Methods and compositions for tagging and identifying polynucleotides
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.
1. A method of estimating the number of target polynucleotides in a sample, comprising:
ligating a discriminatory oligonucleotide tag to a complex nucleic acid sample comprising multiple molecular species so that substantially every target polynucleotide receives a tag that comprises a unique nucleotide sequence, to produce a tagged sample;
amplifying the target polynucleotides in said tagged sample; and
determining the number of different tags that are present in the amplified target polynucleotides, thereby providing an estimate of the number of target polynucleotides that are amplified.
2. The method of claim 1 , wherein said amplifying uses a primer that hybridizes to said oligonucleotide tag.
3. The method of claim 1 , wherein said target polynucleotide is a fragment of genomic DNA.
4. The method of claim 1 , wherein said determining is done by hybridizing the amplified target polynucleotides to an array.
5. The method of claim 1 , wherein said determining is done by DNA sequencing.
6. The method of claim 1 , further comprising sequencing the amplified target polynucleotides.
7. The method of claim 1 , wherein said oligonucleotide tag comprises a degenerate discriminatory sequence of 2 to 18 nucleotides.
8. The method of claim 1 , wherein said target polynucleotides are isolated prior to amplification.
9. The method of claim 8 , wherein said target polynucleotides are isolated by hybridizing said pooled sample to a probe that is immobilized to a solid support.
10. The method of claim 8 , wherein said target polynucleotides are isolated by hybridizing said pooled sample to a biotinylated probe that is captured by a solid support.
11. The method of claim 8 , wherein said isolating is done by exonuclease digestion of non-circularized probes.
12. The method of claim 8 , wherein said isolating is done by template-driven ligation.
13. The method of claim 8 , wherein said isolating is done by template-driven strand extension.