Chemically modified ligase cofactors, donors and acceptors
Inventors
Paul, Natasha • Shum, Jonathan • Lebedev, Alexandre • Zon, Gerald
Assignees
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Publication Number
US-8728725-B2
Publication Date
2014-05-20
Expiration Date
Abstract
Provided herein are methods for ligation of polynucleotides containing modified ligation components, particularly modified ligase cofactors, modified acceptors and modified donors. The methods readily applied to ligation-based assays for detection of a nucleic acid sequence where the use of the modified cofactor improves discrimination between matched and mismatched templates. Furthermore, the use of the modified ligation components reduces or eliminates the ligation in the absence of nucleic acid template.
Core Innovation
The invention relates to methods for detecting a mutation in a target nucleic acid using ligation of donor and acceptor polynucleotides. A reaction mixture includes a cofactor dependent nucleic acid ligase, a modified ATP ligase cofactor, and optionally modified donor polynucleotide and optionally modified acceptor polynucleotide. The method monitors ligation of the donor and acceptor polynucleotides, where the amount of ligation is indicative of the presence or absence of the mutation.
The approach is defined by the effect of including a modified ATP ligase cofactor together with modified donor and acceptor polynucleotides, such that ligation of non-complementary nucleic acid is decreased or inhibited relative to the corresponding reaction wherein the ATP ligase cofactor, donor polynucleotide and acceptor polynucleotide are unmodified. In another aspect, ligation specificity is improved relative to the corresponding reaction with unmodified ATP ligase cofactor, donor polynucleotide and acceptor polynucleotide.
In related embodiments, ligation specificity increases when both the 3′-end of the acceptor polynucleotide and the 5′-end of the donor polynucleotide are matched to the target nucleic acid as compared to when either end comprises a nucleotide mismatch relative to the target nucleic acid. The disclosed components are parameterized using structural embodiments, including Formula IA, Formula II, and Formula III, with modified cofactors beyond ATP including modified NAD+ and labeled modified NAD+ variants.
Claims Coverage
The document contains multiple independent claims directed to detecting a mutation in a target nucleic acid using a ligation-based approach with a modified ATP ligase cofactor and optionally modified donor and acceptor polynucleotides. Across the independent claims, the inventive features focus on mutation indication by monitored donor–acceptor ligation, decreased or inhibited ligation of non-complementary or mismatched nucleic acid relative to matched or unmodified reactions, improved ligation specificity, and matched-efficiency preservation.
Mutation detection via donor–acceptor ligation amount
A method incubating a target nucleic acid in a reaction mixture comprising a cofactor dependent nucleic acid ligase, a modified ATP ligase cofactor, an optionally modified donor polynucleotide and an optionally modified acceptor polynucleotide; monitoring ligation of said donor and acceptor polynucleotides, wherein the amount of ligation is indicative of the presence or absence of said mutation.
Decreased or inhibited non-complementary ligation with modified ATP cofactor and modified donor/acceptor
The presence of said modified ATP ligase cofactor, said donor polynucleotide and said acceptor polynucleotide decreases or inhibits ligation of non-complementary nucleic acid relative to ligation efficiency of the corresponding reaction wherein said ATP ligase cofactor, donor polynucleotide and acceptor polynucleotides are unmodified.
Improved ligation specificity relative to unmodified components
The presence of said modified ATP ligase cofactor, said donor polynucleotide and said acceptor polynucleotide improves ligation specificity relative to the corresponding reaction wherein said ATP ligase cofactor, donor polynucleotide and said acceptor polynucleotides are unmodified.
Increased ligation specificity when acceptor 3′ end and donor 5′ end are matched
Ligation specificity increases when both the 3′-end of said acceptor polynucleotide and the 5′-end of said donor polynucleotide are matched to said target nucleic acid as compared to when either the 3′-end of said acceptor polynucleotide or the 5′-end of said donor polynucleotide comprises a nucleotide mismatch relative to said target nucleic acid.
Inhibition or reduction of mismatched ligation while retaining matched ligation efficiency
The presence of said modified ATP ligase cofactor, said acceptor polynucleotide and said donor polynucleotide inhibits or reduces ligation of mismatched nucleic acid; and has at least about the same ligation efficiency of matched nucleic acid as compared to said reaction mixture wherein said ATP ligase cofactor, donor polynucleotide and acceptor polynucleotides are unmodified.
Across the independent claims, mutation detection is achieved by monitoring donor–acceptor ligation where ligation amount indicates mutation presence or absence. Specificity is obtained by using a modified ATP ligase cofactor in combination with optionally modified donor and acceptor polynucleotides that decrease or inhibit mismatch ligation and improve ligation specificity relative to unmodified counterparts, while maintaining comparable ligation efficiency for matched nucleic acid.
Stated Advantages
Decreases or inhibits ligation of non-complementary nucleic acid relative to ligation efficiency of the corresponding reaction with unmodified ATP ligase cofactor, donor polynucleotide and acceptor polynucleotide.
Improves ligation specificity relative to the corresponding reaction with unmodified ATP ligase cofactor, donor polynucleotide and acceptor polynucleotide.
Increases ligation specificity when both the acceptor 3′-end and the donor 5′-end are matched to the target nucleic acid compared to when either end comprises a nucleotide mismatch.
Inhibits or reduces ligation of mismatched nucleic acid while having at least about the same ligation efficiency of matched nucleic acid compared to a reaction mixture using unmodified ATP ligase cofactor, donor polynucleotide and acceptor polynucleotide.
Documented Applications
Detecting a mutation in a target nucleic acid by monitoring donor–acceptor ligation as indicative of mutation presence or absence.
Using defined ligation modalities for monitoring ligation, including oligonucleotide ligation assay (OLA), ligase chain reaction (LCR), Ligase Mediated PCR (LM-PCR), PCR ligation detection reaction (PCR-LDR), padlock probes, PCR oligonucleotide ligation assay (PCR-OLA), gap LCR, SNPlex, MLPA, GoldenGate Genotyping Assay, Molecular Inversion Probe Assay, proximity ligation, and next-generation sequencing by ligation.
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