Homopolymer mediated nucleic acid amplification
According to some aspects of the invention, provided herein are methods of amplifying nucleic acids using homopolymer-mediated ligation. The methods, in some embodiments, comprise adding a first homopolymer of at least 12 nucleotides to each 3′ end of blunt-ended double-stranded nucleic acid containing a target nucleic acid, thereby producing a partially double-stranded nucleic acid.
1. A method, comprising
(a) adding a first homopolymer of at least 12 nucleotides to the 3′ end of each nucleic acid strand of a blunt-ended double-stranded nucleic acid containing a target nucleic acid by combining in a single reaction vessel the blunt-ended double-stranded nucleic acid, terminal deoxynucleotidyl transferase (TdT), deoxynucleotide triphosphate (dNTP), and a chain terminator, wherein the ratio of dNTP to chain terminator is at least 11 to 1, thereby producing a partially double-stranded nucleic acid having a homopolymer of at least 12 nucleotides at each 3′ end; and
(b) adding a second homopolymer of at least 4 nucleotides to the 5′ end of each strand of the partially double-stranded nucleic acid, wherein the second homopolymer is attached at its 5′ end to a first oligonucleotide and is shorter than and complementary to the first homopolymer.
2. The method of claim 1 , wherein the second homopolymer of at least 4 nucleotides is added to the 5 ′ end of each nucleic acid strand of the partially double-stranded nucleic acid by combining in a single reaction vessel the partially double-stranded nucleic acid, ligase, and the second homopolymer attached to the first oligonucleotide under conditions that permit ligation.
3. The method of claim 1 , further comprising amplifying the target nucleic acid by polymerase chain reaction using a first primer and a second primer,
wherein the first primer comprises, from 5′ to 3′, (a) a second oligonucleotide that is the same as the first oligonucleotide and (b) a third homopolymer that is the same as the second homopolymer, and
wherein the second primer comprises, from 5′ to 3′, (c) a third oligonucleotide that is different from the first and second oligonucleotides, and (d) a fourth homopolymer that is complementary to the first homopolymer, wherein the fourth homopolymer is longer than the second homopolymer.
4. The method of claim 3 , wherein the fourth homopolymer contains at least 12 nucleotides.
5. The method of claim 1 , wherein the first homopolymer contains deoxycytosine nucleotides or deoxyguanosine nucleotides.
6. The method of claim 1 , wherein the second homopolymer contains deoxycytosine nucleotides or deoxyguanosine nucleotides.
7. The method of claim 3 , wherein the third homopolymer and the fourth homopolymer each contains deoxycytosine nucleotides or deoxyguanosine nucleotides.
8. The method of claim 1 , wherein the first homopolymer is about 15 to about 30 nucleotides in length.
9. The method of claim 1 , wherein the first oligonucleotide contains a restriction endonuclease recognition site, a recombination site, or a promoter for in vitro transcription.
10. The method of claim 1 , wherein the chain terminator is a dideoxynucleotide (ddNTP).
11. The method of claim 10 , wherein the dideoxynucleotide is ddCTP, ddGTP or ddATP.
12. The method of claim 10 , wherein the ratio of dNTP to ddNTP is about 11 to 1 to about 29 to 1.
13. A method, comprising:
(a) adding a first homopolymer of at least 12 nucleotides to the 3′ end of each nucleic acid strand of a blunt-ended double-stranded nucleic acid containing, from 5′ to 3′, a known target region contiguous with an unknown target region, by combining in a single reaction vessel the blunt-ended double-stranded nucleic acid, terminal deoxynucleotidyl transferase (TdT), deoxynucleotide triphosphate (dNTP), and a chain terminator, wherein the ratio of dNTP to chain terminator is at least 11 to 1, thereby producing a first partially double-stranded nucleic acid having a homopolymer of at least 12 nucleotides at each 3′ end; and
(b) amplifying the known and unknown target regions by polymerase chain reaction using a first primer and a second primer, wherein the first primer contains, from 5′ to 3′, a first oligonucleotide and a second oligonucleotide that is the same as at least a portion of the known target region; and the second primer contains, from 5′ to 3′, a second oligonucleotide different from the first oligonucleotide, and a second homopolymer complementary to the first homopolymer.