Methods of nucleic acid quantification and detection using anomalous migration

摘要

Described are approaches for the identification, detection, and quantification of nucleic acids in a biological sample. These methods are based, in part, on the elucidation of anomalous migration properties of short nucleic acid molecules when conjugated to a fluorescent label, such as fluorescein labels, such that a smaller nucleic acid reliably migrates slower than a larger nucleic acid under the same conditions of separation.

主权项

1. A method for the detection of a target nucleic acid in a sample, the method comprising the steps of:
a) contacting a nucleic acid sample under hybridizing conditions with an oligonucleotide primer and a FAM-labeled oligonucleotide probe member to create a mixture of hybridized duplexes comprising a primer and a probe member hybridized to the same strand of the same target nucleic acid molecule in said sample, wherein:
i) said primer hybridizes 5′ of said probe member on said target; andii) said probe member hybridizes with said target at the probe's 3′ end and comprises a 5′ overhang sequence of 0 to n nucleotides that do not hybridize to said target nucleic acid; andiii) wherein the FAM-label on said labeled probe is attached to the 5′ terminal nucleotide of said probe when the 5′ overhang=0 or to one of the n non-hybridizing nucleotides when n>0; b) maintaining the mixture of step (a) with a template-dependent nucleic acid polymerase having 5′ to 3′ nuclease activity, under conditions sufficient to permit the 5′ to 3′ nuclease activity of the polymerase to cleave the hybridized probe member to release a labeled detection molecule; c) separating nucleic acids in said mixture by electrophoresis; and d) detecting said FAM-labeled detection molecule in the separated nucleic acids, wherein said FAM-labeled detection molecule indicates the presence of said target molecule, and wherein said FAM-labeled detection molecule is detected in an anomalous migration position for non-hybridizing overhangs 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.