Compositions and methods for detecting single nucleotide polymorphisms
View Patent ↗Described herein are nucleic acid based probes and methods for discriminating and detecting single nucleotide variants in nucleic acid molecules (e.g., DNA). The methods include use of a pair of probes can be used to detect and identify polymorphisms, for example single nucleotide polymorphism in DNA. The pair of probes emit a different fluorescent wavelength of light depending on the association and alignment of the probes when hybridized to a target nucleic acid molecule. Each pair of probes is capable of discriminating at least two different nucleic acid molecules that differ by at least a single nucleotide difference. The methods can probes can be used, for example, for detection of DNA polymorphisms that are indicative of a particular disease or condition.
1. A method of detecting a single nucleotide polymorphism in a KRAS gene, comprising:
(a) hybridizing a set of probes to a DNA sample from a human subject, the set of probes comprising:
a first probe comprising a nucleic acid sequence set forth as SEQ ID NO: 91, wherein the nucleic acid sequence of the first probe comprises a nucleation sequence bound to templated metal nanoclusters that are fluorescent, and
a second probe comprising a nucleic acid sequence set forth as SEQ ID NO: 92, wherein the nucleic acid sequence of the second probe comprises an enhancer sequence that enhances the fluorescence of the templated metal nanoclusters;
and wherein:
the first and second probes hybridized to the DNA sample form a three-way junction wherein the templated metal nanoclusters of the first probe are proximal to the enhancer sequence of the second probe;
the polymorphism is positioned at a branch point of the three-way junction;
the presence of the polymorphism causes a shift in alignment of the templated metal nanoclusters relative to the enhancer sequence compared to a wild-type control, resulting in a detectable change in a fluorescence emission wavelength of the metal nanoclusters; and
wherein the probes do not hybridize to non-target DNA;
(b) measuring the fluorescence emission wavelength of the probes hybridized to the DNA sample in response to ultraviolet light, visible light, and/or near infrared excitation light, and
(c) detecting the presence of the single nucleotide polymorphism in the KRAS gene by detecting the detectable change in fluorescence emission wavelength of the metal nanoclusters compared to the wild-type control.
2. The method of claim 1 , wherein the excitation light comprises a wavelength of from 200 nm to 2000 nm.
3. The method of claim 1 , wherein the association of the enhancer portion and the metal nanoclusters enhances fluorescence emission of the templated metal nanoclusters by at least 2-fold.
4. The method of claim 1 , wherein the difference in the wavelength of the fluorescence emission of the test mixture compared to that of the corresponding control mixture is a visible color difference.
5. The method of claim 1 , wherein the difference in the wavelength of the fluorescence emission of the test mixture compared to that of the corresponding control mixture is at least 1 nm.
6. The method of claim 1 , wherein
(a) the first probe is no more than 75 nucleotides in length;
(b) the second probe is no more than 75 nucleotides in length; or
(c) both (a) and (b).
7. The method of claim 1 , wherein the metal of the templated metal nanoclusters is silver, gold or copper.
8. The method of claim 1 , wherein
(a) the first probe consists of the nucleic acid sequence set forth as SEQ ID NO: 91;
(b) the second probe consists of the nucleic acid sequence set forth as SEQ ID NO: 92; or
(c) the first and second probes consist of the nucleotide sequences set forth as SEQ ID NOs: 91 and 92, respectively.