Compositions and Methods for Intramolecular Nucleic Acid Rearrangement

• US 20120135407A1
• Filed: 08/13/2010
• Published: 05/31/2012
• Est. Priority Date: 08/20/2009
• Status: Active Grant

• Current Assignee: 10X Genomics Incorporated
• Original Assignee: Population Genetics Technologies Ltd.
• Inventors: Brenner, Sydney, Mikawa, Gi, Slatter, Andrew, Osborne, Robert
• Granted Patent: US 8,298,767 B2
• Time in Patent Office: Days
• US Class Current: 435/6.11
• 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: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6855 Ligating adaptors
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2525/143 incorporating a promoter sequence
• CPC Class Codes: C12Q 2525/155 incorporating/generating a new priming site
• CPC Class Codes: C12Q 2525/161 incorporating target specific and non-target specific sites
• CPC Class Codes: C12Q 2535/122 Massive parallel sequencing
• 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

Abstract

Aspects of the present invention are drawn to processes for moving a region of interest in a polynucleotide from a first position to a second position with regard to a domain within the polynucleotide, also referred to as a “reflex method”. In certain embodiments, the reflex method results in moving a region of interest into functional proximity to specific domain elements present in the polynucleotide (e.g., primer sites and/or MID). Compositions, kits and systems that find use in carrying out the reflex processes described herein are also provided.

Claims

1. A method of sample analysis, comprising:

a) annealing a reflex sequence in a polynucleotide strand in a sample with a complement of the reflex sequence present in the polynucleotide strand, wherein the polynucleotide strand comprises the following in a 5′

to 3′

orientation;

a first domain comprising i. a multiplex identifier (MID) whose identity can be used to differentiate polynucleotides in the sample, and ii. a primer site_;

the reflex sequence;

a polynucleotide of interest having a desired site that is distal to the first domain; and

the complement of the reflex sequence, wherein the complement of the reflex sequence is present at the 3′

end of the polynucleotide strand;

b) extending from the 3′

end of the complement of the reflex sequence through the first domain to produce a double stranded region comprising a complement of the first domain at the 3′

end of the extended polynucleotide strand, thereby moving the first domain into proximity with the desired site; and

c) sequencing the MID and the desired site using a primer that complementary to the primer site in the complement of the first domain.

4. The method of claim 1, wherein the reflex sequence is from 10 to 50 nucleotides in length.

5. The method of claim 1, wherein the first domain comprises an RNA polymerase site.

6. The method of claim 1, the method further comprising removing the first domain and the reflex sequence from the 5′

end of the double stranded region.

7. The method of claim 6, wherein the removing step comprises treating the nucleic acid of step b with T7 exonuclease.

8. The method of claim 1, wherein the first domain is present in an adapter region attached to the polynucleotide of interest.

9. The method of claim 8, wherein the reflex sequence is present in the adapter region.

10. The method of claim 8, wherein the reflex sequence is a sequence present within the 5′

region of the polynucleotide of interest.

11. The method of claim 1, wherein the sample is a multiplexed sample, wherein the first domain in each of the polynucleotide strands in the multiplexed sample comprises a MID corresponding to its parental sample.

12. The method of claim 1, wherein the sample comprises multiple polynucleotide strands, and wherein each polynucleotide strand in the sample comprises a unique MID in the first domain.

13. The method of claim 1, wherein the polynucleotide strand is produced by:

annealing a nucleic acid synthesis primer to a parent polynucleotide strand, wherein the parent polynucleotide strand comprises the following in a 5′

to 3′

orientation;

the first domain;

the reflex sequence; and

a second domain comprising the polynucleotide of interest;

wherein the nucleic acid synthesis primer comprises;

a 3′

priming domain that is complementary to a site adjacent to and 3′

of the polynucleotide of interest in the second domain; and

a 5′

reflex sequence;

extending the nucleic acid synthesis primer to produce an extension product; and

replicating the resultant extension product with a second nucleic acid synthesis primer that anneals at the 3′

end of the complement of the first domain, thereby producing the polynucleotide strand.

14. The method of claim 13, wherein the length of the second domain is from 30 bases to 30 kilobases.

15. The method of claim 13, wherein the parent polynucleotide strand is enriched prior to the annealing step based on its sequence.

16. The method of claim 1, further comprising the steps of:

c) replicating the polynucleotide strand of step b with a first nucleic acid synthesis primer that anneals at the 3′

end of the complement of the first domain to produce a first extension product;

d) annealing a second nucleic acid synthesis primer to the first extension product, wherein the second nucleic acid synthesis primer comprises a 3′

priming domain that is complementary to a site within the polynucleotide of interest in the first extension product; and

e) extending the second synthesis primer to produce a second extension product.

17. The method of claim 16, wherein the second synthesis primer comprises a 5′

domain that is not complementary to the polynucleotide of interest in the first extension product.

18. The method of claim 17, wherein the first domain comprises a first primer site and the 5′

domain of the second synthesis primer comprises a second primer site.

19. The method of claim 18, wherein the length of the polynucleotide of interest in the second extension product is from 50 to 10,000 nucleotides.

20. A kit comprising:

a polynucleotide strand comprising the following in a 5′

to 3′

orientation;

a first domain;

a reflex sequence;

a polynucleotide of interest; and

a complement of the reflex sequence, wherein the complement of the reflex sequence is

present at the 3′

end of the polynucleotide strand; and

instructions for performing the method of claim 1.

21. The method of claim 1, wherein polynucleotide of interest is cDNA.

22. The method of claim 1, further comprising repeating said method to obtain the nucleotide sequence of a second desired site in said polynucleotide of interest, and analyzing the sequence obtained in c) and the sequence of the second desired site to obtain linkage information about polymorphisms in said polynucleotide of interest.

2-3. -3. (canceled)