Nucleic acid detection assay
Inventors
Millar, Douglas Spencer • Melki, John Robert • Gabor Miklos, George L.
Assignees
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Publication Number
US-7846693-B2
Publication Date
2010-12-07
Expiration Date
Abstract
A method for determining the methylation status of a potential methylation site in genomic nucleic acid comprising treating genomic nucleic acid with an agent which modifies cytosine bases but does not modify 5methyl-cytosine bases under conditions to form a. modified nucleic acid template containing a potential methylation site; providing a first clamp containing a first capture sequence complementary to a region flanking one side of the potential methylation site in the modified nucleic acid template, providing a second clamp containing a second capture sequence complementary to a region flanking the other side of the potential methylation site in the modified nucleic acid template; allowing the first clamp and the second clamp to hybridise to the modified nucleic acid template; ligating the hybridised first and second clamps to form a probe spanning the potential methylation site in the modified nucleic acid template; digesting the modified nucleic acid template to obtain the probe; and detecting the probe and determining the methylation status of the potential methylation site in the modified genomic nucleic acid.
Core Innovation
The invention provides methods for determining the methylation status of a potential methylation site in genomic nucleic acid using a cytosine-modifying agent that modifies cytosine bases but does not modify 5-methyl-cytosine bases, thereby forming a modified nucleic acid template from two complementary strands. The approach interrogates the potential methylation site after template conversion by hybridizing clamp oligonucleotides to regions flanking the site on each strand and using ligation to create a probe spanning the potential methylation site.
For the linear probe format, a first clamp includes a first capture sequence and a second capture sequence, complementary to regions flanking one side of the potential methylation site on each strand, and a second clamp includes a third capture sequence complementary to the region flanking the other side of the potential methylation site. The hybridised clamps are ligated to form a probe spanning the potential methylation site in the modified nucleic acid template, and the modified nucleic acid template is digested to obtain the probe for detection.
For the circular probe format, clamp sequences are arranged as a second clamp that provides a third capture sequence and a fourth capture sequence complementary to the regions flanking the other side of the potential methylation site on each strand. After hybridisation to the two complementary strands, the clamps are ligated to form a circular probe spanning the potential methylation site, the modified nucleic acid template is digested to obtain the circular probe, and the circular probe is detected to determine the methylation status.
Claims Coverage
The document contains two independent claims directed to clamp-based methylation determination using cytosine conversion and probe generation by clamp ligation, including both linear probe and circular probe geometries. The two independent claims jointly define the workflow from cytosine-modifying conversion to clamp hybridisation, ligation to form a probe, digesting the modified nucleic acid template, and detecting the probe.
Cytosine-modifying conversion preserving 5-methyl-cytosine
Treating genomic nucleic acid with an agent which modifies cytosine bases but does not modify 5-methyl-cytosine bases under conditions to form a modified nucleic acid template from two complementary strands of genomic nucleic acid containing a potential methylation site.
Linear probe via two-clamp ligation to span the potential methylation site
Providing a first clamp containing a first capture sequence and a second capture sequence, first capture sequence being complementary to a region flanking one side of the potential methylation site in the first strand of the modified nucleic acid template, and the second capture sequence being complementary to a region flanking one side of the potential methylation site in the second strand of the modified nucleic acid template; providing a second clamp containing a third capture sequence complementary to a region flanking the other side of the potential methylation site in the modified nucleic acid template; allowing the first clamp and the second clamp to hybridise to the modified nucleic acid template; ligating the hybridised first and second clamps to form a probe spanning the potential methylation site in the modified nucleic acid template.
Digesting the modified template to obtain the probe and detecting to determine methylation status
Digesting the modified nucleic acid template to obtain the probe; and detecting the probe and determining the methylation status of the potential methylation site in the modified genomic nucleic acid.
Circular probe via clamp ligation spanning the potential methylation site
Providing a first clamp containing a first capture sequence and a second capture sequence, first capture sequence being complementary to a region flanking one side of the potential methylation site in the first strand of the modified nucleic acid template, and the second capture sequence being complementary to a region flanking one side of the potential methylation site in the second strand of the modified nucleic acid template; providing a second clamp containing a third capture sequence and a fourth capture sequence, the third capture sequence being complementary to a region flanking the other side the potential methylation site in the first strand of the modified nucleic acid template, and the fourth capture sequence being complementary to a region flanking the other side of the potential methylation site in the second strand of the modified nucleic acid template; allowing the first clamp and the second clamp to hybridise to the two complementary strands of the modified nucleic acid template; ligating the hybridised clamps to form a circular probe spanning the potential methylation site in the complementary strands of the modified nucleic acid template.
Digesting the modified template to obtain the circular probe and detecting to determine methylation status
Digesting the modified nucleic acid template to obtain the circular probe; and detecting the circular probe and determining the methylation status of the potential methylation site in the modified genomic nucleic acid.
Overall, the claim coverage centers on converting genomic nucleic acid with a cytosine-modifying agent that preserves 5-methyl-cytosine, then using clamp hybridisation to flanking regions around a potential methylation site, followed by ligation-based probe formation. The independent claims distinguish a linear probe produced from two clamps ligated to span the site versus a circular probe produced from clamp pairs ligated to span the site, with detection of the resulting probe to determine methylation status.
Stated Advantages
Improved specificity via dual polymerase/ligase fidelity.
Reduced non-specific amplification and reduced PCR contamination.
Optional avoidance of genomic target amplification.
Template removal to reduce background.
High multiplexing using shared universal primer sites plus array addressing.
Reduced PCR bias due to ligation/hybridization dependence.
Documented Applications
Determining methylation status of potential methylation sites in genomic nucleic acid after cytosine base modification that preserves 5-methyl-cytosine, including CpG methylation interrogation.
Micro-array detection using clamp-based probe detection with array addressing and zip code primers/tags.
Southern blot type detection of the probe.
Real-time PCR quantitation-based detection of the probe.
Detection using fluorescent beads, enzyme conjugates, or radioactive beads.
Detection using ligase chain reaction (LCR).
Detection using isothermal DNA amplification, including strand displacement amplification and rolling circle amplification.
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