Double-stranded conformational polymorphism analysis

A method of detecting the presence or absence of at least one mismatch between a nucleic acid probe and a nucleic acid target, wherein said probe and target have sequences which differ by not more than five nucleotide base pair mismatches and wherein said target sequence comprises a nucleic acid molecule of from about 25 to 300 nt and said probe comprising a known polynucleotide sequence of from 15 to 50 nt and a sugar and base free photoactivatable cross-linking coumarinyl glycerol group occupying a nucleotide position between two nucleotides of said known polynucleotide sequence, which when said probe sequence is hybridized to said target sequence, upon photoactivation forms a covalent bond between said probe sequence and said target sequence, said method comprising:

• combining, in a hybridizing medium, a nucleic acid sample comprising said target and said probe under mild stringency hybridizing conditions for a time sufficient for said target and said probe to hybridize;
• irradiating at a wavelength in the range of about 300-400 nm said hybridizing medium to form cross-links between said probe and target sequence to which said probe is hybridized to cross-linked double-stranded nucleic acid;
• separating nucleic acid in said hybridizing medium by denaturing electrophoresis and comparing the migratory rate of said cross-linked double-stranded nucleic acid to a known mismatched or matched cross-linked double-stranded nucleic acid standard, whereby the presence or absence of said at least one mismatch is determined.

A method of detecting the presence or absence of at least one mismatch between a nucleic acid probe and a nucleic acid target, wherein said probe and target have sequences which differ by not more than five nucleotide base pair mismatches, said target sequence comprising a nucleic acid molecule of from about 25 to 300 nt and wherein said probe comprises a known polynucleotide sequence of from 15 to 50 nt and a sugar and base free photoactivatable cross-linking coumarinyl glycerol group occupying a nucleotide position between two nucleotides of said known polynucleotide sequence, which when said probe sequence is hybridized to said target sequence, upon photoactivation forms a covalent bond between said probe sequence and said target sequence, said method comprising:

• combining, in a hybridizing medium, a nucleic acid sample comprising said target and said probe under mild stringency hybridizing conditions equivalent to a temperature in the range of 25-70° C. and with 0.1-1.5 M sodium for a time sufficient for said target and said probe to hybridize;
• irradiating at a wavelength in the range of about 300-400 nm said hybridizing medium to form cross-links between said probe and target sequence to which said probe is hybridized to from cross-linked double-stranded nucleic acid;
• separating nucleic acid in said hybridizing medium by denaturing gel electrophoresis and comparing the migratory rate of said cross-linked double-stranded nucleic acid to a known mismatched or matched cross-linked double-stranded nucleic acid standard, whereby the presence or absence of said at least one mismatch is determined.

A method of detecting the presence or absence of at least one mismatch between a nucleic acid probe and a nucleic acid target, wherein said probe and target have sequences which differ by not more than five nucleotide base pair mismatches, said target sequence comprising a nucleic acid molecule of from about 25 to 300 nt and said probe comprising a known polynucleotide sequence of from 15 to 50 nt and a sugar and base free photoactivatable cross-linking coumarinyl (glycerol group occupying a nucleotide position between two nucleotides of said known polynucleotide sequence, which when said probe sequence is hybridized to said target sequence, upon photoactivation forms a covalent bond between said probe sequence and said target sequence, said method comprising:

• combining, in a hybridizing medium, a nucleic acid sample comprising said target and said probe under high stringency hybridizing conditions for a time sufficient for said target and said probe to hybridize, where a probe complementary to said target results in at least about a 2-fold greater amount of hybridization than a mismatched probe;
• irradiating at a wavelength in the range of about 300-400 nm said hybridizing medium to form cross-links between said probe and target sequence to which said probe is hybridized to cross-linked double-stranded nucleic acid;
• separating nucleic acid in said hybridizing medium by denaturing electrophoresis and determining the amount of cross-linked double-stranded nucleic acid, where the amount of cross-linked double-stranded nucleic acid is related to the presence or absence of mismatches between said probe and said target.

A method of detecting the presence or absence of at least one mismatch between a nucleic acid probe and a nucleic acid target, wherein said probe and target have sequences which differ by not more than five nucleotide base pair mismatches and wherein said probe comprises a known polynucleotide sequence and photoactivatable cross-linking coumarinyl glycerol group occupying a nucleotide position between two nucleotides of said known polynucleotide sequence and said coumarinyl glycerol group is not appended to a sugar or base of said known polynucleotide sequence, which when said probe sequence is hybridized to said target sequence, upon photoactivation forms a covalent bond between said probe sequence and said target sequence, said method comprising:

• combining, in a hybridizing medium, a nucleic acid sample comprising said target and said probe under mild stringency hybridizing conditions for a time sufficient for said target and said probe to hybridize;
• irradiating said hybridizing medium to form cross-links between said probe and target sequence to which said probe is hybridized to from cross-linked double-stranded nucleic acid;
• separating nucleic acid in said hybridizing medium by denaturing electrophoresis and comparing the migratory rate of said cross-linked double-stranded nucleic acid to a known mismatched or matched cross-linked double-stranded nucleic acid standard, whereby the presence or absence of said at least one mismatch is determined.