Compositions and methods for intramolecular nucleic acid rearrangement
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 multiplex indentifier). Compositions, kits and systems that find use in carrying out the reflex processes described herein are also provided.
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;
the reflex sequence;
a polynucleotide of interest that is from 100 bases to 30 kilo bases in length and 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) amplifying the desired site from the polynucleotide strand of step b) using a first nucleic acid synthesis primer that anneals at the 3′ end of the complement of the first domain and a second nucleic acid synthesis primer that comprises a 3′ priming domain that is complementary to a site within the polynucleotide of interest.
2. The method of claim 1 , wherein the first domain comprises a multiplex identifier (MID).
3. The method of claim 1 , wherein the first domain comprises a primer site.
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 that identifies the sample from which the polynucleotide strand is derived.
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 parent polynucleotide strand is enriched prior to the annealing step based on its sequence.
15. The method of claim 1 , wherein the second synthesis primer comprises a 5′ domain that is not complementary to the polynucleotide of interest in the first extension product.
16. The method of claim 15 , wherein the first domain comprises a first primer site and the 5′ domain of the second synthesis primer comprises a second primer site.
17. The method of claim 1 , further comprising sequencing the desired site.