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 - 8 . (canceled)
9 . A method of quantifying methylated DNA, comprising:
a) contacting a DNA sample with a methylation-sensitive reagent, wherein said DNA sample comprises a major population and a minor population;
b) partitioning said DNA sample into a plurality of spatially isolated partitions;
c) detecting a first quantity of a first locus within said DNA sample;
d) detecting a second quantity of a second locus within said DNA sample; and
e) comparing said first and second quantities, to obtain a value indicative of a percentage of methylated DNA in the sample.
10 . The method of claim 9 , wherein said plurality of spatially isolated partitions are emulsified droplets.
11 . The method of claim 9 , wherein said methylation-sensitive reagent is a methylation-sensitive enzyme.
12 . The method of claim 9 , wherein said major population comprises maternal DNA and said minor population comprises fetal DNA
13 . The method of claim 12 , wherein said first locus is hypermethylated in fetal DNA
14 . The method of claim 9 , wherein said first locus comprises a sequence selected from the group consisting of: RASSFlA, CASP8, RARB, SCGB3Al, DAB2IP, PTPN6, THYl, TMEFF2, APC, and PYCARD.
15 . The method of claim 9 , wherein said second locus does not comprise a restriction site recognized by said methylation-sensitive reagent.
16 . The method of claim 9 , wherein said second locus is methylated in (a) maternal DNA and (b) fetal DNA.
17 . The method of claim 15 , wherein said second locus comprises a sequence from the group consisting of: RNASE P and TERT.
18 . The method of claim 9 , further comprising detecting a signal associated with a third locus within said DNA sample.
19 . The method of claim 18 , wherein said third locus is not significantly methylated in fetal DNA.
20 . The method of claim 18 , wherein said third locus is cleaved by said methylation-sensitive reagent, wherein said methylation-sensitive reagent is an enzyme.
21 - 23 . (canceled)
24 . The method of claim 18 , wherein said third locus comprises a sequence of the Y chromosome, a Rh blood type gene, a RhD blood type gene, a RhC blood type gene, a RhE blood type gene, an ABO blood type gene, a HLA-type gene, BETAACTIN, or SRY.
25 . (canceled)
26 . The method of claim 18 , 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.
27 . (canceled)
28 . The method of claim 9 , further comprising, prior to step (a), isolating a subsample from said DNA sample, wherein said subsample is not contacted with said methylation-sensitive reagent, and detecting said first or second locus in said subsample, and/or detecting a third locus within said subsample.
29 - 30 . (canceled)
31 . The method of claim 9 , wherein said methylation-sensitive reagent is selected from the group consisting of: bisulfate, hydrogen sulfite and disulfite.
32 . The method of claim 9 , wherein said detecting of said first and second quantities comprises an amplification reaction.
33 . (canceled)
34 . The method of claim 12 , 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.
35 . The method of claim 34 , wherein said pregnancy-associated disorder is selected from the group consisting of: preeclampsia, preterm labor, and intrauterine growth retardation (IUGR).
36 . The method of claim 9 , 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.
37 - 108 . (canceled)