This invention relates to methods and compositions useful for the analysis of nucleic acid sequences. In particular, the invention provides methods and compositions useful for the analysis of mixtures of multiple nucleic acid sequences.
Nucleic acid analysis techniques capable of large-scale multiplex analysis of a plurality of polymorphic loci are needed for a variety of practically important applications, e.g., for the identification of individuals, e.g., paternity testing or in forensic analysis, for organ transplant donor-recipient matching, for genetic disease diagnosis, prognosis and genetic counseling, for characterization of infectious agents, and for the study of oncogenic mutations. Many of these applications depend on the ability to discriminate single-nucleotide differences in a target nucleic acid sequence at a multiplicity of loci.
One solution to the problem of multiplex analysis of nucleic acids is to use arrays of different-sequence nucleic acids attached to a solid support, where the sequence of the attached nucleic acids are related to their location in the array (e.g., Kucherlapti and Childs, Nature Genetics, 21: 15-19 (1999); Lipshutz et al., Nature Genetics, 21: 2024 (1999)). However, nucleic acid arrays have several significant practical drawbacks including (1) issues associated with the fabrication of arrays, e.g., the expense and complexity of the fabrication and testing of arrays, e.g., by in situ synthesis or spotting; (2) characterization of arrays once they are fabricated, e.g., confirmation that the proper sequence is located at the proper location; (3) the integrity the nucleic acid at each array location, e.g., there may be intra-strand cross-linking and/or multiple constraining contacts between the solid support and the bound nucleic acid; and (4) difficulties associated with detecting hybridization events on a solid support, e.g., fluorescence background and/or non-specific binding of a labeled entity to the support.
In an alternative approach, multiple probes are analyzed by electrophoretically separating probes that include mobility-modifying tails that correlate a particular probe with a particular mobility address (e.g., Grossman et al., Nucl. Acids Res., 22: 4527-4534 (1994); Grossman et al., U.S. Pat. No. 5,624,800). However, this approach has the drawback of requiring the synthesis of a large number of tailed probes.
Thus, there remains a continuing need for a nucleic acid analysis technique that permits highly multiplexed analysis without the drawbacks associated with nucleic acid arrays or with existing mobility-based methods.
The present invention is directed towards methods and compositions useful for the multiplex analysis of nucleic acids.
In a first aspect, the present invention provides a binary composition for detecting one or more target nucleic acid sequences in a mixture. The binary composition includes a probe having a target-specific portion for sequence-specific hybridization to a target nucleic acid sequence, and a tag. The binary composition further includes a mobility modifier having a tail and a tag complement for binding to the tag. When the probe and the mobility modifier are bound through the tag and tag complement, a probe/mobility modifier complex is formed. In a preferred embodiment, the probe comprises a polynucleotide.
In a second aspect, the present invention provides a method for detecting one or more target nucleic acid sequences present in a sample. In the method, a sample potentially containing one or more target nucleic acid sequences is contacted with one or more probes under conditions effective for sequence-dependent hybridization of the probe and the target nucleic acid sequence, where each probe includes a target-specific portion and a tag. Next, the hybridized probe is treated to form a modified probe, where the treatment is effective to distinguish probes that have bound to the target nucleic acid from those that have not. Before or after the probes are contacted with the target nucleic acid, the probe are contacted with one or more mobility-modifiers to form a probe/mobility modifier complex, each mobility modifier having a La complement for binding selectively to the tag of an associated probe, and a tail. Finally, the probe/mobility modifier complex is analyzed using a mobility-dependent analysis technique.