Nucleic acid analysis using sequence tokens

• US 20070259357A1
• Filed: 01/22/2007
• Published: 11/08/2007
• Est. Priority Date: 01/23/2006
• Status: Active Grant

• Current Assignee: Personal Genome Diagnostics, Inc. (Laboratory Corporation Of America Holdings)
• Original Assignee: Population Genetics Technologies Ltd.
• Inventors: Brenner, Sydney
• Granted Patent: US 7,544,473 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• 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 2525/186 incorporating a non-extendable or blocking moiety
• CPC Class Codes: C12Q 2537/113 Heteroduplex formation
• CPC Class Codes: C12Q 2563/179 the label being a nucleic acid

Abstract

The present invention provides methods and compositions for tagging nucleic acid sequence fragments, e.g., a set of nucleic acid sequence fragments from a single genome, with one or more unique members of a collection of oligonucleotide tags, or sequence tokens, which, in turn, can be identified using a variety of readout platforms. As a general rule, a given sequence token is used once and only once in any tag sequence. In addition, the present invention also provides methods for using the sequence tokens to efficiently determine variations in nucleotide sequences in the associated nucleic acid sequence fragments.

Claims

1. A method of tagging a plurality of populations of polynucleotides each with a unique sequence token, comprising:

generating a plurality of unique sequence token tags;

dividing polynucleotides of each population into at least two non-overlapping nucleic acid segments; and

ligating a unique sequence token tag to each non-overlapping nucleic acid segment of each population of polynucleotides to provide a plurality of populations of polynucleotides each tagged with a unique sequence token tag,

wherein each unique sequence token tag is used to tag only one population of polynucleotides.

2. The method of claim 1, wherein said predetermined number of said non-overlapping segments is all non-overlapping segments of said polynucleotide.

3. The method of claim 2, wherein each of said non-overlapping segments has a length that is the same for all such segments.

4. The method of claim 1, wherein each population of polynucleotides comprises genomic DNA from a single subject.

5. The method of claim 4, wherein the subject is a human.

6. The method of claim 1, wherein said dividing is by restriction enzyme digestion of the polynucleotides.

7. A method of tagging a plurality of genomic DNA samples from a plurality of subjects each with a unique sequence token, comprising:

generating a plurality of unique sequence token tags;

dividing each genomic DNA sample from each subjects into at least two non-overlapping nucleic acid segments; and

ligating a unique sequence token tag to each non-overlapping nucleic acid segment of each genomic DNA sample to provide a plurality of genomic DNA samples from a plurality of subjects each tagged with a unique sequence token tag,

wherein each unique sequence token tag is used to tag only the genomic DNA from one subject.

8. The method of claim 7, wherein said predetermined number of said non-overlapping segments is all non-overlapping segments of said polynucleotide.

9. The method of claim 8, wherein each of said non-overlapping segments has a length that is the same for all such segments.

10. The method of claim 7, wherein the subject is a human.

11. The method of claim 7, wherein said dividing is by restriction enzyme digestion of the polynucleotides.

12. A method of screening for the presence or absence of a rare nucleotide allele of a polymorphism in a population of enriched genomic DNA segments from a population of genomic DNA samples, the method comprising the steps of:

incubating a reaction mixture under polymerization conditions, comprising;

(i) an enriched population of non-overlapping genomic DNA segments, wherein each genomic DNA segment comprises a unique sequence token,

(ii) an oligonucleotide probe complementary to a region of the genomic DNA segment upstream of a polymorphism, wherein the polymorphism comprises a rare nucleotide allele and a frequent nucleotide allele, and

(iii) dideoxy nucleotide triphosphate corresponding to the rare nucleotide allele and the frequent nucleotide allele, wherein the nucleotide triphosphate corresponding to the rare nucleotide allele is conjugated to a first member of a binding pair;

dividing the reaction mixture into at least two groups by exposing the reaction mixture to the second member of the binding pair to provide a first group comprising oligonucleotide probes having the nucleotide triphosphate corresponding to the rare nucleotide hybridized to the genomic DNA segments to provide a group of bound genomic DNA segments; and

determining the unique sequence tokens of the bound genomic DNA segments to identify genomic DNA samples having the rare nucleotide allele of the polymorphism.

13. The method of claim 12, wherein each of said non-overlapping genomic DNA segments have a length that is the same for all such segments.

14. The method of claim 12, wherein said first binding member is biotin, avidin, strepavidin, or a magnetic bead.

15. The method of claim 12, wherein said second binding member is biotin, avidin, strepavidin, or a magnetic bead.

16. The method of claim 12, wherein said step of determining includes specifically hybridizing said generated sequence tokens with complements thereof attached to one or more solid phase supports.

17. The method of claim 12, wherein said step of determining includes sequencing the sequence token or a portion thereof.

18. A sieving device, comprising:

at least one substrate support having an bottom surface and a top surface, wherein the bottom surface comprises a plurality linear elements and wherein each linear element comprises a unique oligonucleotide immobilized thereon; and

a receiving unit having top surface comprising a microfluidic channel, wherein the bottom surface of the substrate is positioned on the top surface of the receiving unit and the linear elements of the substrate extend into the microfluidic channel.

19. The sieving device of claim 18, further comprising a plurality of substrate supports.

20. A kit comprising:

a sieving device comprising at least one substrate having a top surface and a bottom surface, wherein the bottom surface comprises a plurality linear elements and wherein each linear element comprises a unique oligonucleotide immobilized thereon, and a receiving unit having top surface comprising a microfluidic channel, wherein the bottom surface of the substrate is positioned on the top surface of the receiving unit and the linear elements of the substrate extend into the microfluidic channel; and

instructions for using the sieving device to identify sequence tokens in a population.

21. The kit of claim 20, further comprising a plurality of substrate supports.