Derivatized versions of ligase enzymes for constructing DNA sequences
View Patent ↗A method of making very long, double-stranded synthetic poly-nucleotides. A multiplicity of short oligonucleotides is provided. The short oligonucleotides are sequentially hybridized to each other. Enzymatic ligation of the oligonucleotides provides a contiguous piece of PCR-ready DNA of predetermined sequence.
1. A method of constructing poly-nucleotides, comprising the steps of:
a) providing a mixture comprising a nucleic acid template chemically coupled to a protein ligase and single stranded DNAs complementary to the template, for ligation,
b) ligating strands of DNA using a protein ligase and a complementary sequence as a template, wherein said step of ligating utilizes hybridization to a complementary template which has been chemically coupled to a protein ligase enzyme.
2. The method of constructing polynucleotides of claim 1 including repeatedly adding single-stranded DNA to a growing piece of double-stranded DNA which is tethered to a protein ligase enzyme.
3. The method of constructing polynucleotides of claim 1 including repeatedly adding double-stranded DNA to a growing piece of double-stranded DNA which is tethered to a protein ligase enzyme.
4. A method of constructing very long, double-stranded synthetic poly-nucleotides comprising the steps of:
a) providing a mixture comprising a nucleic acid template chemically coupled to a protein ligase and single stranded DNAs complementary to the template, for ligation,
b) providing a multiplicity of oligonucleotides,
c) sequentially hybridizing said oligonucleotides to each other, and
d) enzymatic ligating said oligonucleotides to provide a contiguous piece of DNA of predetermined sequence, wherein said step of enzymatic ligating utilizes hybridization to a complementary template which has been chemically coupled to a protein ligase enzyme.
5. The method of constructing polynucleotides of claim 4 including repeatedly adding single-stranded DNA to a growing piece of double-stranded DNA which is tethered to a protein ligase enzyme.
6. The method of constructing polynucleotides of claim 4 including repeatedly adding double-stranded DNA to a growing piece of double-stranded DNA which is tethered to a protein ligase enzyme.
7. The method of constructing polynucleotides of claim 1 including repeatedly adding either single-stranded DNA or double-stranded DNA to a growing piece of double-stranded DNA which is tethered to a protein ligase enzyme.
8. A method of constructing very long, double-stranded synthetic polynucleotides comprising the steps of:
a) providing a mixture comprising a nucleic acid template chemically coupled to a protein ligase and single stranded DNAs complementary to the template, for ligation,
b) providing a multiplicity of short single-stranded oligonucleotides,
c) sequentially hybridizing said short single stranded oligonucleotides to each other, and
d) enzymatic ligating said short single-stranded oligonucleotides to provide a contiguous piece of double stranded DNA of predetermined sequence, wherein said step of ligating utilizes hybridization to a complementary template which has been chemically coupled to a protein ligase enzyme.
9. The method of constructing polynucleotides of claim 8 including repeatedly adding single-stranded DNA to a growing piece of double-stranded DNA which is tethered to a protein ligase enzyme.
10. The method of constructing polynucleotides of claim 8 including repeatedly adding double-stranded DNA to a growing piece of double-stranded DNA which is tethered to a protein ligase enzyme.