Nucleotide compositions comprising photocleavable markers and methods of preparation thereof
Labelled nucleotides and polynucleotides useful in the sequencing of nucleic acids are described. Methods of preparing photocleavable marker nucleotides and photocleavable marker-polynucleotide conjugates are described. Such photocleavable markere nucleotides can be incorporated into nucleic acid so as to create photocleavable marker-polynucleotide conjugates.
1. A method of preparing a marker-photocleavable linker-nucleotide conjugate comprising:
a) providing i) a photocleavable linker comprising a protective group, ii) an amino-nucleotide analog, and iii) an activated marker molecule selected from the group consisting of BODIPY-FL-SF and Cy5-NHS;
b) operably linking said photocleavable linker to said amino-nucleotide analog to produce a photocleavable linker-nucleotide conjugate;
c) removing said protective group from said photocleavable linker-nucleotide conjugate under conditions such that an activated photocleavable linker-nucleotide conjugate is created, wherein said activated photocleavable linker-nucleotide conjugate has an exposed reactive site on the linker portion of the conjugate; and
d) contacting said activated marker molecule with said activated photocleavable linker-nucleotide conjugate under conditions such that a marker-photocleavable linker-nucleotide conjugate is produced.
2. The method of claim 1 , wherein said protective group is selected from the group consisting of Fmoc and Bpoc.
3. The method of claim 1 , wherein said amino-nucleotide analog is 5-(3-aminoallyl)-2′-deoxyuridine 5′-triphosphate.
4. The method of claim 1 further comprising: p 1 e) contacting said marker-photocleavable linker-nucleotide conjugate of step (d) with a modifying enzyme, in the presence of nucleic acid, under conditions such that said marker-photocleavable linker-nucleotide conjugate is incorporated into said nucleic acid to produce a labeled nucleic acid.
5. The method of claim 4 , further comprising f) detecting said incorporated marker of said marker-photocleavable linker-nucleotide conjugate in said labeled nucleic acid.
6. The method of claim 5 , further comprising, prior to step f), the step of separating unincorporated marker-photocleavable linker-nucleotide coiijugate from said labeled nucleic acid.
7. The method of claim 4 , further comprising the step of removing the incorporated photocleavable marker from said labeled nucleic acid by exposing said labeled nucleic acid to electromagnetic radiation so as to create a treated nucleic acid.
8. The method of claim 7 , further comprising subjecting said treated nucleic acid to a further subsequent labeling reaction with a different marker-photocleavable linker-nucleotide conjugate after said removing step.
9. The method of claim 4 , wherein said nucleic acid-modifying enzyme is a terminal deoxynucleotidyl transferase.
10. The marker-photocleavable linker-nucleotide conjugate produced according to claim 1 .
11. A marker-photocleavable linker-nucleotide conjugate of the structure:
wherein F is a fluorophore marker and PPP is: