Methods and Compositions for Tagging and Identifying Polynucleotides

• US 20140011708A1
• Filed: 03/28/2013
• Published: 01/09/2014
• Est. Priority Date: 02/10/2005
• Status: Active Grant

• Current Assignee: Agilent Technologies, Inc.
• Original Assignee: Population Genetics Technologies Ltd.
• Inventors: Brenner, Sydney
• Granted Patent: US 9,018,365 B2
• Time in Patent Office: Days
• US Class Current: 506/26
• CPC Class Codes: C12N 15/1065 Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags
• CPC Class Codes: C12N 15/1093 General methods of preparing gene libraries, not provided for in other subgroups
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
• CPC Class Codes: C12Q 2563/179 the label being a nucleic acid
• CPC Class Codes: C12Q 2565/514 characterised by the use of the arrayed oligonucleotides as identifier tags, e.g. universal addressable array, anti-tag or tag complement array
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

The invention provides methods and compositions for attaching oligonucleotide tags to polynucleotides for the purpose of carrying out analytical assays in parallel and for decoding the oligonucleotide tags of polynucleotides selected in such assays. Words, or subunits, of oligonucleotide tags index submixtures in successively more complex sets of submixtures (referred to herein as “tiers” of submixtures) that a polynucleotide goes through while successive words are added to a growing tag. By identifying each word of an oligonucleotide tag, a series of submixtures is identified including the first submixture that contains only a single polynucleotide, thereby providing the identity of the selected polynucleotide. The analysis of the words of an oligonucleotide tag can be carried out in parallel, e.g. by specific hybridization of the oligonucleotide tag to its tag complement on an addressable array; or such analysis can be carried out serially by successive specific hybridizations of labeled word complements, or the like.

Claims

1-21. -21. (canceled)

22. A composition of nucleic acid molecules made by the following method:

ligating a discriminatory oligonucleotide tag to fragmented genomic DNA so that substantially every target polynucleotide in said fragmented genomic DNA receives a tag that comprises a unique nucleotide sequence, to produce a tagged sample; and

amplifying the target polynucleotides in said tagged sample by polymerase chain reaction to produce a plurality of amplicons that each comprise a unique tag.

23. The method of claim 22, wherein said fragmented genomic DNA is fragmented by digestion with a restriction enzyme.

24. The method of claim 22, wherein said fragmented genomic DNA is fragmented mammalian genomic DNA.