Asymmetric adapter library construction
View Patent ↗The present invention provides methods and compositions for asymmetrically tagging a nucleic acid fragment using asymmetric adapters.
1. A hairpin adaptor comprising:
a) a double stranded stem region; and
b) a loop region that comprises a cleavable linkage that is positioned between two non-complementary regions, wherein the non-complementary region that is 5′ of the cleavable linkage comprises a primer binding site that is in the range of 8 to 100 nucleotides in length.
2. The hairpin adaptor of claim 1 , wherein primer binding site is in the range of 10-50 nucleotides in length.
3. The hairpin adaptor of claim 1 , wherein the hairpin adaptor comprises one or more of the following: a unique identifier (UID), an RNA polymerase promoter region, a restriction enzyme site, and a recombination site.
4. The hairpin adaptor of claim 1 , wherein the adaptor comprises a 5′ overhang.
5. The hairpin adaptor of claim 1 , wherein the adaptor comprises a 3′ overhang.
6. The hairpin adaptor of claim 1 , wherein the adaptor comprises a single nucleotide 3′ overhang.
7. The hairpin adaptor of claim 6 , wherein the single nucleotide is a dT.
8. The hairpin adaptor of claim 1 , wherein the cleavable linkage is enzymatically cleavable.
9. The hairpin adaptor of claim 8 , wherein the cleavable linkage is cleavable by an RNase.
10. The hairpin adaptor of claim 9 , wherein the RNAse is RNase 1.
11. The hairpin adaptor of claim 1 , wherein the cleavable linkage is a disulfide bond, and the cleaving is done using a reducing agent.
12. A kit comprising:
the hairpin adaptor of claim 1 ; and
a primer that hybridizes to the primer binding site in the non-complementary region that is 5′ of the cleavable linkage.
13. The kit of claim 12 , wherein the primer is in the range of 10-50 nucleotides in length.