Looped probe design to control hybridization stringency

Abstract

The present invention provides a method for the generation of novel libraries of encoded magnetic particles from sub-libraries of by the generation of novel sub- libraries of magnetic nanoparticles and encoded particles. The sub-libraries are functionalized on demand are useful in the formation of arrays. The present invention is especially useful for performing multiplexed (parallel) assays for qualitative and/or quantitative analysis of binding interactions of a number of analyte molecules in a sample.

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         41 . A method of controlling molecular stringency in the hybridization of target nucleic acid sequences and a probe, wherein hybridization is detected to identify the presence of said target nucleic acid sequence in the sample, comprising: 
 providing a probe having a first segment complementary with at least a portion of said target nucleic acid sequences and a second segment, separated from said first segment by a linker segment complementary to said first segment; and    controlling which target nucleic acid sequences in a sample hybridize to the first segment or to the second segment by selecting the length of the second segment such that the stability of a duplex formed by first and the second segment is adjusted to intentionally reduce the number of different target nucleic acid sequences which will hybridize to the probe during the assay.    
     
     
         42 . The method of  claim 41  wherein the assay is conducted under conditions wherein the second segment and the target nucleic acid sequences competitively hybridize to the first segment.  
     
     
         43 . The method of  claim 41  wherein the first segment and the target nucleic acid sequences competitively hybridize to the second segment.  
     
     
         44 . The method of  claim 41  wherein the probe is attached to a bead.  
     
     
         45 . The method of  claim 41  wherein the bead is encoded to permit identification of the probe attached thereto.  
     
     
         46 . A method of assaying for a target nucleic acid sequence in a sample wherein target nucleic acid sequences in a sample hybridize with a probe and the hybridization is detected to identify the presence of said target nucleic acid sequences in the sample, comprising: 
 providing a probe having a first segment complementary with at least a portion of said target nucleic acid sequences and a second segment, separated from said first segment with a linker segment, which is complementary to said first segment;    placing the probe and the sample in contact under conditions wherein the second segment and the target nucleic acid segment competitively hybridize to the first segment; and detecting hybridization of the target nucleic acid segment to the first segment.    
     
     
         47 . The method of  claim 46  wherein hybridization is detected by detecting the open-loop configuration in which first and second segments are not hybridized to one another.  
     
     
         48 . The method of  claim 47  wherein the open loop configuration is detected by means of detecting fluorescence from a pair of fluorescent moieties by fluorescence energy transfer.  
     
     
         49 . The method of  claim 47  wherein the open loop configuration is detected by means of detecting fluorescence from the donor in a donor-acceptor pair of fluorescent moieties respectively associated with the first and second segments of the probe (the members of the pair capable of interacting, in the closed loop configuration of the probe) by fluorescence energy transfer.  
     
     
         50 . The method of  claim 46  wherein the hybridization of the target to the second segment is detected by elongation of said second segment of the probe;  
     
     
         51 . The method of  claim 46  wherein the probe is attached to a bead.  
     
     
         52 . The method of  claim 47  wherein the bead is encoded to permit identification of the probe attached, where more than one type of probe is used in the assay.  
     
     
         53 . The method of  claim 47  wherein the nucleic acid sequence is DNA or RNA.  
     
     
         54 . An oligonucleotide for use in assays for target nucleic acid sequences, comprising a first segment complementary with at least a portion of said target nucleic acid sequences and a second segment, separated from said first segment with a linker segment, which is complementary to and capable of hybridizing with said first segment.  
     
     
         55 . The oligonucleotide of  claim 53  which is DNA or RNA.