Methods of nucleic acid quantification and detection using anomalous migration

A method for the detection of a target nucleic acid in a sample, the method comprising:

• a) contacting a nucleic acid sample under hybridizing conditions with a pair of oligonucleotide primers and a FAM-labeled oligonucleotide probe to create a mixture of hybridized duplexes of said pair of oligonucleotide primers and said FAM-labeled probe annealed to a target nucleic acid present in said sample,
• wherein the pair of oligonucleotide primers comprises a first oligonucleotide primer that hybridizes to the antisense of a 5′ region of the target nucleic acid and a second oligonucleotide primer that hybridizes to a 3′ region of the target nucleic acid,
• wherein said first oligonucleotide primer and said FAM-labeled oligonucleotide probe hybridize to the same strand of said target nucleic acid,
• wherein said 5′ and 3′ regions of said target sequence flank a region of said target sequence to which said FAM-labeled oligonucleotide probe hybridizes, and
• wherein said FAM-labeled oligonucleotide probe comprises a 5′ overhang sequence of 0 to n nucleotides that does not hybridize to said target nucleic acid, and wherein the FAM label on said labeled probe is attached to the 5′ terminal nucleotide of said probe when 5′ overhang=0 or to one of the n non-hybridizing nucleotides when n>0;
• b) PCR (polymerase chain reaction) amplifying target nucleic acid molecules present in said sample by a plurality of cycles of:
• i) extending annealed oligonucleotide primers using a thermostable nucleic acid polymerase having 5′ to 3′ exonuclease activity, under conditions sufficient to permit primer extension of annealed oligonucleotide primers and to permit the 5′ to 3′ nuclease activity to cleave annealed oligonucleotide probe to thereby release a labeled detection molecule;
• ii) heating to separate nucleic acid strands generated in step (i);
• and
• iii) maintaining under temperature conditions that permit annealing of oligonucleotide primer pairs and labeled oligonucleotide probes to target nucleic acids present in said sample;
• c) separating nucleic acids generated in step (b);
• and
• d) detecting a released FAM-labeled detection molecule in the separated nucleic acids to thereby detect the presence and/or amount of said target nucleic acid present in said sample, wherein FAM-labeled detection molecule is detected in an anomalous migration position for a non-hybridizing overhang of n=0 to 6, with n=1 migrating faster than n=0, n=2 migrating faster than n=1, n=3 migrating faster than n=2, n=4 migrating faster than n=3, n=5 migrating faster than n=4, and n=6 migrating faster than n=5, and where the FAM-labeled detection molecules released from probes with non-hybridizing overhangs greater than n=6 show little change in migration with increasing overhang length, until migration migrate progressively slower with increasing overhang length.

A method for the multiplex detection of target nucleic acids in a sample, the method comprising:

• a) contacting a nucleic acid sample under hybridizing conditions with a pair of oligonucleotide primers and a fluorescently labeled oligonucleotide probe for each member of a plurality of different nucleic acid targets to be detected in said sample, to create a mixture of hybridized duplexes of said pair of oligonucleotide primers and said fluorescently labeled probe specific for each said target nucleic acid member present in said sample, wherein:
• i) for each nucleic acid target, the pair of oligonucleotide primers comprises a first oligonucleotide primer that hybridizes to the antisense of a 5′ region of the target nucleic acid and a second oligonucleotide primer that hybridizes to a 3′ region of the target nucleic acid;
• ii) for each nucleic acid target, said 5′ and 3′ regions of said target sequence flank a region of said target sequence which is complementary to said fluorescently labeled oligonucleotide probe,
• iii) said first oligonucleotide primer and said fluorescently labeled oligonucleotide probe for each nucleic acid target hybridize to the same strand of said target nucleic acid, and
• iv) at least one fluorescently labeled probe is FAM-labeled, and wherein when the fluorescently labeled probe is FAM-labeled, said probe comprises a 5′ overhang sequence of 0 to n nucleotides that do not hybridize to the target nucleic acid, where the FAM label on the labeled probe is attached to the 5′ terminal nucleotide of the probe when the overhang=0 or to one of the n non-hybridizing nucleotides when n>0, and wherein each different FAM-labeled probe has a different length of 5′ overhang;
• and
• v) the length n of 5′ overhang sequence and/or the identity of the label differs for each different target sequence to be detected;
• b) PCR amplifying target nucleic acid molecules present in said sample by a plurality of cycles of:
• i) extending annealed oligonucleotide primers using a nucleic acid polymerase having 5′ to 3′ exonuclease activity, under conditions sufficient to permit primer extension of annealed oligonucleotide primers and to permit the 5′ to 3′ nuclease activity to cleave annealed oligonucleotide probes and thereby release a fluorescently labeled detection molecule specific for each of the plurality of target nucleic acids present in said sample;
• ii) heating to separate nucleic acid strands generated in step (i);
• and
• iii) annealing oligonucleotide primer pairs and fluorescently labeled oligonucleotide probes to target nucleic acids present in said sample;
• c) separating nucleic acids generated in step (b) such that fluorescently labeled detection molecules specific for each of said target nucleic acids present in said sample can be identified;
• and
• d) detecting each of said fluorescently labeled detection molecules released, whereby the presence and/or amount of each of said plurality of target nucleic acids present in said sample is indicated, wherein FAM-labeled detection molecules are detected in an anomalous migration position for a non-hybridizing overhang of n=0 to 6, with n=1 migrating faster than n=0, n=2 migrating faster than n=1, n=3 migrating faster than n=2, n=4 migrating faster than n=3, n=5 migrating faster than n=4, and n=6 migrating faster than n=5, and where the FAM-labeled detection molecules released from probes with non-hybridizing overhangs greater than n=6 show little change in migration with increasing overhang length, until migration migrate progressively slower with increasing overhang length.