Methods and devices for single-molecule whole genome analysis
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Publication Number
US-11939627-B2
Publication Date
2024-03-26
Expiration Date
Abstract
Provided are methods and devices for single-molecule genomic analysis. In one embodiment, the methods entail processing a double-stranded nucleic acid and characterizing said nucleic acid. These methods are useful in, e.g. determining structural variations and copy number variations between individuals.
Core Innovation
The document describes characterizing a first DNA and a second DNA by labeling a plurality of sequence-specific locations on each DNA with a labeling technique that does not cut or nick a strand of DNA. The labeled DNAs are then linearized so that recognizable patterns of labels are detected along the linearized DNAs. The detected labels are indicative of sequence characteristics of the first DNA and the second DNA, enabling comparison of similarity or difference in those sequence characteristics.
A central aspect is the use of sequence-specific labeling agents that generate label patterns usable as barcodes associated with genomic structural variants and copy number variations. The observed barcode signal patterns are linked to reference signatures, including reference DNA patterns and in silico barcode signatures, to infer sequence characteristic differences between samples and reference DNA. The approach further includes systems for nanochannel or nanotrack linearization to support detection of label patterns along long DNA.
The document also discloses labeling and detection using flap/gap generation on double-stranded DNA via sequence-specific nicking, with polymerase extension and filled-in gaps or extended strands, followed by labeling of flaps, filled gaps, or extended strands with tag species. Tag species are described as including fluorophores and quantum dots and also molecular labels associated with methyltransferase and binding proteins or antibodies. The detected label patterns are then used for mapping and correlation of barcode signals to sequenced segments for genome scaffolding assembly.
Claims Coverage
Two independent claims are identified. The claims center on non-cutting, non-nicking labeling of multiple sequence-specific locations, linearization, detection of recognizable label patterns indicative of sequence characteristics, and comparison either between a first and second DNA sample or between the first DNA and a reference DNA label pattern.
Labeling sequence-specific locations without cutting or nicking
Label a plurality of sequence-specific locations on a first DNA and a second DNA with a labeling technique that does not cut or nick a strand of DNA;
Linearizing labeled DNAs for pattern detection
Linearizing at least a portion of the labeled first DNA and a portion of the labeled second DNA;
Detecting recognizable label patterns indicative of sequence characteristics
Detecting a recognizable pattern of labels on the linearized first DNA and on the linearized second DNA, wherein the labels are indicative of a sequence characteristic of the first DNA and a sequence characteristic of the second DNA;
Comparing patterns to ascertain similarity or difference
Comparing the recognizable patterns of the first and second DNAs to ascertain a similarity or difference in a sequence characteristic of the first and second DNAs.
Reference-pattern comparison for DNA characterization
Comparing the recognizable pattern of labels on the linearized first DNA to a pattern of labels indicative of a known sequence characteristic of a reference DNA to ascertain a similarity or difference in a sequence characteristic of the first DNA and the reference DNA.
The inventive concept centers on non-cutting, non-nicking labeling of multiple sequence-specific locations, linearization, detection of recognizable label patterns indicative of sequence characteristics, and comparison between DNA samples or between a DNA sample and a reference DNA label pattern.
Stated Advantages
Documented Applications
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