Processes for directionally ligating double stranded nucleic acids
View Patent ↗Amplification mixtures, kits, amplicons, kits and processes are provided for amplifying a nucleic acid. In particular, provided are processes which utilize an amplification mixture comprising a polymerase, a deoxynucleotidetriphosphate (dNTP) mixture which contains modified dNTPs, a first primer and a second primer. Further provided are dNTP mixtures which contain modified dNTPs for at least two of the four nucleotide triphosphates, which when incorporated into a polynucelotide, impart resistance to enzymatic degradation by an exonuclease at the sites of incorporation of the modified dNTPs. Also provided are the amplicons and vectors which incorporate the modified nucleotides.
1. A process for directionally ligating a double-stranded nucleic acid to a first adaptor sequence, the process comprising:
a. forming an amplification product from the double-stranded nucleic acid using a mixture comprising
(i) a polymerase,
(ii) a deoxynucleotidetriphosphate (dNTP) mixture, the dNTP mixture comprising non-modified dNTPs for at least one of the four nucleotide triphosphates comprising dATP, dGTP, dCTP, dTTP and analogs thereof which, when incorporated into a polynucleotide, do not impart resistance against enzymatic degradation by an exonuclease at the site of incorporation of the non-modified dNTPs, and modified dNTPs for at least one of the four nucleotide triphosphates comprising dATP, dGTP, dCTP, dTTP and analogs thereof which, when incorporated into a polynucleotide, impart resistance against enzymatic degradation by an exonuclease at the site of incorporation of the modified dNTPs,
(iii) a first primer complementary to a first strand of the double-stranded nucleic acid, said first primer having a first terminus complementary to a first ligation site sequence of the first adaptor sequence, and
(iv) a second primer complementary to a second strand of the double-stranded nucleic acid, said second primer having a second terminus complementary to a second ligation site sequence of a second adaptor sequence, wherein the first terminus of the first primer and the second terminus of the second primer are not identical;
b. treating the amplification product with the exonuclease to form a digested amplicon having a first overhang sequence at a first termini and a second overhang sequence at a second termini, wherein the first and second termini of said digested amplicon terminate at the sites of incorporation of said modified dNTPs, and wherein the first overhang sequence at the first termini is complementary to the first ligation site sequence of the first adaptor sequence and the second overhang sequence at the second termini is complementary to the second ligation site sequence of the second adapter; and
c. ligating the first overhang sequence of the digested amplicon to the first ligation site sequence of the first adaptor sequence.
2. The process of claim 1 wherein the first adaptor sequence comprises a nucleotide sequence encoding at least one epitope tag.
3. The process of claim 1 wherein the process further comprises ligating the second overhang sequence of the digested amplicon to the second ligation site sequence of the second adaptor sequence.
4. The process of claim 3 wherein the first ligation site sequence and the second ligation site sequence are ends of a cloning vector.
5. The process of claim 1 wherein the modified dNTPs are alpha phosphate modified dNTPs.
6. The process of claim 5 wherein the alpha phosphate modified dNTPs are alpha phosphate thio-substituted dNTPs or alpha phosphate borano-substituted dNTPs.
7. The process of claim 1 wherein the amount of the four nucleotide triphosphates in the dNTP mixture is determined by the ratio of the concentration of modified dNTP 1 relative to the concentration of non-modified dNTP 1 .
8. The process of claim 1 wherein the dNTP mixture comprises modified dNTPs for two of the four nucleotide triphosphates.
9. The process of claim 8 wherein the amount of the four nucleotide triphosphates in the dNTP mixture is determined by the ratio of the concentration of modified dNTP 1 to the concentration of modified dNTP 2 relative to the concentration of non-modified dNTP 1 to the concentration of non-modified dNTP 2 , wherein dNTP 1 and dNTP 2 is dATP, dCTP, dGTP or dTTP, provided that dNTP 1 and dNTP 2 are not identical.
10. The process of claim 8 wherein one of the modified dNTPs is an alpha thiophosphorano dNTP and another of the modified dNTPs is an alpha boranophosphorano dNTP.
11. The process of claim 8 wherein each of the modified dNTPs is an alpha thiophosphorano dNTP.
12. The process of claim 1 wherein the first ligation site sequence is an Acc65I, AflII, AgeI, AcaI, ApoI, AvrII, BamHI, BglII, BsiWI, EagI, EcoRI, HindIII, NcoI, NgoMIV, NheI, NotI, SalI, XbaI, XhoI or XmaI recognition sequence, and the second ligation site sequence is an Acc65I, AflII, AgeI, AcaI, ApoI, AvrII, BamHI, BglII, BsiWI, EagI, EcoRI, HindIII, NcoI, NgoMIV, NheI, NotI, SalI, XbaI, XhoI or XmaI recognition sequence.
13. A process for cloning a nucleic acid into a vector, the process comprising:
a. forming an amplification product from the double-stranded nucleic acid using a mixture comprising
(i) a polymerase,
(ii) a deoxynucleotidetriphosphate (dNTP) mixture, the dNTP mixture comprising non-modified dNTPs for at least one of the four nucleotide triphosphates comprising dATP, dGTP, dCTP, dTTP and analogs thereof which, when incorporated into a polynucleotide, do not impart resistance against enzymatic degradation by an exonuclease at the site of incorporation of the non-modified dNTPs, and modified dNTPs for at least two of the four nucleotide triphosphates comprising dATP, dGTP, dCTP, dTTP and analogs thereof which, when incorporated into a polynucleotide, impart resistance against enzymatic degradation by an exonuclease at the site of incorporation of the modified dNTPs,
(iii) a first primer complementary to a first strand of the double-stranded nucleic acid, said first primer having a first terminus complementary to a first ligation site sequence of the vector, and
(iv) a second primer complementary to a second strand of the double-stranded nucleic acid, said second primer having a second terminus complementary to a second ligation site sequence of the vector;
b. treating the amplification product with the exonuclease to form a digested amplicon, wherein both termini of said digested amplicon terminate at the sites of incorporation of said modified dNTPs, and wherein a first termini of said digested amplicon is complementary to the first ligation site sequence of the vector and a second termini of said digested amplicon is complementary to the second ligation site sequence of the vector; and
c. ligating the digested amplicon to a first and second ligation site sequence of the vector.
14. The process of claim 13 wherein said process further comprises:
a. transforming a host cell with said vector; and
b. identifying a clone of host cells that contains the digested amplicon in said vector.
15. The process of claim 13 wherein the first and second ligation site of the cloning vector are not identical.
16. The process of claim 15 wherein at least 80% of the digested amplicons are ligated in only one direction to the first and second ligation site sequences of said vector.