Methods and compositions for enriching nucleic acids
The present invention relates to methods of enriching target single-stranded nucleic acids in a mixed population of single-stranded nucleic acids. The method involves protecting the target single-stranded nucleic acids and using a 5′ exonuclease to digest the non-target single-stranded nucleic acids. The invention also relates to methods of cloning target single-stranded nucleic acids into vectors, and to associated compositions and kits.
1. A method for enriching target single-stranded nucleic acids in a mixed population of single-stranded nucleic acids, said method comprising:
a) providing a mixed population of single-stranded nucleic acids containing target single-stranded nucleic acids and non-target single-stranded nucleic acids, wherein the target single-stranded nucleic acids comprise a target sequence at or within 21 bp of their 5′ ends;
b) contacting the mixed population of single-stranded nucleic acids with a blocking oligonucleotide, wherein the blocking oligonucleotide is capable of hybridising to the target sequence of the target single-stranded nucleic acids, resulting in the target nucleic acid lacking an affinity binding domain attached to a capture domain;
c) contacting the mixed population of nucleic acids of step b) with a single-strand specific 5′ exonuclease; and
d) incubating the mixed population of nucleic acids of step c) under conditions for the 5′ exonuclease to digest the non-target single-stranded nucleic acids, such that the target single-stranded nucleic acids are enriched,
wherein the blocking oligonucleotide has a melting temperature (Tm) of at least 45° C., at least 47° C., or at least 49° C.
2. The method of claim 1 , wherein the single-stranded nucleic acids are DNA or synthesized oligonucleotides.
3. The method of claim 1 , wherein the mixed population of single-stranded nucleic acids is a library that comprises members that have repetitive sequences or members that share regions of sequence identity.
4. The method of claim 1 , wherein the target single-stranded nucleic acids are from 50 to 1000 nucleotides in length, from 100 to 1000 nucleotides in length, or from 150 to 750 in nucleotides in length.
5. The method of claim 1 , wherein the target sequence is from 10 to 50 nucleotides in length.
6. The method of claim 1 , wherein the target sequence is located at the 5′ end of target single-stranded nucleic acid.
7. The method of claim 1 , wherein the blocking oligonucleotide is RNA.
8. The method of claim 1 , further comprising the step of partially or completely eliminating the blocking oligonucleotide.
9. The method of claim 8 , wherein eliminating is done by digesting the blocking oligonucleotide with a ribonuclease.
10. The method of claim 1 , wherein the single-strand specific 5′ exonuclease is an RecJ enzyme.
11. The method of claim 1 , further comprising, after step d), the step of inactivating and/or removing the single-strand specific 5′ exonuclease.
12. The method of claim 1 , further comprising providing a single-stranded binding protein (SSBP).
13. The method claim 1 , further comprising the step of incorporating the target single-stranded nucleic acids into a vector.
14. The method of claim 13 , wherein the target single-stranded nucleic acids are converted to target double-stranded nucleic acids prior to incorporation into the vector.
15. The method of claim 13 , wherein the target nucleic acids are to be incorporated into the vector using enzymatic assembly of overlapping DNA fragments.
16. The method of claim 13 , wherein the target single- or double-stranded nucleic acids and vector have corresponding overlapping regions configured to permit incorporation of the target single- or double-stranded nucleic acids into the vector by enzymatic assembly of overlapping DNA fragments.
17. The method of claim 13 , wherein incorporating includes the steps of:
i) providing a linear double-stranded DNA vector which comprises sequences at its 5′ and 3′ ends that overlap with sequences at the 5′ and 3′ ends of the target single- or double-stranded nucleic acids;
ii) contacting the linear double-stranded DNA vector with an exonuclease, which chews back the ends of the double-stranded DNA vector to produce a vector having single-stranded overhangs;
iii) contacting said vector having single-stranded overhangs with the target single-stranded nucleic acids or target double-stranded nucleic acids having single-stranded overhangs;
iv) annealing complementary sequences of the target single- or double-stranded nucleic acids and the overhangs of vector to form an annealed vector product;
v) contacting said annealed vector product with a DNA polymerase that extends the 3′ ends to fill gaps in the annealed product; and
vi) contacting the product of step v) with a ligase to heal nicks.