US 2007/0184468 A1Published application

Methods for detecting and localizing DNA mutations by microarray

AssigneeThe State of Oregon Acting by and through the State Board of Higher Education on Behalf of the University of Oregon

InventorGuowen Liu

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Abstract

This disclosure provides methods for detecting and localizing DNA mutations by DNA microarray. In various embodiments, the described methods include use of restriction endonuclease(s) and/or mismatch-recognition nuclease(s) to detect and/or localize mutations. In one representative method, reference and target DNA are digested using one or more restriction endonucleases, resultant DNA strands are labeled (e.g., using a DNA polymerase), and the labeled mixture of DNAs is hybridized to a microarray. In another representative method, reference and target DNA are denatured and annealed to form a mixture containing heteroduplex DNA, one or more mismatch-recognition nuclease(s) are used to nick or cleave at least a portion of the heteroduplex DNA, resultant DNA strands are labeled (e.g., using a DNA polymerase) and the labeled mixture of DNAs is hybridized to a microarray.

Claims12

Dependent claims

A method for detecting and localizing one or more DNA mutations in a target DNA as compared to a reference DNA, the method comprising the steps of:

a) using a restriction endonuclease(s) to digest a mixture of the reference and target DNA or to digest the reference and target DNA separately and then mix, wherein the restriction endonuclease(s) will digest the reference and target DNA into different DNA fragments around the position of any mutation that causes the elimination or creation of a restriction site of the endonuclease(s):
two short restriction fragments flanking the position of the mutation and a large fragment spanning the position of the mutation.
b) labeling DNA strands flanking mutations by one or more cycles of denaturation, annealing and labeling with a DNA polymerase(s), wherein denaturation and annealing will allow one single strand from the short fragments flanking a mutation to anneal with one strand of the large fragment spanning the mutation to form such a partially double-stranded DNA that a DNA polymerase(s) could then use the short strand of this DNA as a primer and the long strand as a template to label the short strand DNA by incorporating modified nucleotides into newly synthesized DNA. c) detecting and localizing DNA mutations by hybridizing a combination of the above labeled DNA and a labeled control DNA to a DNA microarray(s) and identifying the sequence of each DNA whose spot has significantly higher ratio of the label signal from the mixture of reference and target DNA to the label signal from the control DNA than some of the other spots, wherein a labeled control DNA is the reference DNA alone or the target DNA alone treated in the same way as the mixture of reference and target DNA in steps a) and b) but labeled differently from the mixture of reference and target DNA.

A method for detecting and localizing DNA mutations in a target DNA as compared to a reference DNA, the method comprising the steps of:

a) denaturing and annealing of a mixture of the reference and target DNA to form heteroduplex DNA, its corresponding homoduplex DNA and other homoduplex DNA, wherein the mixture of the reference and target DNA is a mixture of fragmented reference and target DNA, obtained by fragmenting the mixture of the reference and target DNA with a restriction endonuclease(s) or fragmenting the reference and target DNA separately and then mixing them together.
b) using a mismatch-recognition nuclease(s) to nick or cleave the heteroduplex DNA, wherein the mismatch-recognition nuclease(s) will nick or cleave the heteroduplex DNA at or around the position of any mutation that causes a mismatch detectable by the nuclease(s), but will not specifically nick or cleave its corresponding homoduplex DNA and other homoduplex DNA.
c) labeling DNA strands flanking mutations by one or more cycles of denaturation, annealing and labeling with a DNA polymerase(s), wherein denaturation and annealing will allow one short single strand from the nicked or cleaved heteroduplex DNA to anneal with one long strand from its corresponding homoduplex DNA or one intact strand from the nicked heteroduplex DNA to form such a partially double-stranded DNA that a DNA polymerase(s) could then use the short strand of this DNA as a primer and the long strand as a template to label the short strand DNA by incorporating modified nucleotides into newly synthesized DNA.
d) detecting and localizing DNA mutations by hybridizing a combination of the above labeled DNA and a labeled control DNA to a microarray(s) and identifying the sequence of each DNA whose spot has a significantly higher ratio of the label signal from the mixture of reference and target DNA to the label signal from the control DNA than some of the other spots, wherein a labeled control DNA is the reference DNA alone or the target DNA alone treated in the same way as the mixture of reference and target DNA in steps a), b) and c) but labeled differently from the mixture of reference and target DNA.

1 more element

Dependencies are inferred from the claim text.

Facts

Application: 11/605,826Filed 28 Nov 2006
Priority: 6 Sep 2002
Published: 9 Aug 2007
Term ends: Not estimatedThe source records don’t support an estimate.

Classification4 codes

C12Q 1/6837
Enzymatic or biochemical coupling of nucleic acids to a solid phase: using probe arrays or probe chips
C12Q 1/683
For detection of mutation or polymorphism: involving restriction enzymes, e.g. restriction fragment length polymorphism [RFLP]
C12Q 1/6858
Nucleic acid amplification reactions: allele-specific amplification
C12Q
Measuring or testing processes involving enzymes, nucleic acids or microorganisms

Family

Application10/236,598Continuation · filed 6 Sep 2002
Application11/605,826Continuation · filed 28 Nov 2006
US 2004/0048257 A1Published application
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US 2007/0184468 A1This patent

From the US filing history. Foreign family members aren’t listed.