Method for reducing adapter-dimer formation
Methods are provided for ligating a 3′ adapter and a 5′ adapter to a target polynucleotide so as to avoid adapter dimer formation. Embodiments of the methods include adding a blocking oligonucleotide after the first ligation in which a 3′ adapter is ligated to the target polynucleotide so that the blocking oligonucleotide is capable of hybridizing to excess 3′ adapter and the ligated 3′ adapter. Subsequently, a 5′ adapter is ligated to the target polynucleotide thus avoiding adapter dimer formation.
1. A kit comprising:
(a) a 3′ adapter oligonucleotide having a non-natural 3′ end that prevents ligation of the 3′ end of the 3′ adapter oligonucleotide to another polynucleotide;
(b) a blocking oligonucleotide that has a 3′ hydroxyl group and at least 10 nucleotides of complementary to the 3′ adapter, wherein the blocking oligonucleotide:
hybridizes to the 3′ adaptor oligonucleotide to produce a duplex having a blunt end comprising the 3′ hydroxyl group of the blocking oligonucleotide; and
primes cDNA synthesis of an RNA polynucleotide when the 3′ adaptor oligonucleotide is ligated to the 3′ end of the RNA polynucleotide; and
(c) a buffer comprising a non-naturally occurring buffering agent.
2. A kit according to claim 1 , further comprising a first ligase.
3. A kit according to claim 1 further comprising a second ligase.
4. A kit according to claim 3 , wherein the second ligase is a T4 mutant ligase.
5. A kit according to claim 1 , wherein the blocking oligonucleotide is a DNA.
6. A kit according to claim 1 further comprising a 5′ adapter.
7. A kit according to claim 1 , further comprising a reverse transcriptase.
8. A kit according to claim 1 , wherein the 3′ adapter has a length of 10-50 nucleotides.
9. A kit according to claim 1 , wherein the 3′ adapter is pre-adenylated.
10. A reaction mixture, comprising:
(a) a 3′ adapter oligonucleotide having a non-natural 3′ end that prevents ligation of the 3′ end of the 3′ adapter oligonucleotide to another polynucleotide ;
(b) a buffer comprising a non-naturally occurring buffering agent; and
(c) a polynucleotide; and
(d) a blocking oligonucleotide has a 3′ hydroxyl group and at least 10 base pairs of complementary to the 3′ adapter oligonucleotide, wherein:
i. some of the 3′ adapter oligonucleotide is ligated to the 3′ end of the polynucleotide and some of the 3′ adapter oligonucleotide is not ligated to the 3′ end of the polynucleotide; and
ii. the blocking oligonucleotide is hybridized to at least the 3′ adapter oligonucleotide that is not ligated to the 3′ end of the polynucleotide to produce a duplex having a blunt end comprising the 3′ hydroxyl group of the blocking oligonucleotide.
11. A reaction mixture according to claim 10 , wherein the polynucleotide is an RNA and the 3′ adapter is a DNA.
12. A reaction mixture according to claim 10 , further comprising a 5′ adapter ligated to the polynucleotide.
13. A reaction mixture according to claim 10 , further comprising a reverse transcriptase.
14. A reaction mixture according to claim 10 , wherein the polynucleotide is a member of a polynucleotide library wherein the polynucleotides in the library are ligated to 3′ adapters.
15. A reaction mixture according to claim 10 , wherein the 3′ adapters contain a barcode.
16. A reaction mixture comprising:
(a) a 3′ adapter oligonucleotide having a non-natural 3′ end that prevents ligation of the 3′ end of the 3′ adapter oligonucleotide to another polynucleotide;
(b) a buffer comprising a non-naturally occurring buffering agent; and
(c) a polynucleotide; and
(d) a blocking oligonucleotide has a 3′ hydroxyl group and at least 10 nucleotides of complementary to the 3′ adapter oligonucleotide, wherein:
i. some of the 3′ adapter oligonucleotide is ligated to the 3′ end of the polynucleotide and some of the 3′ adapter oligonucleotide is not ligated to the 3′ end of the polynucleotide; and
ii. the blocking oligonucleotide is hybridized to at least the 3′ adapter oligonucleotide that is not ligated to the 3′ end of the polynucleotide,
wherein different polynucleotides ligated to a 3′ adapter are pooled from multiple samples and the blocking oligonucleotide is hybridized to excess 3′ adapter oligonucleotide.
17. A reaction mixture according to claim 10 , wherein the polynucleotide is a member of a library of polynucleotides.