Compositions for sorting polynucleotides

Abstract

The invention provides a method of tracking, identifying, and/or sorting classes or subpopulations of molecules by the use of oligonucleotide tags. Oligonucleotide tags of the invention each consist of a plurality of subunits 3 to 6 nucleotides in length selected from a minimally cross-hybridizing set. A subunit of a minimally cross-hybridizing set forms a duplex or triplex having two or more mismatches with the complement of any other subunit of the same set. The number of oligonucleotide tags available in a particular embodiment depends on the number of subunits per tag and on the length of the subunit. An important aspect of the invention is the use of the oligonucleotide tags for sorting polynucleotides by specifically hybridizing tags attached to the polynucleotides to their complements on solid phase supports. This embodiment provides a readily automated system for manipulating and sorting polynucleotides, particularly useful in large-scale parallel operations, such as large-scale DNA sequencing, mRNA fingerprinting, and the like, wherein many target polynucleotides or many segments of a single target polynucleotide are sequenced simultaneously.

1. A composition of matter comprising:
 a solid phase support having one or more spacially discrete regions; and  
 a uniform  population of substantially  identical oligonucleotide tag complements covalently attached to the solid phase support in at least one of the one or more spacially discrete regions, the oligonucleotide tag complements comprising a plurality of subunits, each subunit consisting of an oligonucleotide having a length from three to six nucleotides and each subunit being selected from a minimally cross-hybridizing set, wherein a subunit of the set and a component of any other subunit of the set would have at least two mismatches.  
 
     
     
       2. The composition of matter of  claim 1  wherein said plurality of said subunits is in the range of from 4 to 10. 
     
     
       3. The composition of matter of  claim 2  wherein said solid phase support is a microparticle having a single spacially discrete region. 
     
     
       4. The composition of matter of  claim 3  wherein said microparticles  is selected from the group consisting of glass microparticles, magnetic beads, and polystyrene microparticles. 
     
     
       5. A composition of matter comprising a plurality of from ten thousand to a hundred thousand different polynucleotides, selected from cDNA molecules or fragments of a target polynucleotide to be analyzed or sequenced, said composition including a mixture of microparticles,
 wherein each microparticle has identical polynucleotides of the plurality attached thereto,    and wherein substantially all different polynucleotides in the plurality are attached to different microparticles.    
     
     
       6. The composition of  claim 5  wherein each microparticle has about  10   5 identical polynucleotides attached thereto.    
     
     
       7. A composition of matter comprising a plurality of different polynucleotides, selected from cDNA molecules or fragments of a target polynucleotide to be analyzed or sequenced, said composition including a mixture of microparticles,
 wherein tag complements are attached to each said microparticle,    and wherein each said cDNA molecule or fragment has an oligonucleotide tag attached, such that substantially all the same molecules have the same oligonucleotide tag attached and substantially all different molecules have different oligonucleotide tags attached,    such that perfectly matched duplexes are formed between the tag complements of said microparticles and the oligonucleotide tags of said cDNA molecules or fragments,    whereby, each microparticle has identical polynucleotides of the plurality attached thereto, and substantially all different polynucleotides in the plurality are attached to different microparticles.