Composition, kits and methods for the detection of point mutations and SNPs
Inventors
Adlerstein, Daniel • Amicarelli, Giulia • Minnucci, Giulia
Assignees
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Publication Number
US-9404149-B2
Publication Date
2016-08-02
Expiration Date
Abstract
The present invention refers to a method for detecting a point mutations of a nucleotide sequence by an improvement of the LAMP (loop amplification mediated polymerization) amplification method, as well as to a set of primers and kit therefor. As a non limitative embodiment, the invention refers to the G1849T mutation of the JAK2 gene.
Core Innovation
The disclosed invention provides a set of primers for detecting, by loop mediated isothermal amplification, the presence of a point mutation in a target nucleic acid molecule in a background of wild type nucleic acid molecules. The primer set includes outer primers F3 and B3 and inner primers FIP and BIP, with defined nucleic acid sequence regions on opposite strands and with the point mutation located relative to non-overlapping complementary regions designated as F2c and B2c.
The primer set further includes a stem-loop mutant extensible primer. The stem-loop mutant extensible primer comprises a central loop sequence complementary to a region of the target nucleic acid molecule comprising the point mutation, and a 5′ end sequence and a 3′ end sequence complementary to each other to form a stem upon intramolecular hybridization, with the hybridization affinity of the central loop sequence to the mutation-containing region higher than the intramolecular hybridization affinity of the 5′ sequence to the 3′ sequence.
In addition, the invention provides a non-extensible moiety capable of selectively hybridizing to wild type nucleic acid molecules and of blocking amplification thereof. The invention is discussed as an improved LAMP-based assay for detecting single-base point mutations/SNPs using an outer/inner primer scheme, the mutant-selective stem-loop/self-annealed extensible loop primer, and a wild-type-blocking non-extensible moiety, with embodiments including peptide nucleic acid (PNA).
Claims Coverage
The provided independent claim covers a primer set for loop mediated isothermal amplification of point mutations in a wild type background. The claim includes three core inventive elements: an outer/inner LAMP primer architecture with defined mutation-relative placement, a stem-loop mutant extensible primer with an affinity relationship, and a non-extensible moiety that selectively blocks amplification of wild type.
Outer and inner LAMP primers with mutation-relative regions
A set of primers comprising first outer primer F3 and second outer primer B3, and first inner primer FIP and second inner primer BIP, with defined regions on opposite strands and the point mutation located according to an upstream/downstream relationship relative to non-overlapping complementary regions F2c and B2c.
Stem-loop mutant extensible primer with higher mutation-target affinity
A stem-loop mutant extensible primer comprising a central loop sequence complementary to a region of a target nucleic acid molecule comprising the point mutation, and a 5′ end sequence and a 3′ end sequence complementary to each other to form a stem upon intramolecular hybridization, wherein the hybridization affinity of the central loop sequence to the mutation-containing region is higher than the intramolecular hybridization affinity of the 5′ sequence to the 3′ sequence.
Non-extensible wild-type blocking moiety
A non-extensible moiety capable of selectively hybridizing to the wild type nucleic acid molecules and of blocking amplification thereof.
Overall, the claimed subject matter combines an outer/inner LAMP primer layout with specified non-overlapping mutation-relative regions, a stem-loop mutant extensible primer requiring a central-loop-to-stem affinity hierarchy, and a wild-type-selective non-extensible blocking moiety.
Stated Advantages
Improved detection of point mutations/SNPs in a wild type background using LAMP-based amplification with mutant selection and wild-type blocking.
Sensitivity down to about 0.01% mutant sequences in a wild type background.
Ability to estimate mutant allele proportion using calibrators above or below 50%.
Clinical comparison includes performance assessment against ARMS with discordant cases resolved by confirmatory sequencing.
Documented Applications
Detection of single-base point mutations/SNPs in a wild-type background, exemplified by JAK2 G1849T/V617F.
Use in clinical sample testing and comparison versus ARMS, with discordant cases resolved by confirmatory sequencing.
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