US 10,590,473 B2Granted

Rapid quantitative detection of single nucleotide polymorphisms or somatic variants and methods to identify malignant neoplasms

AssigneesThe Broad Institute, Inc., Dana-Farber Cancer Institute, Inc., The General Hospital Corporation

InventorsMatthew Meyerson, Ganesh M. Shankar, Joshua M. Francis, Daniel P. Cahill, Mikael L. Rinne

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Abstract

Provided are systems, kits, and methods for the quantitative detection of single nucleotide polymorphisms or variants to identify malignant neoplasms. The methods include use of modified oligonucleotide blockers with peptide nucleic acid backbones that hybridize to and block logarithmic amplification of the wild-type alleles of a target, and incorporation of locked nucleic acids into probes that are complementary to a mutant allele of the target sequence to increase specificity. The methods include detection of variants in sequences with high GC content and/or low complexity, such as the TERT promoter, IDH1, BRAF, NRAS, GNAQ, GNA11 and H3F3 A gene variants. The methods include sensitive detection and staging of cancers with low cellularity, and can be used intraoperatively such as for glioma, or to detect cell-free circulating tumor DNA, such as for melanoma.

Claims23

Dependent claims

A method for establishing a molecular diagnosis in at least one cancer in a subject, the method comprising:

(a) isolating DNA from a sample from the subject;
(b) denaturing the DNA isolated from the sample and a control DNA template;
(c) annealing a forward primer that hybridizes to a first region on a sense strand of a first target nucleic acid, a reverse primer that hybridizes to a second region on an antisense strand of the first target nucleic acid, a locked nucleic acid (LNA) probe comprising an oligonucleotide with at least one LNA modification on at least one nucleotide, wherein the probe comprises a nucleic acid sequence that is complementary to a mutant allele of the first target nucleic acid, and a peptide nucleic acid (PNA) blocker that hybridizes to a wild-type allele of the first target nucleic acid;
(d) amplifying a DNA amplicon comprising the mutant allele in the first target nucleic acid;
(e) detecting the mutant allele in the first target nucleic acid in the sample;
and
(f) quantifying the amount of the mutant allele in the first target nucleic acid in the sample relative to the amount in a control, wherein a higher prevalence of the amount of mutant allele in the sample relative to the control indicates presence and/or stage of the cancer in the subject.

4 more elements

A system for detection or amplification of a single nucleotide polymorphism (SNP) or somatic nucleotide alteration in a target nucleic acid in a sample, the system comprising:

(a) a sample comprising a target nucleic acid;
(b) a forward primer that hybridizes to a first region on a sense strand of the target nucleic acid sequence in the sample, wherein the target nucleic acid comprises a single nucleotide polymorphism (SNP) or somatic nucleotide alteration compared to a corresponding wild-type nucleic acid which does not comprise the SNP or somatic nucleotide alteration;
(c) a reverse primer that hybridizes to a second region on an antisense strand of the target nucleic acid;
(d) a locked nucleic acid (LNA) probe comprising an oligonucleotide with at least one LNA modification on at least one nucleotide, wherein the LNA probe comprises a nucleic acid sequence that is complementary to the target nucleic acid comprising the SNP or somatic nucleotide alteration and is located within the nucleic acid region amplified by the forward primer and the reverse primer;
(e) a peptide nucleic acid (PNA) blocker that hybridizes to a wild-type allele of the target nucleic acid, the blocker comprising peptide nucleic acid oligonucleotides that block amplification of the wild-type allele, and do not block amplification of the target nucleic acid comprising the SNP or somatic nucleotide alteration, wherein the PNA blocker hybridizes to a nucleic acid sequence located within the region amplified by the forward primer and the reverse primer;
and
(f) a reaction buffer;
wherein the forward primer, the reverse primer, the LNA probe, and the PNA blocker recognize their target nucleic acids under same temperature conditions.

5 more elements

Dependencies are inferred from the claim text.

Facts

Application: 15/538,382Filed 22 Dec 2015 · us provisional application
Priority: 22 Dec 2014
Published: US 2017/0369939 A128 Dec 2017
Granted: 17 Mar 2020
Term ends: Not estimatedThe source records don’t support an estimate.

Classification6 codes

C12Q 1/6853
Nucleic acid amplification reactions: using modified primers or templates
C12Q 1/6827
Hybridisation assays: for detection of mutation or polymorphism
C12Q 2525/107
Modifications characterised by: incorporating a peptide nucleic acid
C12Q 2531/113
The purpose being amplify/increase the copy number of target nucleic acid: pcr