Detection of allelic expression imbalance
The present invention provides compositions and methods for the detection and characterization of allelic expression imbalance from a heterozygous gene locus. More particularly, the present invention provides compositions, kits, and methods for the determination of allelic expression imbalance by the comparison of expression levels from each of two alleles of a given gene locus through the use of an invasive cleavage structure assay (e.g. the INVADER assay).
1 . A method of detecting the presence or absence of allelic expression imbalance from a heterozygous gene locus, comprising;
a) providing a sample comprising a population of target nucleic acid sequences, wherein said target nucleic acid sequences comprise: i) mRNA transcripts produced from said heterozygous gene locus, ii) cDNA products produced from said mRNA transcripts; or iii) amplified products produced from said cDNA products;
b) contacting said sample with invasive cleavage assays under conditions such that a quantitative signal for a first allele and a second allele in said population of target nucleic acid sequences is determined; and
c) comparing said quantitative signal for said first and second alleles to determine the presence or absence of allelic expression imbalance from said heterozygous gene locus in said sample.
2 . The method of claim 1 , wherein said invasive cleavage assays comprise first and second oligonucleotides, wherein said first and second oligonucleotides are configured to form invasive cleavage structures with said target nucleic acid sequences.
3 . The method of claim 2 , wherein said first oligonucleotides comprise a 5′ portion and a 3′ portion, wherein said 3′ portion is configured to hybridize to said target nucleic acid sequences, and wherein said 5′ portion is configured to not hybridize to said target nucleic acid sequences.
4 . The method of claim 2 , wherein said second oligonucleotides comprise a 5′ portion and a 3′ portion, wherein said 5′ portion is configured to hybridize to said target nucleic acid sequences, and wherein said 3′ portion is configured to not hybridize to said target nucleic acid sequences.
5 . The method of claim 1 , wherein said sample is a biological sample from a subject.
6 . The method of claim 5 , further comprising step d) identifying said subject as having a particular condition based on the presence or absence of allelic expression imbalance from said heterzygous gene locus in said sample.
7 . The method of claim 6 , wherein said particular condition is selected from the group consisting of: breast cancer, brain cancer, pancreatic cancer, loss of heterozygosity, loss of imprinting, and proper imprinting.
8 . The method of claim 1 , wherein said quantitative signal from said first allele is at least two percent greater than said quantitative signal from said second allele, and wherein said comparing determines the presence of allelic expression imbalance from said heterozygous gene locus in said sample.
9 . The method of claim 1 , wherein said invasive cleavage assays are configured to detect single nucleotide polymorphisms in said target nucleic acid sequences in order to generate said quantitative signal for said first allele and said second allele.
10 . A method of detecting the presence or absence of allelic expression imbalance from a heterozygous gene locus, comprising;
a) providing a sample comprising;
i) a first nucleic acid population comprising first target nucleic acid molecules selected from: i) genomic DNA molecules comprising said heterozygous gene locus, or ii) a first amplified product produced from said genomic DNA molecules; and
ii) a second nucleic acid population comprising second target nucleic acid molecules selected from: i) mRNA transcripts produced from said heterozygous gene locus, ii) cDNA products produced from said mRNA transcripts; or iii) a second amplified product produced from said cDNA products;
b) contacting said sample with invasive cleavage assays under conditions such that a quantitative signal for a first allele and a second allele in said first and in said second nucleic acid populations is determined; and
c) comparing said quantitative signal for said first and second alleles in said second nucleic acid population to each other and to said quantitative signal for said first and second alleles in said first nucleic acid population to determine the presence or absence of allelic expression imbalance from said heterozygous gene locus in said sample.
11 . The method of claim 10 , wherein said invasive cleavage assays comprise first and second oligonucleotides, wherein said first and second oligonucleotides are configured to form invasive cleavage structures with said target nucleic acid sequences.
12 . The method of claim 11 , wherein said first oligonucleotides comprise a 5′ portion and a 3′ portion, wherein said 3′ portion is configured to hybridize to said target nucleic acid sequences, and wherein said 5′ portion is configured to not hybridize to said target nucleic acid sequences.
13 . The method of claim 11 , wherein said second oligonucleotides comprise a 5′ portion and a 3′ portion, wherein said 5′ portion is configured to hybridize to said target nucleic acid sequences, and wherein said 3′ portion is configured to not hybridize to said target nucleic acid sequences.
14 . The method of claim 10 , wherein said sample is a biological sample from a subject.
15 . The method of claim 14 , further comprising step d) identifying said subject as having a particular condition based on the presence or absence of allelic expression imbalance from said heterozygous gene locus in said sample.
16 . The method of claim 15 , wherein said particular condition is selected from the group consisting of: breast cancer, brain cancer, pancreatic cancer, loss of heterozygosity, loss of imprinting, and proper imprinting.
17 . The method of claim 10 , wherein said invasive cleavage assays are configured to detect single nucleotide polymorphisms in order to generate said quantitative signal for said first allele and said second allele in said first and second nucleic acid populations.
18 . A kit comprising first and second invasive cleavage assays configured for detecting the presence or absence of allelic expression imbalance from a heterozygous gene locus, wherein said first invasive cleavage assay is configured to generate a quantitative signal for a first allele of said heterozygous gene locus, and said second invasive cleavage assay is configured to generate a quantitative signal for a second allele of said heterozygous gene locus.
19 . The kit of claim 18 , wherein said first and second invasive cleavage assays comprise first and second oligonucleotides, wherein said first and second oligonucleotides are configured to form invasive cleavage structures with target nucleic acid sequences.
20 . The kit of claim 19 , wherein said first oligonucleotides comprise a 5′ portion and a 3′ portion, wherein said 3′ portion is configured to hybridize to said target nucleic acid sequences, and wherein said 5′ portion is configured to not hybridize to said target nucleic acid sequences.