the load of fetal polynucleotides in a biological sample, e.g., the load of fetal polynucleotides (e.g., DNA, RNA) in maternal plasma. The present disclosure provides methods and compositions for detecting cellular processes such as cellular viability, growth rates, and infection rates. This disclosure also provides compositions and methods for detecting differences in copy number of a target polynucleotide. In some embodiments, the methods and compositions provided herein are useful for diagnosis of fetal genetic abnormalities, when the starting sample is maternal tissue (e.g., blood, plasma). The methods and materials described apply techniques for allowing detection of small, but statistically significant, differences in polynucleotide copy number.
1. A method of detecting methylated DNA, comprising:
a) contacting a DNA sample comprising a major population and a minor population with a methylation-sensitive reagent;
b) partitioning said DNA sample into a plurality of spatially-isolated partitions;
detecting a first locus within said DNA sample, wherein said-first locus is c) hypermethylated in the minor population and wherein the first locus is selected from the group consisting of CASP8, RARB, SCGB3A1, DAB2IP, PTPN6, THY1, TMEFF2, and PYCARD; and
d) quantifying a first quantity of said first locus, thereby detecting methylated DNA.
2. The method of claim 1 , further comprising detecting a second locus within said DNA sample, wherein said second locus is present in both the major population and the minor population and wherein said second locus is not significantly cleaved by said methylation-sensitive reagent.
3. The method of claim 1 , further comprising detecting a third locus within said DNA sample, wherein said third locus is present in both the major population and the minor population and wherein said third locus is significantly cleaved by said methylation-sensitive reagent.
4. The method of claim 1 , further comprising amplifying a sequence associated with said first locus to produce a detectable signal.
5. The method of claim 4 , wherein said signal is a fluorescence signal.
6. A The method claim 1 , further comprising:
e) detecting a second quantity of a second locus within said DNA sample; and
f) comparing said first and second quantities, to obtain a value indicative of a percentage of methylated DNA in the sample.
7. The method of claim 6 , wherein said major population comprises maternal DNA and said minor population comprises fetal DNA.
8. The method of claim 6 , wherein said second locus does not comprise a restriction site recognized by said methylation-sensitive reagent.
9. The method of claim 6 , wherein said second locus is methylated in (a) the major population and (b) the minor population.
10. The method of claim 6 , further comprising detecting a signal associated with a third locus within said DNA sample.
11. The method of claim 10 , wherein said value indicative of the percentage of methylated DNA in said sample is adjusted by a value associated with the presence of said third locus within said DNA sample.
12. The method of claim 6 , further comprising calculating the amount of total DNA in said sample.
13. The method of claim 6 , wherein said detecting of said first and second quantities comprises an amplification reaction.
14. The method of claim 7 , further comprising comparing said value indicative of the percentage of methylated DNA in said DNA sample with a value at an earlier gestational timepoint, thereby detecting a pregnancy-associated disorder.
15. The method of claim 6 , wherein said value indicative of the percentage of methylated DNA in the sample is calculated using a detectable signal from at least a third locus within said DNA sample, wherein said third locus comprises a sequence that is not significantly cleaved by said methylation-sensitive reagent.
16. The method of claim 1 , wherein said spatially-isolated partitions are wells.
17. The method of claim 6 , wherein said spatially-isolated partitions are wells.
18. The method of claim 1 , wherein said methylation-sensitive reagent is a methylation-sensitive enzyme.