Methods of determining nucleic acid structural information
Inventors
Saghbini, Michael G. • Sadowski, Henry B. • Pljevaljcic, Goran • Hastie, Alex R. • Cao, Han
Assignees
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Publication Number
US-11959134-B2
Publication Date
2024-04-16
Expiration Date
Abstract
Methods of double-stranded nucleic acid sequence determination and assembly that are able to identify insertions, deletions, repeat region sizes and genomic rearrangements, for example, are disclosed herein, which can use relatively large labeled nucleic acid fragments to analyze the structure of even larger genetic regions. In some embodiments these methods involve the use of certain parameters which unexpectedly improve overall method performance. In some embodiments these methods involve sample labeling that does not result in the formation of single-stranded nucleic acid fragment labeling intermediaries.
Core Innovation
The invention analyzes nucleic acid molecules by providing a plurality of nucleic acid molecules comprising occurrences of DNA repeated motifs. The DNA repeated motifs comprise methyltransferase recognition sequences and are selected to have an average repeat frequency of about 5 sites to about 35 sites per 100 Kb, with an input coverage of 50-fold or less. The method covalently labels the occurrences of the DNA repeated motifs in a sequence-specific fashion while maintaining the strand phosphodiester bond integrity of the covalently labeled nucleic acid molecules.
Covalent labeling is effected with a methyltransferase in the presence of a modified cofactor, where the modified cofactor comprises modified S-adenosyl methionine (SAM). The modified cofactor further comprises a tag that is detectable, transferable, or both, and the detecting step identifies patterns of the DNA repeated motif across the plurality of nucleic acid molecules. In configurations with two DNA repeated motifs, labeling is performed in a CpG-methylation-insensitive fashion at a first repeated motif and in a CpG-methylation-dependent fashion at a second repeated motif, and both labels are detected to identify patterns of both motifs.
The invention maintains strand phosphodiester bonds intact by using covalently labeled nucleic acid molecules having a length of at least 150 kb. Detected label patterns are used to assemble a map of a genomic region from the motif patterns and to compare the assembled map to a digitally stored reference genome map using in silico analysis. The approach is described as enabling long double-stranded nucleic acids mapping/assembly while avoiding compromising strand integrity.
Claims Coverage
The document provides two independent claims centered on analyzing nucleic acid molecules using covalent, sequence-specific methyltransferase-based labeling of DNA repeated motifs with modified S-adenosyl methionine cofactor tags, while preserving intact strand phosphodiester bonds. The second independent claim adds dual-motif labeling distinguishing CpG-methylation-insensitive versus CpG-methylation-dependent labeling and detecting patterns of both motifs. Each independent claim includes key quantitative constraints including input coverage of 50-fold or less and labeling at nucleic acid length at least 150 kb, together with motif repeat frequency constraints of about 5 sites to about 35 sites per 100 Kb.
Methyltransferase-based sequence-specific covalent motif labeling with modified SAM tags while preserving intact strand phosphodiester bonds
providing a plurality of nucleic acid molecules with occurrences of a DNA repeated motif, wherein the DNA repeated motif comprises a methyltransferase recognition sequence and is selected to have an average repeat frequency of about 5 sites to about 35 sites per 100 Kb with input coverage 50-fold or less; covalently labeling the plurality of nucleic acid molecules in a sequence-specific fashion at the DNA repeated motif with a methyltransferase in the presence of a modified cofactor comprising modified S-adenosyl methionine (SAM) and comprising a tag that is detectable, transferable, or both; detecting the label to identify patterns of the DNA repeated motif; wherein the covalently labeled nucleic acid molecules have a length of at least 150 kb and the strand phosphodiester bonds are intact
Dual DNA repeated motif labeling distinguishing CpG-methylation-insensitive and CpG-methylation-dependent motif patterns with intact strand phosphodiester bonds
providing a plurality of nucleic acid molecules with occurrences of a first DNA repeated motif and a second DNA repeated motif, wherein the first DNA repeated motif, the second DNA repeated motif, or both have an average repeat frequency of about 5 sites to about 35 sites per 100 Kb and input coverage is 50-fold or less; covalently labeling the plurality of nucleic acid molecules in a sequence-specific and CpG-methylation-insensitive fashion at the first DNA repeated motif with a first label effected with a CpG-methylation-insensitive methyltransferase in the presence of a first modified cofactor; covalently labeling the plurality of nucleic acid molecules in a sequence-specific and CpG-methylation-dependent fashion at the second DNA repeated motif with a second label effected with a CpG-methylation-dependent methyltransferase in the presence of a second modified cofactor; detecting the first label and the second label to identify patterns of the first DNA repeated motif and the second DNA repeated motif respectively; wherein the covalently labeled nucleic acid molecules have a length of at least 150 kb and the strand phosphodiester bonds are intact
Across the independent claims, the core claim coverage is the use of methyltransferase-catalyzed covalent, sequence-specific labeling of DNA repeated motifs using modified SAM cofactor tags to detect motif patterns while maintaining intact strand phosphodiester bonds. The second independent claim further covers simultaneous pattern identification from two motifs differentiated by CpG-methylation-insensitive versus CpG-methylation-dependent labeling.
Stated Advantages
Analyzing nucleic acid molecules without compromising strand phosphodiester bond integrity.
Documented Applications
No documented applications found
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