Method and substances for isolation and detection of small polynucleotides

Abstract

A capture probe suitable for use with methods for isolating, labeling or detecting small polynucleotides. A method for isolating a small polynucleotide of interest from a sample comprising hybridizing the small polynucleotide to the capture probe and lengthening the small polynucleotide by primer extension or ligation. A method for detecting a small polynucleotide of interest following isolation by amplification of the primer extension products and/or hybridization and subsequent cleavage of dual labeled detector probes.

What is claimed is: 
     
         1 . A capture probe comprising a polynucleotide, the polynucleotide comprising:
 (a) a spacer segment comprising a spacer segment sequence, the spacer segment comprising a 3′ end and a 5′ end;   (b) a template segment comprising a template segment sequence, the template segment comprising a 3′ end and a 5′ end; and   (c) a small polynucleotide binding segment comprising a small polynucleotide binding segment sequence;   wherein the small polynucleotide binding segment is substantially complementary to, and capable of hybridizing to, one or more than one small polynucleotide of interest by Watson-Crick base pairing, wherein the small polynucleotide of interest is selected from the group consisting of a RNA polynucleotide, a DNA polynucleotide and a combination thereof;   wherein the 5′ end of the spacer segment is connected to the 3′ end of the small polynucleotide binding segment; and   wherein the 3′ end of template segment is connected to the 5′ end of the small polynucleotide binding segment.   
     
     
         2 . The capture probe of  claim 1 , further comprising a solid phase binding segment of a molecular composition capable of binding to a solid phase. 
     
     
         3 . The capture probe of  claim 1 , wherein the spacer segment includes a RNA polymerase termination site. 
     
     
         4 . The capture probe of  claim 1 , wherein the small polynucleotide binding segment is substantially complementary to, and capable of hybridizing to, a miRNA of interest. 
     
     
         5 . The capture probe of  claim 1 , wherein the template segment includes one or more than one sequence selected from the group consisting of:
 (a) a polynucleotide polymerase recognition site or a sequence that is complementary to a polynucleotide polymerase recognition site;   (b) one or more than one sequence that is a restriction enzyme recognition motif; and   (c) one or more than one sequence that is complementary to a RNA-cleaving catalytic nucleic acid.   
     
     
         6 . A composition comprising two or more capture probes according to  claim 1 , the composition comprising:
 (a) a first capture probe comprising a first spacer segment, a first small polynucleotide binding segment and a first template segment; and   (b) a second capture probe comprising a second spacer segment, a second small polynucleotide binding segment and a second template segment, where the second small polynucleotide binding segment comprises a different polynucleotide binding segment sequence than the first polynucleotide binding segment and the second template segment comprises a different template segment sequence than the first template segment.