Enzymatic nucleic acid synthesis: compositions and methods for inhibiting pyrophosphorolysis
View Patent ↗Nucleotide triphosphate probes containing a molecular and/or atomic tag on a a γ and/or β phosphate group and/or a base moiety having a detectable property are disclosed, and kits and method for using the tagged nucleotides in sequencing reactions and various assay. Also, phosphate and polyphosphate molecular fidelity altering agents are disclosed.
1. A composition comprising a purified polymerase, a nucleic acid template molecule, and at least one nucleotide compound selected from the group consisting of compounds of the following general formulas:
Z—OPO 2 —(OP(EE′)O) n —PO 2 O —Z′ (e)
Z—OPO 2 —(OP(EE′)O) n PO 2 —Z′ (f)
Z—PO 2 —(OP(EE′)O) n PO 2 O—Z′ (g)
Z—PO 2 —(OP(EE′)O) n PO 2 —Z′ (h)
where Z and Z′ are each independently a hydrogen atom or a thermally stable substituent, E and E′ are each independently an oxygen atom or a thermally stable substituent, and n is an integer having a value of at least two.
2. A method for performing a nucleotide incorporation, comprising:
forming a nucleotide polymerization reaction mixture including a polymerase, a template nucleic acid molecule, and a nucleotide analog selected from the group consisting of nucleotide compounds having the following general formulas:
Z—OPO 2 —(OP(EE′)O) n —PO 2 O —Z′ (e)
Z—OPO 2 —(OP(EE′)O) n PO 2 —Z′ (f)
Z—PO 2 —(OP(EE′)O) n PO 2 O—Z′ (g)
and
Z—PO 2 —(OP(EE′)O) n PO 2 —Z′ (h)
where Z and Z′ are each independently a hydrogen atom or a thermally stable substituent, E and E′ are each independently an oxygen atom or a thermally stable substituent, and n is an integer having a value of at least 2; and
incorporating the nucleotide analog into a nascent nucleic acid molecule in a template-dependent manner using the polymerase.
3. A method for nucleotide incorporation, comprising: forming a sequencing reaction mixture including a template nucleic acid molecule, a polymerase and at least one nucleotide compound having a formula selected from the group consisting of:
Z—OPO 2 —(OP(EE′)O) n —PO 2 O —Z′ (e)
Z—OPO 2 —(OP(EE′)O) n PO 2 —Z′ (f)
Z—PO 2 —(OP(EE′)O) n PO 2 O—Z′ (g)
and
Z—PO 2 —(OP(EE′)O) n PO 2 —Z′ (h)
where Z and Z′ are each independently a hydrogen atom or a thermally stable substituent, E and E′ are each independently an oxygen atom or a thermally stable substituent, and n is an integer having a value of at least 2;
incorporating one or more nucleotide compounds of the sequencing reaction mixture into a nascent nucleic acid molecule in a template-dependent manner using the polymerase;
detecting a signal that indicates the incorporation of at least one of the nucleotide compounds into the nascent nucleic acid molecule by the polymerase.
4. The method of claim 3 , further including identifying at least one of the incorporated nucleotide compounds, thereby obtaining sequence information from the template nucleic acid molecule.
5. The composition of claim 1 , wherein n is 2.
6. The composition of claim 1 , wherein n is at least 3.
7. The composition of claim 1 , wherein n is at least 4.
8. The composition of claim 1 , wherein n is at least 5.
9. The method of claim 3 , wherein n is 2.
10. The method of claim 3 , wherein n is at least 3.
11. The method of claim 3 , wherein n is at least 4.
12. The method of claim 3 , wherein n is at least 5.
13. A method for performing a nucleotide incorporation, comprising: contacting a polymerase with a template nucleic acid molecule and a nucleotide including a polyphosphate chain including at least four phosphate groups, and incorporating the nucleotide into a nascent nucleic acid molecule in a template-dependent manner using the polymerase.
14. The method of claim 13 , further including identifying the nucleotide that is incorporated into the nascent nucleic acid molecule.
15. The method of claim 13 , further including: detecting a signal indicating the template-dependent incorporation of the nucleotide into the nascent nucleic acid molecule by the polymerase.
16. The method of claim 15 , wherein the nucleotide includes an optically detectable label that generates an optically detectable emission indicating the template-dependent incorporation.
17. The method of claim 16 , wherein detecting the signal further includes detecting a change in the intensity, the frequency or the duration of the optically detectable emission.
18. The method of claim 16 , wherein the polymerase tag includes a fluorescent resonance energy transfer (FRET) donor and the label of the nucleotide includes a FRET acceptor, and wherein the incorporating further includes performing energy transfer between the FRET donor and the FRET acceptor.
19. The method of claim 13 , wherein the polyphosphate chain includes at least five phosphate groups.
20. The method of claim 17 , further including identifying the nucleotide that is incorporated by analyzing the signal.
21. The method of claim 20 , wherein the identifying the nucleotide includes determining the base identity of the nucleotide.