Oligonucleotide tags for sorting and identification
First Claim
1. A method of sorting polynucleotides from a population of polynucleotides onto one or more solid phase supports, the method comprising the steps of:
- (a) attaching an oligonucleotide tag from a repertoire of tags to each polynucleotide in a population of polynucleotides such that each oligonucleotide tag from the repertoire is selected from a single minimally cross-hybridizing set of oligonucleotides having a length between 12 and 30 nucleotides, wherein each oligonucleotide of the set is separately synthesized and differs from every other oligonucleotide of the set by at least six nucleotides;
(b) sampling the population of polynucleotides such that substantially all different polynucleotides in the population have different oligonucleotide tags attached; and
(c) sorting the polynucleotides from the population by specifically hybridizing the oligonucleotide tags with their respective complements, the respective complements being attached as uniform populations of substantially identical oligonucleotides in spatially discrete regions on the one or more solid phase supports.
5 Assignments
0 Petitions
Accused Products
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 comprise oligonucleotides selected from a minimally cross-hybridizing set. Preferably, such oligonucleotides each consist of a plurality of subunits 3 to 9 nucleotides in length. 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.
631 Citations
33 Claims
-
1. A method of sorting polynucleotides from a population of polynucleotides onto one or more solid phase supports, the method comprising the steps of:
-
(a) attaching an oligonucleotide tag from a repertoire of tags to each polynucleotide in a population of polynucleotides such that each oligonucleotide tag from the repertoire is selected from a single minimally cross-hybridizing set of oligonucleotides having a length between 12 and 30 nucleotides, wherein each oligonucleotide of the set is separately synthesized and differs from every other oligonucleotide of the set by at least six nucleotides; (b) sampling the population of polynucleotides such that substantially all different polynucleotides in the population have different oligonucleotide tags attached; and (c) sorting the polynucleotides from the population by specifically hybridizing the oligonucleotide tags with their respective complements, the respective complements being attached as uniform populations of substantially identical oligonucleotides in spatially discrete regions on the one or more solid phase supports. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12)
-
-
13. A method of sorting polynucleotides from a population of polynucleotides onto one or more solid phase supports, the method comprising the steps of:
-
(a) attaching an oligonucleotide tag from a repertoire of tags to each polynucleotide in a population of polynucleotides such that each oligonucleotide tag from the repertoire is selected from a single minimally cross-hybridizing set of double stranded oligonucleotides having a length between 18 and 40 nucleotides, wherein each double stranded oligonucleotide of the set is separately synthesized and differs from every other double stranded oligonucleotide of the set by at least six nucleotides; (b) sampling the population of polynucleotides such that substantially all different polynucleotides in the population have different oligonucleotide tags attached; and (c) sorting the polynucleotides from the population by specifically hybridizing the oligonucleotide tags with their respective complements, the respective complements being attached as uniform populations of substantially identical oligonucleotides in spatially discrete regions on the one or more solid phase supports. - View Dependent Claims (14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 28, 29, 30, 31, 33)
-
-
25. The method of characterizing a population of mRNA molecules, the method comprising the steps of:
-
forming a population of cDNA molecules from the population of mRNA molecules such that each cDNA molecule has an oligonucleotide tag attached, the olignucleotide tags being selected from a single minimally cross-hybridizing set of olignucleotides having a length of 12 and 30 nucleotides, wherein each olignucleotide of the set is separately synthesized and differs from every other olignucleotide of the set by at least six nucleotides; sampling the population of cDNA molecules such that substantially all different cDNA molecules have different olignucleotide tags attached; sorting the cDNA molecules by specifically hybridizing the oligonucleotide tags with their respective complements, the respective complements being attached as uniform populations of substantially identical complements in spatially discrete regions on one or more solid supports; determining the nucleotide sequence of a portion of each of the sorted cDNA molecules; and characterizing the population of mRNA molecules by the frequency distribution of the portion of sequences of the cDNA molecules. - View Dependent Claims (26, 27)
-
-
32. The method of claim 32 wherein said number of loaded microparticles is at least 500,000.
Specification